Harnessing the power of enzymes for environmental stewardship

JBA-06412; No of Pages 21 Contents lists available at Biotechnology Advances Research review paper Harnessing the power of enzymes for environmental stewardship Philippe Demarche, Charles Junghanns, Rakesh R. Nair, Spiros N. Agathos Earth & Life Institute, Laboratory of Bioengineering, Université Catholique de Louvain, Place Croix du Sud 2/19, 1348 Louvain-la-Neuve, Belgium Available online xxxx Enzymes are versatile catalysts with a growing number of applications in biotechnology. Their propertiesrender them also attractive for waste/pollutant treatment processes and their use might be advantageous over conventional treatments. This review highlights enzymes that are suitable for waste treatment, with a Pollutant biodegradation focus on cell-free applications or processes with extracellular and immobilized enzymes. Biological wastes are treated with hydrolases, primarily to degrade biological polymers in a pre-treatment step. Oxidoreductases and lyases are used to biotransform speciﬁc pollutants of various nature. Examples from pulp and paper, textile, food and beverage as well as water and chemical industries illustrate the state of the art of enzymatic pollution treatment. Research directions in enzyme technology and their importance for future development Directed evolution in environmental biotechnology are elaborated. Beside biological and biochemical approaches, i.e. enzyme Enzyme immobilization prospection and the design of enzymes, the review also covers efforts in adjacent research ﬁelds such as insolubilization of enzymes, reactor design and the use of additives. The effectiveness of enzymatic processes, especially when combined with established technologies, is evident. However, only a limited number ofenzymatic ﬁeld applications exist. Factors like cost and stability of biocatalysts need to be addressed and thecollaboration and exchange between academia and industry should be further strengthened to achieve thegoal of sustainability.
2011 Elsevier Inc. All rights reserved.
Pollution control via enzyme technology in industries . . . . . . . . . . . . . . . . . . . . . .
Abbreviations: 4-CP, 4-chlorophenol; ABTS, 2,2′-azino-bis-3-ethylbenzothiozoline-6-sulphonic acid; AOP, Advanced oxidation process; BOD, Biochemical oxygen demand; BPA, Bisphenol A; CDH, Cellobiose dehydrogenase; CLEA®, Crosslinked enzyme aggregate; COD, Chemical oxygen demand; CYP450, Cytochrome P450; CSTR, Continuous stirred-tankreactor; DEHP, Di-(2-ethylhexyl)-phthalate; DHA, Haloalkane dehalogenase; DMP, Dimethyl phthalate; EDC, Endocrine disrupting chemical; EE2, 17α-ethinylestradiol; E1, Estrone;E2, 17β-estradiol; E3, Estriol; FBR, Fluidized bed reactor; FMR, Flat membrane reactor; HAA, Haloacetic acids; Hb, Hemoglobin; HBT, N-hydroxybenzotriazole; HRP, Horseradishperoxidase; LME, Lignin-modifying enzyme; MBR, Membrane bioreactor; MnP, Manganese peroxidase; MOW, Mixed ofﬁce waste; NHase, Nitrile hydratase; NP, Nonylphenol; OP,Organophosphorus compound; OPAA, Organophosphorus acid anhydrolase; OPH, Organophosphorus hydrolase; O&G, Oil and grease; PAH, Polycyclic aromatic hydrocarbons; PBR,Packed bed reactor; PEG, Polyethylene glycol; PeMP, Pentyl methyl phthalate; PTT, Poly(trimethylene terephthalate); RT, Residence time; TAH, Tannase; TCS, Triclosan; Tyr,Tyrosinase; U, Unit of enzyme activity; WRF, White rot fungi; WWTP, Wastewater treatment plant.
⁎ Corresponding author at: Group of Bioengineering, Earth & Life Institute, Université Catholique de Louvain, Croix du Sud 2/19, 1348 Louvain-la-Neuve, Belgium. Tel.: +32 10473644; fax: + 32 10473062.
E-mail addresses: (P. Demarche), (C. Junghanns), (R.R. Nair), (S.N. Agathos).
0734-9750/$ – see front matter 2011 Elsevier Inc. All rights reserved.
doi: Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011), P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx Additives: redox mediators, cosubstrates and protecting agents Reactor and process design side-reactions can occur) and energy consuming. Chemical processesoften use harsh chemicals which are unsafe for workers and the Over the past century, there has been an increased awareness of environment. Oxidoreductases may represent an alternative for the effects of pollution, and public pressure has inﬂuenced both pollution control through oxidation of organic contaminants.
industries and governments. Environmental pollution is no longer Two types of copper-containing enzymes, laccase and tyrosinase unavoidable. There are increasing demands to replace traditional (Tyr), and three kinds of heme enzymes, namely peroxidases, industrial processes by less- or non-polluting ones. During this hemoglobin (Hb) and cellobiose dehydrogenase (CDH) are intro- gradual shift which is a major challenge for our and the coming duced below. In particular, fungal lignin modifying enzymes (LME), generations, the treatment of wastes from current human activities laccases and peroxidases, have been extensively investigated for and as heritage of our industrial history remains a problem to be potential biotechnological applications and are hence well repre- solved. Only as these treatments will gradually merge with environ- sented in this review ( ). In addition, the new basic and applied mentally benign industrial processes, a truly sustainable economy will knowledge represented by publications and patents on these enzymes become a reality.
has an upward trend (). Lignin, a highly complex, stable and The use of enzymes in industrial processes is usually linked to a irregular polymer, requires enzymes with the ability to nonspeciﬁ- reduced consumption of energy as well as chemicals and thus cally oxidize substrates with high redox potential ( beneﬁcial for the environment. The enzyme world market grew at a double-digit rate in the last seven years and was about $5.1 billion in Laccases EC 1.10.3.2 [benzenediol:oxygen oxidoreductases] are 2009 ). Enzymes catalyze speciﬁc re- widely distributed multi-copper proteins capable of oxidizing a actions and mostly act under moderate conditions (temperature, pH, variety of phenolic and non-phenolic compounds solvents and ionic strength). Hence enzymes represent a promising . They are extensively studied since the mid- tool for the selective removal of pollutants from waste streams.
seventies of the last century (Laccases have Enzyme speciﬁcity also precludes undesired side-reactions, which various physiological functions depending on the producing species would otherwise increase reactant consumption and correspondingly and their cellular location. They are involved in virulence processes raise the cost of treatment — a great advantage over conventional (yeasts, bacteria, pathogenic fungi), lignin degradation (white rot chemical treatment processes. The application of enzymes to waste fungi) or deposition (plants), pigment synthesis (fungi, bacteria) and treatment was already proposed in the 1930s (however cuticle sclerotization in insects ). Laccases catalyze one- the degradation of a pollutant (parathion) by enzymes was ﬁrst electron oxidations by transferring one electron from four substrate illustrated in the late 1970s ().
molecules to one molecule of molecular oxygen which is reduced to The purpose of this review is to highlight enzymes that are water (Oxidized substrate radicals can suitable for waste treatment applications, to summarize waste undergo nonspeciﬁc polymerization reactions.
treatment situations in which the use of enzymes is feasible, and todeﬁne the issues/perspectives and potential bottlenecks relevant to the development of enzyme-based waste treatment processes.
In this review, only pollutant transformations mediated by cell-free or immobilized enzymes are considered. Processes with whole cells have been included only when extracellular enzymes areinvolved.
1.1. Oxidoreductases EC 1 Laccases contain four copper atoms in different sites which are classiﬁed according to their spectroscopic and functional properties: Oxidoreductases catalyze the electron transfer from one substrate Type 1 (blue), Type 2 (normal) and Type 3 (coupled binuclear). Type 2 to another, where the oxidized substrate is referred to as electron and 3 represent the active site for the binding and the reduction of donor in contrast to the reduced substrate, the electron acceptor. The oxygen. For more detailed information on reaction mechanism and recommended name according to the EC classiﬁcation system is physiological role, readers can refer to reviews (e.g. "donor:acceptor oxidoreductase" (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC- Tyrosinase EC 1.10.3.1, EC 1.14.18.1 [catechol:oxygen oxidoreduc- IUBMB)). Commonly used names are "donor dehydrogenase", tase, monophenol:oxygen oxidoreductase] is another copper-contain- "acceptor reductase" and, when molecular oxygen is the electron ing protein known as a monophenol oxidase or catecholase, acceptor, "donor oxidase".
ubiquitously distributed in organisms Oxidation reactions are essential in current waste treatment It shares most of its substrates with strategies. Most of them are based on physicochemical principles, laccases, but the ability to oxidize tyrosine is exclusive to Tyr. In having the typical disadvantages of being nonspeciﬁc (i.e. undesirable animals, fungi and plants, this trans-membrane protein is involved in Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi: P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx Oxidoreductases Laccase (EC 1.10.3.2) Phenols and halogenated phenols Emerging pollutants; drugs, hormones, personal care products, plasticizers, Polycyclic aromatic hydrocarbons (EC 1.10.3.1/1.14.18.1) Peroxidase (EC 1.11.x) Phenols and halogenated phenols Emerging pollutants drugs, hormones, personal care products, plasticizers, micropollutants, etc.
Kraft pulping efﬂuent Aromatic amines, polycyclic aromatic Cellobiose dehydrogenase Pulp mill efﬂuent Cutinase (EC 3.1.1.74) Phthalate derivatives Malathion (pesticide) Lipase (EC 3.1.1.3) Phthalate derivatives Pet food processing efﬂuent Waste cooking oil Tannase (EC 3.1.1.20) Olive mill wastes Tannery efﬂuents Phosphoric triester hydrolases Pesticides and nerve agents Pulp and paper sludge Amylases (EC 3.2.x) Cassava starch detoxiﬁcation Paper recycling (deinking) Alcohol distillery efﬂuent Biodegradable plastics Chitinases (EC 3.2.1.x) Shellﬁsh wastes Citrus processing waste (EC 3.2.1.x + EC 4.2.2.x) Protease (EC 3.4); keratinases, Silk manufacturing efﬂuent collagenases, pepsins and papain Keratin-rich wastes (feathers, skins) Seafood processing efﬂuent Alcohol distillery efﬂuent Mercury abatement Nitrile degrading enzymes (EC 3.5); nitrilase, aliphatic nitrilaseand amidaseHaloalkane dehalogenase (EC Nitrile hydratases (EC 4.2.1.84) Cyanide hydratase (EC 4.2.1.66) lyase (EC 4.2.2.x) Citrus processing waste Refer to pectinases (EC 3.2.1.x + EC 4.2.2.x) a Pectinases and pectin lyases are often co-expressed by organisms and/or used in combination for the treatment of pectic substances.
the synthesis of melanin and other pigments (e.g. browning of sliced subsequently oxidized to o-diquinones and the oxidation of o- fruits and vegetables). Tyr contains a Type 3 copper center and diphenols to o-quinones. The latter can undergo non-enzymatic catalyzes two distinct reactions using molecular oxygen; the polymerizations. Thus, Tyr is an oxidase with a monophenolase hydroxylation of monophenols to form o-diphenols which are activity (cresolase) and diphenolase activity (catecholase) ( Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),

P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx ), leading to the The ferriprotoporphyrin IX prosthetic group is a common feature assignment of two EC-numbers.
of all heme peroxidases. Ferriprotoporphyrin consists of four pyrrolerings linked by methylene bridges with iron (III) as central atom (The catalytic cycle of peroxidases is a three-stepreaction. First, the native enzyme (Fe3+) is oxidized by hydrogen peroxide. This produces water and the oxidized form of peroxidase, called Compound I (Fe4+–R+•). Second, Compound I oxidizes onemolecule of substrate, resulting in a substrate radical and CompoundII (Fe4+). Compound II oxidizes a second substrate molecule, leading to a second substrate radical and the native peroxidase (reducedstate Fe3+) (. The balance of the catalytic cycle is given in.
→ 2 oxidized donors þ H Broad substrate speciﬁcity of different peroxidases is related to their high redox potential (1000 mV) and structural properties.
Mainly non-animal heme-peroxidases were intensively investigated as potential biocatalysts for the removal of various pollutants, among While tyrosinase exerts hydroxylase activity on monophenols, them horseradish peroxidase (HRP, Class III) and manganese laccase oxidizes them through a radical mechanism peroxidase (MnP, Class II). In recent years, efforts have been made ). Both enzymes are thus classiﬁed as phenol oxidases.
to obtain cheap peroxidases from diverse sources such as bitter gourd, The redox potentials of laccases and Tyr vary between 400 and turnip and soybean ( Hemoglobin (Hb) can exert peroxidase activity under speciﬁc Peroxidases EC 1.11.x [donor: hydrogen peroxide oxidoreductases] redox conditions, and was examined as a potential and inexpensive are predominantly heme proteins which utilize hydrogen peroxide (when derived from slaughterhouse wastes) substitute for main- stream (heme) peroxidases. Hb is a macromolecular heme-protein 2O2) or organic hydroperoxides as cosubstrate to oxidize a variety of organic and inorganic substrates. They are found in all ﬁve kingdoms of present in bacteria, plants and animals ( life. Based on the EC nomenclature, peroxidases are divided into two ). Unlike heme peroxidases, Hb contains a subclasses, peroxidases (EC 1.11.1) with currently 19 entries and ferroprotoporphyrin IX with an iron(II)-center which is responsible peroxygenases (EC 1.11.2) with 4 entries. This review will concentrate for its ability to bind molecular oxygen, oxygen transport being its on "non-animal heme-peroxidases" including intracellular peroxi- physiological role. Peroxidase can bind molecular oxygen when the dases of bacterial and eukaryotic origin (Class I, e.g. yeast cytochrome c iron (III) is reduced to iron (II) and Hb likewise exhibits an intrinsic peroxidase), secreted fungal peroxidases (Class II, e.g. lignin and peroxidase activity ().
manganese peroxidases), and classic secretory plant peroxidases Cellobiose dehydrogenase (EC.1.1.99.18) is an extracellular fungal (Class III, e.g. horseradish and bitter gourd peroxidases). As the enzyme of interest because of its ability to produce radicals. CDH is an classiﬁcation of peroxidases is currently a highly discussed topic, the enzyme containing both ﬂavin and b-type heme (ferriprotoporphyrin IX).
reader may follow dedicated literature ( It is secreted by wood-degrading fungi, including white-rot and soft-rot fungi. Its probable physiological function is cellulose and lignindepolymerization and prevention of cellulose repolymerization Cellobiose dehydrogenaseoxidizes, in a ping-pong mechanism, various substrates such as cellobiose,cellodextrins, mannodextrins and lactose, to their corresponding lactoneswith the concomitant reduction of the ﬂavin (FAD to FADH2). The ﬂavin issubsequently reoxidized by the heme group ).
Quinones, Fe3+, Cu2+ and I− are suitable ﬁnal electron acceptors (). Hydroxyl radicals are indirectly generatedthrough the reduction of Fe3+ to Fe2+ and O2 to peroxide (Fentontype reaction), even if oxygen is a poor substrate for CDH. Thoseradicals can react subsequently with diverse compounds ( Oxidation of various substrates by the above-mentioned oxidative enzymes generates intermediates which can undergo nonenzymaticreactions like polymerization (i.e. intermediates react with each otherand/or with parent compounds), depolymerization (i.e. cleavage ofexisting polymers) and ring cleavage of aromatics (nitroaromatics,synthetic dyes, aromatic structures in lignin). All of these reactions canbe valuable in biotechnological processes, from biodegradation to foodprocessing, wood pulp biobleaching and biosensors Fig. 1. Publications and patents on enzymes used in "waste treatment and disposal" although there are (Chemical Abstract section title in Sciﬁnder®) as representation of the interest in only a few applications of Tyr described, attributed to difﬁculties in enzymes for pollution management strategies. Publications and patents are drawn in production of the enzyme from eukaryotic sources ( black and blue, respectively. The dotted lines show publications and patents for all Likewise, development of CDH applications has been hampered enzymes listed in (excluding hemoglobin), plain lines represent oxidoreduc-tases and dashed lines represent hydrolases.
by the high cost of its production. Among them, amperometric Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi: P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx biosensors for lactose and diphenol detection are the most tangible 1.2. Hydrolases EC 3 ).
These sensors can be operated for considerable periods of time due to Hydrolases are classiﬁed as EC 3 and further divided into 13 pH tolerance and stability of the constitutive CDH subcategories according to the type of bonds hydrolyzed (NC-IUBMB).
). proposed to exploit CDH under They catalyze the hydrolysis of various compounds according to the particular reductive conditions for biodegradation purposes. They showed that oxalate, when it serves as iron chelator, is oxidized byhydroxyl radicals to form the strongly reducing carboxylate anion A−B þ H2O→A−OH þ B−H radical. With this cascade, CDH could catalyze the reduction ofbromotrichloromethane to trichloromethyl radicals.
The systematic name is always formed by adding "hydrolase" to the One of the largest research ﬁelds for applications of phenol oxidases substrate (substrate hydrolase). The accepted name in most cases is and heme proteins is the treatment of wastewaters containing phenolic formed by the name of the substrate with the sufﬁx -ase. Many compounds. Examples of phenolic compounds treated with peroxidases hydrolases, regardless of their phylogenetic origin or catalytic function, under model wastewater conditions are phenol share an α/β sheet of eight β-sheets connected by α-helices as ﬁrst described by This structural element is well conserved ), p-bromophenol ), α-naphthol in order to keep three catalytic residues in a functional position.
phenylenediamines, catechol, resorcinol, hydroqui- Exempliﬁed on a trypsin-like protease, this so called catalytic triad is none (and chlorocatechols formed by an aspartate, a histidine and a serine. The serine hydroxyl recently reviewed potential group is deprotonated within the triad forming a very nucleophilic peroxidase applications. Systems developed using synthetic wastewaters alkoxide group (―O−) which may attack a suitably positioned substrate are potentially transferable to the treatment of phenol-rich efﬂuent carbonyl carbon atom to form an acyl intermediate which is subse- streams from e.g. pulp and paper plants, olive mills, petroleum reﬁning quently attacked by water to release the products. facilities, resins and plastics manufacturing. A drop in the toxicity of classiﬁed hydrolases based on their catalytic site, phenolics is reported to mainly occur through polymerization processes i.e. the residue forming a covalent bond with the substrate. Beside the above mentioned serine-hydrolases, metal-dependant hydrolases and ). Oxidation of phenols carboxyl (aspartyl and glutamyl) hydrolases are major groups based on leads to phenoxy radicals which undergo non-enzymatic polymerization.
this classiﬁcation scheme. Hydrolases are involved in virtually all The insoluble polymer can be easily removed from the efﬂuent biochemical processes. Hydrolase-catalyzed reactions take part in afterwards (ﬁltration or sedimentation).
metabolism, signal transduction, development, etc. However, this review Among the relevant lab-scale systems for the treatment of model will focus on catabolic hydrolases, as they are of outstanding industrial efﬂuents are continuously operated membrane bioreactors (MBR) interest – approximately 75% of the industrial enzymes are hydrolases and packed bed bioreactors ) – and already have found numerous applications, including (PBR) (These conﬁgurations save space environmental ones (Catabolic hydrolases depolymerize and energy, compared to e.g. continuously stirred tank reactors macromolecules such as proteins, carbohydrates and nucleic acids for (CSTR) in series with ﬁltration or sedimentation units. further metabolism. Because of the need to break down a wide range of operated a packed-bed reactor, ﬁlled with white nutrients, hydrolases usually have broad substrate speciﬁcity.
radish peroxidase immobilized on diethylaminoethyl cellulose, for the Esterases (EC 3.1) act on ester bonds. Selected representatives in treatment of 0.5 mM α-naphtol-spiked water over one month (57% this review are cutinases, lipases, tannases and organophosphate hydrolases. Esterases such as fungal cutinases (EC 3.1.1.74) can Other potential applications of peroxidases in water treatment hydrolyze xenobiotic polymers, e.g. polyamides, polyesters and poly include oil removal in case of oil leakage in process water ( (lactic acid), due to analogies to their natural substrate, cutin.
and polymerization of dissolved organic matter (humic and Molecular aspects of microbial degradation of xenobiotic polymers fulvic acids) which acts as precursor of disinfection by-products in and possible implications of microorganisms, enzymes and genes in water supplies ().
environmental biotechnology are developed by .
Degradation studies are often conducted on synthetic wastewaters.
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are widely However, assessment in relevant matrices should also be systematically distributed and catalyze hydrolysis and synthesis of ester bonds in considered at lab-scale. In a study of peroxidase-based removal of long chain triglycerides (Their physiological role estrogens ), a complete elimination was observed in involves the breakdown and mobilization of lipids inside cells as well as distilled and ultra-ﬁltered water (Milli-Q system) with a few units of transfers of lipids between organisms . Microbial peroxidase, while 250-fold more enzyme activity was necessary to lipases have biotechnological applications in organic synthesis, food and achieve similar results in ﬁltered wastewater.
ﬂavor industries, detergent manufacturing, removal of oil and grease Taking only redox potential and substrate speciﬁcity into considera- (O&G) in wastewaters and biodiesel reﬁning. Aspects of lipase tion, a vast number of oxidoreductases are eligible for an application in production, characterization, immobilization and applications are environmental biotechnology. More than 2700 enzymes of the comprehensively examined by cytochrome P450 (CYP450) superfamily were identiﬁed in all domains of life and have the ability to oxidize a large variety of organic technologically interesting dimension of lipases is their tolerance substances, often through their monooxygenase activity, i.e. insertion of against organic solvents ( an oxygen atom into a carbon–hydrogen bond leading to a hydroxyl Tannase (tannin acyl hydrolase (TAH), EC 3.1.1.20) is involved in the group However, industrial modiﬁcation of complex tannins (e.g. during fruit ripening). Tannic scale enzyme production, the requirement for cofactors (e.g. FAD, NAD), substances are the most important plant phenolics after lignin-related turnover number, and stability of multicomponent enzymes are criteria phenols and the main chemical group of natural anti-microbial which limit the number of suitable enzymes ( substances produced by plants ). Tannins are These decisive factors can also be seen as severe polyphenols of varying molecular weight able to form complexes with constraints to be eliminated before envisaging cost-effective applica- proteins and with others macromolecules such as sugars and cellulose, tions. Possibilities to overcome such constraints are discussed hereafter thereby precipitating them. TAHs transform tannic acid, methyl gallate, (refer to ).
ethyl gallate, n-propylgallate, and isoamyl gallate to gallic acid and Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011), P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx glucose. Therefore, they may be used to degrade tannins contained in 100,000 tons per year, for the production of enzymes could decrease industrial waste streams, such as tanneries TAHs the cost of enzyme production and solve an environmental issue can be obtained from plants, animals and microbes, and commercial TAH is produced using ﬁlamentous fungi, mainly Aspergillus sp., in Pectinases include polygalacturonases (EC 3.2.1.15), pectinesterases submerged cultures The ﬁrst (EC 3.1.1.11), pectin lyases (EC 4.2.2.10) and pectate lyases (EC 4.2.2.2), evidence of bacterial strains isolated from olive mill waste and using based on their mode of action on the heterogeneous structure of pectic tannic acid as sole source of carbon and energy was recently reported substances. Pectins are polymers of D-galactopyranosyluronic acids (. The highest tannase activity was detected in joined by α-D-(1→4) glycosidic linkages. The main chain can be Pantoea species.
modiﬁed in various ways (ramiﬁcation with neutral sugars, esteriﬁca- Hydrolysis of organophosphorus compounds (OP), used as pesti- tion, acetylation) (). The main-chain-degrading enzymes cides and warfare agents, can be achieved using phosphoric triester are esterases and depolymerases ). The hydrolases. Aryldialkylphosphatase (EC 3.1.8.1) – also known as latter group is further divided into hydrolases and lyases.
organophosphorus hydrolase (OPH) or phosphotriesterase – and Pectic substances are ubiquitous in plants and are the major diisopropyl-ﬂuorophosphatase (EC 3.1.8.2) – commonly called organo- components of the middle lamella, thus constituting the majority of phosphorus acid anhydrolase (OPAA) – are the most studied subgroups.
fruit processing wastes (especially from citrus fruits) and represent- OPH hydrolyzes esters of phosphoric acid (P―O bond), while OPAA ing a potential ﬁeld of application for pectinases.
acts preferably on P―F or P―CN bonds. Biotechnological applications Peptidases (EC 3.4) constitute another group of enzymes with great of OP enzymes for environmental cleaning and personal protection economical implication. Proteases, peptidases or proteinases are materials have been reviewed in synonyms for enzymes conducting proteolysis, or cleavage of peptide addressed hydrolases involved in the detoxiﬁca- bonds. Numerous proteases are commercially produced and used in tion of OP but also carbamate and pyrethroid insecticides. Catabolic (laundry) detergent formulations, protein synthesis, brewing, food pathways for some OPs with emphasis on biochemical and molecular industry, leather and dairy processing aspects of OP degradation by microbes, along with the evolution and Biodegradation/biotransformation applications include treat- distribution of related genes/enzymes were deeply reviewed by ment of various protein-rich efﬂuents from food and beverage There, they also report putative industries. Keratinases, collagenases and pepsins are promising tools applications of OP-degrading microbes and their enzymes for to enhance the low biodegradability of structural animal proteins like bioremediation and treatment of OP poisoning.
keratin and collagen. Keratin for example is compact and strongly Glycosylases (EC 3.2) are economically the most important stabilized by hydrogen bonds, hydrophobic interactions and disulﬁde hydrolases. They hydrolyze bonds between a carbohydrate and bonds which render it resistant to proteolytic degradation ( another group (including other carbohydrates), and are thus essential Papain or cysteine protease (EC 3.4.22.2) is an enzyme found in for breaking down carbohydrate polymers, such as cellulose, papaya having numerous current applications (e.g. cell culture hemicellulose and starch as well as chitin. Glycosylases – amylases, preparations, meat tenderizer, medical debridement) which may cellulases, xylanases and chitinases as prominent representatives – substitute more expensive proteases.
are used in food, brewery and wine, animal feed, textile and laundry, Nitrile degrading enzymes (EC 3.5) act on carbon–nitrogen bonds pulp and paper industries, as well as in agriculture and for research other than peptide bonds.
purposes. Their applications are diverse; treatment/recycling of Hydrolysis is the common way for the microbial catabolism of nitriles wastes, production of fermentable products, production of renewable (. The biological degradation of nitriles fuels (bioethanol) and ﬁne chemicals. For a general overview on the proceeds through two enzymatic routes Nitrilases (EC 3.5.5.1) use of carbohydrolases please refer to Glycosylases are and aliphatic nitrilases (EC 3.5.5.7) catalyze the direct hydrolysis of nitriles rarely used for a direct biotransformation of pollutants, but are very to the corresponding carboxylic acids, forming ammonia without free useful to pre-treat polymeric wastes in order to decrease the waste amide intermediates (Nitrilases exhibit a volume, to increase the bioavailability of attached or linked pollutants selectivity that cannot be achieved with acid- or base-catalyzed chemical or to improve the digestibility of wastes for subsequent treatment hydrolysis in addition to enantioselectivity, producing single stereoiso- processes, exempliﬁed by studies on the sacchariﬁcation of municipal mers, and regioselectivity, hydrolyzing a single nitrile group in a waste leading to an increased conversion to ethanol after hydrolysis compound with two or more nitrile groups Typically, they are moderately thermostable enzymes with bacterial nitrilases Glycosylases are also important in integrated waste management possessing a higher temperature stability compared to fungal nitrilases strategies, for example when the life cycle of biodegradable plastics is (Amidases (EC 3.5.1.4) sequentially hydrolyze considered (Amylase can degrade nitriles in a two step reaction with amides as intermediate product starch blend polyethylene (and recently a thermo- (Amidases are often co-expressed along with nitrile hydratases stable α-amylase from a marine bacterium was described as a which are classiﬁed as lyases (NHase, EC 4.2.1.84). Oxygenases (EC 1.13) powerful biocatalyst for the treatment of synthetic biodegradable oxidize nitriles to hydroxyl nitriles which then decompose either by spontaneous hydrolysis or by the action of hydroxylnitrilases (EC 4.1.2.37) Chitin is one of the most abundant compounds produced by marine to yield cyanide and the corresponding aldehydes or ketones. The further invertebrates, insects, fungi and algae. It is a polymer of β-1,4-N- degradation of cyanide is described in (Lyase).
acetylglucosamine, whereas chitosan is the deacetylated counterpart.
Dehalogenases (EC 3.8) act on halide bonds. Volatile halogenated Chitin is seldom found pure in nature, most of producing organisms organic compounds are widely used as pesticides, solvents and organic modify chitin by linking it to other polymers, proteins, etc. synthesis intermediates. Haloalkane dehalogenase (DHA, E.C. 3.8.1.5) is ). Chitinolytic enzymes or chitinases (EC 3.2.1.x) perform the an enzyme found in e.g. Rhodococcus erythropolis, which can directly hydrolysis of chitin to its residues N-acetylglucosamine, exo- and hydrolyze short chained 1-haloalkanes (C2 to C8) to primary alcohols endochitinases acting on different moieties of chitin ( without requiring a coenzyme ).
). Chitinase-producing strains usually use chitin/chitosan as carbonsources. () selected an Aspergillus sp. strain among 220 isolates based on its chitinase production. The enzyme wasproduced only when the cultivation medium contained shrimp shells.
Lyases are enzymes that catalyze the cleavage of chemical bonds The bioconversion of shellﬁsh waste, with an estimated volume of (C―C, C―O, C―N and others) by other means than hydrolysis and Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi:

P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx oxidation (NC-IUBMB). Furthermore, they are capable of forming issue. A short treatment of cellulosic sludge with cellulases facilitates multiple bonds or new ring structures. Lyases are divided into 7 the dewatering with belt presses because it shortens the ﬁbers leading classes according to the bond broken. The systematic name is formed to a higher solid content in the resulting cakes ().
according to the pattern substrate group-lyase. Carbon–oxygen lyases Subsequent treatments are therefore facilitated. It should be noted (EC 4.2) catalyze the cleavage of carbon–oxygen bonds leading to that already in proposed the ethanol fermentation of primary clariﬁer sludge produced in sulﬁte pulping operations to Class EC 4.2.1 contains hydro-lyases or dehydratases that cleave reduce the amount of waste. Simultaneous cellulase hydrolysis and carbon–oxygen bonds by elimination of water molecules. Nitrile yeast fermentation could reduce the sludge by 60% wt and release hydratases (NHase, EC 4.2.1.84) are metalloenzymes that contain fermentable sugar for ethanol production without end-product either a non-heme Fe(III)-ion or non-corrin Co(III)-ion, leading to a inhibition of the cellulases.
classiﬁcation into ferric and cobalt NHases. They catalyze the Wood pulping – the separation of cellulose ﬁbers from lignin hydration of diverse nitriles to their corresponding amides ).
structures – is generally based on mechanical (cooking, grinding) NHase is a key enzyme in the enzymatic pathway for the and/or chemicals technologies (acid sulﬁte or Kraft process).
conversion of nitriles to acids. A large number of microorganisms Cholesteryl esters may form pitch deposits in pulp processing. Sterol were isolated expressing NHases which have been puriﬁed and esterase covalently immobilized on polyacrylate-based epoxy-activated characterized ). Although the structural carriers (Dilbeads™) was tested for the continuous hydrolysis characterization of NHases has provided some insights, the exact catalytic mechanism of NHases is still not fully elucidated. Cyanide By the end of the last century, an alternative was introduced to hydratase or formamide dehydratase or formamide hydro-lyase (EC reduce the use of chemicals: the laccase-mediator-system applied to 4.2.1.66) is a primarily fungal enzyme which participates in wood pulping (Lignozym®-process), similar to wood deligniﬁcation cyanoamino acid metabolism. HCN, the best substrate for this group in nature, namely the white-rot of wood and compatible with the pulp of enzymes, is transformed into formamide. Cyanide hydratase was technologies at that time ( ﬁrst partially puriﬁed from Stemphylium loti Oxidoreductases and hydrolases were considered for the manage- Class EC 4.2.2 contains lyases which cleave C―O bonds by the ment of liquid and solid residues (). For instance, horseradish elimination of an alcohol from a polysaccharide. Pectate lyase (EC peroxidase was used for the efﬁcient detoxiﬁcation of phenol- 4.2.2.2) requires the presence of calcium ions and catalyzes the containing condensates of evaporators from Kraft pulping cleavage of (1 → 4)-α-D-galacturonan to oligosaccharides with 4- deoxy-α-D-galacto-4-enuronosyl groups at their non-reducing ends.
For waste water treatment, an acidic and a basic CDH were It is not only involved in the maceration and soft-rotting of plant assessed for their ability to decolorize the acidic and the caustic pulp tissue but also in the activation of defense systems presumably mill efﬂuents from a bleach plant Both through the mechanism of elicitation ).
enzymes were reasonably effective in color removal at their Pectate lyase activity was ﬁrst discovered in cultures of Erwinia respective optimum pH. In addition, CDH-treatment is relevant for carotovora and Bacillus polymyxa softwood and hardwood processing efﬂuents. Even though technical give an overview of so far identiﬁed bacterial species feasibility was shown, the high cost of CDHs discourages their producing pectate lyase. Pectate exo-lyase (EC 4.2.2.9) catalyzes the exploitation for pulp bleaching at industrial scale.
Paper recycling is the cornerstone of further developments in paper from the reducing end of pectate, i.e. de-esteriﬁed pectin, and endo- industry. Recycling includes sorting, dissolving, deinking, etc. before the pectin lyase (EC 4.2.2.10) catalyzes the cleavage of (1 → 4)-α-D- paper making process takes place. The major contaminants in "mixed galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O- ofﬁce wastes" (MOW) and photocopy prints are inks and the most methyl-α-D-galacto-4-enuronosyl groups at their non-reducing ends difﬁcult step in recycling is the deinking. Physicochemical treatments are not always successful to remove ink. Alkaline cellulase is believed todeink paper by releasing short ﬁbers of 20 to 100 μm from the surface of 2. Pollution control via enzyme technology in industries paper substrates examinedenzymatic (cellulase/hemicellulase) versus chemical deinking for MOW 2.1. Pulp and paper industry and photocopy prints. Their conclusions were ambivalent. Althoughenzymes are suitable for deinking, their efﬁcacy critically depends on Pulp and paper industries convert wood and recycled cellulose feed characteristics. Moreover, their action can affect paper strength ﬁbers into pulp and various forms of paper. Pulp and paper mills use properties, which is consistent with the assumed mechanism.
signiﬁcant volumes of water and strong chemicals and generate In the paper making process, starch from corn, maize or potato – enormous amounts of hazardous wastes containing e.g. phenolics and depending on local resources – is used to improve strength and chlorinated compounds. Millions of tons of dried sludge are annually printing properties of paper. Cassava is an important source of starch generated by pulp and paper mills in the US alone. Beside in Africa and might be exploited in paper manufacturing. However, environmental aspects, their handling represents an economical cassava leaves and roots contain a cyanogenic glucoside called Fig. 2. Different pathways of nitrile metabolism modiﬁed from Banerjee et al. (2002).
Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011), P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx linamarin and by action of naturally present linamarases, poisonous dione ). Laccases were shown to act on a variety of hydrogen cyanide is released. Recently, a mixture of cellulase and synthetic dyes; Acid Blue 62 ), Acid Blue 81, xylanase were shown to detoxify cassava starch by releasing Reactive Blue 19 (, Malachite Green ( ), Reactive Black 5, Reactive Yellow 15, Reactive The authors recommend two books which comprehensively cover Red 239 (), Reactive Red 120 ), enzyme technology in pulp and paper industry: methyl orange crystal violet are some examples. Peroxidases were also assayed for dye degradation, e.g.
methylene blue (), 2.2. Textile industry bromophenol blue and methyl orange ). evaluated the potential of a cheap peroxidase from bitter gourd Production of textiles can be divided in two parts; the ﬁber and for dye decolorization over a broad range of 21 industrial dyes with fabric manufacturing and the dyeing process. Only a few possible various chemical structures. The tested peroxidase was able to degrade applications of environmental biotechnology were found for the ﬁrst, nine of the dyes without mediators, and all of them when catalysis was in connection with the low number and amounts of byproducts mediated with 1-hydroxybenzotriazole (HBT). Beside peroxidases, CDHs participate synergistically with laccase in the decolorization of described a protease-based bioprocess for the the anthraquinonic dye Acid Blue 62 ). In a production of bioactive peptides derived from sericin found in silk study of , culture broth containing laccase and industry efﬂuent. Sericin hydrolysate produced with a commercial CDH was used to degrade synthetic dyes. They showed that CDH could protease exhibited good antioxidant activity and tyrosinase-inhibiting be used instead of HBT to enhance decolorization of the dyes Direct activity particularly appreciated in food, cosmetic and pharmaceutical Violet 51 and Ponceau xylidine due to hydroxyl radicals generated (refer industries. Another prospective application refers to poly(trimethylene terephthalate) (PTT), a linear aromatic polyester used in textile industry Coupling reactions lead to less soluble polymers of azo and as ﬁber, but also in ﬁlms, ﬁlaments and plastics manufacturing. A higher anthraquinonic dyes and often occur upon application of LMEs hydrophilicity of such a material is appreciated for further processing (e.g. dyeing) and generally obtained through alkaline and plasma The predominance of those treatments. Enzymatic hydrolysis may represent an alternative for chromophores in industry, together with a reaction mechanism surface modiﬁcations. Polymers and oligomers of PTT were treated involving free radical cascades often leading to polymers, is a clear using diverse polyesterases (cutinases and lipases) ).
justiﬁcation why membrane technologies associated with an enzyme The ability of polyesterases to functionalize PTT, which is poorly treatment are extensively studied, and considered as a promising biodegradable, might also be of interest to increase its bioavailability textile efﬂuent treatment process. For instance, during waste processing. Finally, biotechnological degumming of bast combined ﬁltration and enzymatic treatment by efﬁciently immobi- ﬁbers of ramie and sunn hemp (found in East India) using pectinases lizing and stabilizing laccases on a chitosan membrane. More than 95% and xylanases after a mild chemical treatment presents an option to of an azo dye (Acid Black 10 BX, 20 ppm) could be degraded in 21 manufacture textile materials ).
cycles of operation. Enzyme immobilization is an effective way to The dyeing process – conferring color to textile materials – is a major decrease the treatment cost since it allows several reactor activity in textile industries which requires large amounts of water fordyeing, rinsing and cleaning. The removal of dyes from generatedefﬂuents is a matter of concern considering the progressively stringentenvironmental legislations. Indeed, textile efﬂuents are not only unaesthetic (colored) but toxic and partly carcinogenic. Dyes used intannery and textile manufacturing are mainly synthetic and categorized according to physical and chemical properties (e.g. solubility andcharge) as acidic, basic, direct, disperse, reactive, sulfur ). As these properties are diverse, and because dyes were originallydesigned to be resistant to chemicals, water, light and microbialdegradation, efﬂuents containing dyes are inherently difﬁcult to clean.
Consequently, existing chemical processes, e.g. coagulation, advanced oxidation processes (AOPs), adsorption, chlorination, are partiallyineffective and not economical for the treatment of dye mixtures().
Furthermore, textile efﬂuents exhibit bacterial toxicity and a lowbiochemical oxygen demand (BOD)/chemical oxygen demand (COD) ratio (N0.1), i.e. low biodegradability. As a result, a combination ofchemical coagulation and oxidation followed by aerobic biologicaloxidation is an often employed process ), which may be fused in an integrated biochemical treatment. So far, white rot fungi (WRF) with their lignin-modifying system are the most efﬁcient aerobicorganisms to break down colored pollutants. reviewed the decolorization and detoxiﬁcation capacity of WRF.
However, drawbacks associated with whole cell reactors like uncon-trolled biomass production are difﬁcult to overcome. Here again, theapplication of suitable enzymes might be beneﬁcial.
Azo and anthraquinonic structures account for more than 80% of all textile dyestuff produced ). Azodyes are characterized by nitrogen–nitrogen double bonds (phenyla- Fig. 3. Simpliﬁed process diagram of enzyme technology as applied to the pulp and zobenzene) and anthraquinonic dyes are based on anthracene-9,10- paper industry.
Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi: P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx conﬁgurations together with catalyst reuse. used be retained when lipase-based hydrolysis was applied beforehand.
laccases immobilized on silanized alumina pellets activated with Lipase immobilized on alginate beads was also used to hydrolyze O&G glutaraldehyde and achieved the complete degradation of the resistant in pet food industrial efﬂuents. diazo dye Reactive Black 5 (500 ppm) in a ﬂuidized bed reactor (FBR) evaluated the anaerobic treatability of such efﬂuents with a hybrid and different CSTRs as well as in a continuously operated tubular reactor reactor system consisting of a packed bed reactor with immobilized with this biocatalyst. exploited turnip lipase followed by an upﬂow anaerobic sludge bioreactor. The system peroxidase immobilized on wood shavings for the decolorization of a was successfully operated over a period of 100 days with higher COD, direct dye, Direct Red 23. Their continuously operated packed bed fat removal and methane production rates as compared to a control reactor, in series with an activated silica ﬁlter, was operated over reactor without enzymatic pretreatment.
4 months with satisfactory results (64% removal efﬁciency retained).
Waste cooking oil (e.g. from potato processing plants) can be treated Hematin – a hydroxylated form of heme – has been considered as an with lipase to produce biodiesel of superior quality not competing with economical alternative for some peroxidase-based catalysis. food resources. Enzymatic transesteriﬁcation of free fatty acids studied the catalytic degradation of model dyes Eriochrome Blue contained in waste oil (methanolysis) using immobilized lipase has Black R and Fluorescein with HRP/H2O2 in comparison with a mimetic led to a signiﬁcant titer of fatty acid methyl esters system consisting in hematin/H2O2. More than 85% elimination of both Due to the high interest in lipases for biodiesel reﬁning, it is dyes was achieved with HRP in contrast to 30% of Eriochrome Blue Black expected that their cost will decrease signiﬁcantly, making it feasible to R with only hematin.
apply them to a wider array of degradation processes.
Recently, compared the efﬁciency of nanoﬁltration, coagulation/ﬂocculation and commercial laccases 2.3.2. Protein transformation (Denilite®IIS, Novozyme) for the treatment of a model efﬂuent — Wastes generated by the meat industry are mostly bones, organs and Blue Bezaktiv S-GLD 150 and Black Novacron R plus typical salts.
hard tissues containing animal proteins of poor biodegradability. The Beside the very effective nanoﬁltration (N99% color removal), majority of hard-to-degrade proteins are extracellular matrix proteins enzymatic catalysis was shown to be technically competitive since (collagen, elastin, proteoglycans) and keratins. Keratin-rich wastes better results (N98% color removal) were obtained compared to the including feathers, hair, nails, horns, etc. are typical byproducts of most efﬁcient coagulants and ﬂocculants (N93% color removal).
poultry slaughtering and the leather industry. Currently, incineration is Enzyme inhibition by high salt concentrations and metal ions may the main route for the disposal of such wastes. potentially hamper the feasibility of an enzymatic treatment for investigated thermophilic proteases (keratinase, collagenase, elastase) textile efﬂuents. Marine fungi and their laccases were considered for with broad speciﬁcity and high activity for their use in the treatment of their ability to decolorize black liquor and textile dyes in salty hardly biodegradable proteins. treated porcine efﬂuents containing carbonates, sulﬁdes, sulfates, chlorides, etc.
skin, as an in vitro model of human skin, with fungal keratinase. Their results suggest that keratinase speciﬁcity renders it suitable for leather studied the inﬂuence of Ca2+, Co2+, Zn2+, Cr6+, Cu2+ and Fe2+ on the processing or in vivo applications as cosmetics. Despite numerous laccase redox-mediated decolorization of Remazol Black-B and potential applications of microbial keratinases (animal feed and Remazol Brilliant Blue R (50 ppm). Except for Fe2+ that highly fertilizers production, leather and textile processing, in detergent inhibited enzyme activity, their presence did not exert much effect.
formulations, adjuvant in cosmetics, etc.), industrial usage and market In textile efﬂuents, a bacterial reductive cleavage of the azo dye demand are still in their infancy (A promising bonds may occur and release colorless amines ). Aromatic process is the keratinase-based conversion of agroindustrial wastes into amines are highly toxic pollutants also released from other anthropo- amino acids and soluble proteins of nutritional value for animal feed. For genic activities like rubber manufacturing, chemical, plastic and paper instance, observed a production of more processing. have investigated the potential of than 5 g L−1 of soluble proteins along with 90% wt reduction of the bitter gourd peroxidase for the degradation of aniline, m-chloroaniline, initial feather content (0.7% feather waste as a sole carbon and nitrogen N,N-dimethylaniline, diphenylamine, m-toluidine and p-aminobenzoic source) after 72 h of incubation with protease-producing actinomycete acid. All tested aromatic amines were recalcitrant to enzymatic treatment but when peroxidase activity was mediated by o-dianisidine, Seafood industry efﬂuent streams are usually discarded directly in satisfactory results were obtained with most of them (50 to 95% of the sea without any treatment or valorization. proposed to valorize them for the production of media for Alongside oxidoreductases, a hydrolase was evaluated for the microorganism cultivation through enzymatic hydrolysis. Peptones remediation of tannery efﬂuents. Tannase from Aspergillus candidus obtained from papain, pepsin and trypsin pretreatments of industrial entrapped in alginate beads efﬁciently removed efﬂuent color and octopus processing efﬂuents could efﬁciently promote the growth of reduced tannin concentrations below the discharge limit ( lactic acid bacteria and the production of bacteriocins. Marine . Unlike whole cell treatments with A.
peptones produced after 4 to 10 h of protease treatment may compete candidus, enzymatic treatment did not affect other physicochemical with expensive commercial media and partially solve this pollution properties of raw efﬂuents, nor did it decrease the BOD. Cell-free tannase may thus be applied in a pre-treatment step for efﬂuentdetoxiﬁcation and decolorization before a conventional biological 2.3.3. Sugar transformation Various processes have been investigated for the treatment of pectin-rich efﬂuents from vegetable and fruit processing industries: 2.3. Food and beverage industry physical dewatering, chemical coagulation, activated sludge, sprayirrigation onto lands, etc. These have several disadvantages such as the 2.3.1. Oil and grease transformation low efﬁciency due to the recalcitrance of pectins, environmental Wastewaters from food and feed processing manufacturing may pollution from the use of chemicals and long treatment times in have a high COD arising from fats and may require a pretreatment addition to high costs (A cost-effective and before discharge in sewer systems. demonstrated environmentally friendly method is the (pre)treatment with pectinases the effectiveness of a lipase-based hydrolysis of dairy efﬂuents before from bacteria, which selectively remove pectic substances from the an activated sludge treatment. At 800 mg L−1 O&G, the COD removal wastewater Alkaline pectinase and alkalophilic efﬁciency of activated sludge fell to zero while a removal of 82% could pectinolytic microbes facilitate the removal of pectinaceous material Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011), P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx and render it suitable for decomposition by activated sludge ( represent around 50% and carbohydrates and humic substances up to In citrus processing industries, peels 20%. The solubilized material may be re-fed into the wastewater and centrifugation pulp (semi-solid residue after fruit centrifugation) stream and favor nitrogen and phosphorus removal as mentioned are important byproducts. These fermentable wastes are an economic and environmental problem ). Pectin can be So-called "emerging pollutants" are a heterogeneous group of extracted from citrus peel using pectinase as was shown with substances which are causing increasing concern. Emerging pollutants polygalacturonase secreted by Kluveromyces fragilis yeasts can be deﬁned as organic compounds in the aquatic environment – ). The treatment of centrifugation pulp with surface, ground- and waste-waters – that are not covered by commercial pectinolytic enzymes (Citrozym-LS; 80% polygalacturonase established water quality regulations. They have direct or indirect and 20% pectinesterase) was very efﬁcient in improving pulp pressing adverse effects for humans and wildlife at environmentally relevant and drying of residues. The end-product obtained showed good in vitro concentrations (ng/L–μg/L range). These contaminants include digestibility and its protein content was comparable to agro-industrial pharmaceuticals, personal care products, fragrances, plasticizers, waste products currently used as animal feed ( steroids and hormones, illicit drugs, gasoline additives, ﬂame Disposal of liquid residues (e.g. spent wash, pot ale) remaining retardants, etc. ( after alcohol distillation is of concern for distilleries. Cellulase is A comprehensive study about their presence in attractive for the pre-treatment of alcohol distillery efﬂuents.
wastewaters was conducted between 1999 and 2000, by sampling Enzymatic hydrolysis was shown to substantially increase the rate 139 wastewater streams all over the United States ( of subsequent biological oxidation (). An ). Eighty percent of the samples were tested positive for the anaerobic digestion of yeast cells from Scotch distilleries is not presence of at least one of the ninety-ﬁve monitored organic feasible. The hydrolysis of intact cells was found to be the rate-limiting pollutants. Frequency of detection (f) was 41% for bisphenol A step ). Protease and β-glucanase exhibited a (BPA), 51% for 4-nonylphenol (NP), 58% for triclosan (TCS) and synergistic activity in cell disruption, and papain was also appropriate between 6 and 21% for sex hormones (estrone, estriol, estradiols) to for cell digestion in pot ale (N90% disruption). Subsequent anaerobic highlight compounds used in below mentioned studies of enzymatic digestion of pretreated yeast residue (pot ale) showed 87% COD degradation. Urban wastewater samples were collected on the reduction compared to 13% without enzyme treatment.
eastern shore of San Francisco Bay and analyzed for their endocrine Chitinase can be applied to chitin-containing wastes from aquatic disrupting chemical (EDC) content ). 95% food industry, leading to a release of bioactive products (e.g.
of the samples contained at least one of the monitored EDCs.
antioxidants, carotenoids) from the chitin alongside with its hydrolysis Phthalates (f = 91%) were widely detected but also triclosan products These soluble (f = 43%) and bisphenol A (f = 24%). Conventional wastewater and sugars (N-glucosamine) can also promote bacterial fermentation to drinking water systems were found relatively inefﬁcient for the produce lactic acid as demonstrated by .
removal of pharmaceuticals and hormones ). Theseresults are a call for prevention and for new remediation strategies.
2.3.4. Detoxiﬁcation Enzymes may be exploited in a tertiary – polishing – treatment in studied the detoxiﬁcation of a cyanide- WWTP. The ability of laccases and peroxidases to degrade EDCs was containing extract from dibittering apricot seeds by cyanidase™.
reviewed by . Studies have shown that natural Cyanidase™ (Novo Nordisk, Denmark) is a preparation of immobi- and synthetic hormones are major contributors to estrogenicity of lized cyanide dihydratase from Alcaligenes denitriﬁcans capable of wastewaters. Among them, estrone (E1), 17β-estradiol (E2), 17α- converting cyanide to ammonia and formate in a single-step reaction ethinylestradiol (EE2) and estriol (E3) can be efﬁciently degraded by (It is characterized by a high afﬁnity toward peroxidases (). Commercial laccases cyanide and a high stability. It is able to remove this anion down to were also able to transform these hormones and the common anti- very low levels, i.e. b0.02 mg L−1 CN−. The diffusional-type ﬂat inﬂammatory diclofenac ).
membrane reactor (FMR) operated with immobilized cyanidase™ Even though peroxidases and laccases have similar afﬁnity for the had superior performance compared to stirred tank reactor and ﬁxed targeted estrogens, HRP for instance seems to be more affected by bed reactor conﬁgurations. The advantage of employing a FMR is a wastewater constituents (unidentiﬁed) than laccase ).
protection of the enzyme and the immobilization support from shear Genistein is an isoﬂavonoid present in bleached wood pulp mill damage. Cyanide diffuses through a semi-permeable membrane to efﬂuent and classiﬁed as an endocrine disruptor. Apparently, it is also react with the entrapped enzyme and reaction products diffuse across widely distributed in sewage efﬂuents and very resistant to the membrane to the solution.
wastewater treatment ). completely removed genistein from a synthetic solution 2.4. Water industry using either MnP or laccase. Beside natural and synthetic hormones,laccase is also able to degrade BPA and NP. used Biological nutrient removal (nitrogen and phosphorus) facilities enzymes from lignin-degrading basidiomycetes for the removal of installed in modern WWTP have a low efﬁciency when wastewater is BPA and NP. They showed a decrease of estrogenic activity and the deﬁcient in organic matter (i.e. low BOD/nitrogen ratio). Addition of production of stable polymers of BPA and NP after laccase treatment.
cheap external carbon sources might solve the problem. This was conﬁrmed by the results of assayed the feasibility to reuse food waste, acid fermented . Cabana et al. also developed a reactor after an enzymatic pretreatment using a mixture of commercial system for BPA and NP treatment. They applied cross-linked laccase glycosylase, protease and lipase. Despite a high content of carbohy- aggregates in a continuously operated perfusion basket reactor. With drates, they noticed that protease among the three enzymes was the this system, 85% of tested EDCs could be continuously eliminated over most efﬁcient for solubilization and the production of volatile fatty a 7-day period ). The enzymatic degradation of acids. They hypothesized that proteases can release carbohydrates TCS and a substantial decrease of bacterial toxicity in the treated from complex biopolymers stabilized by lectin-like proteins. A similar solution were shown by several teams ( enzyme mixture also offers opportunities in sludge dewatering ). TCS is a broad-spectrum antimicrobial agent processes by improving sewage sludge settling and reducing used in personal care products and frequently found in surface water.
disposable solid content (). Indeed, organic Under exposure to sunlight, it is transformed into chlorinated, highly materials account for 60% of solid content in sludge where proteins toxic and persistent products (Recently, laccases were Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi: P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx conjugated with chitosan in order to improve their stability and industrialization has left a legacy of polluted land, water and air.
reusability, and the conjugates were applied to TCS degradation and Petrochemical, agrochemical, smelting, mineral extraction, polymers detoxiﬁcation In the same study, not only and rubber as well as explosives industries are linked to a long list of polymerization but also dechlorination of TCS by laccase was evidenced, polluted sites due to the release of chemicals during processing, use and as previously reported by upon use of laccase accidental spills.
supplemented with mediators. applied Pd–nFe Dimethyl phthalate (DMP) is one of numerous phthalic esters used nanoparticles as a catalyst for TCS dechlorination under anaerobic as plasticizers in plastics manufacturing. Its degradation intermediates conditions and polymerized the resulting 2-phenoxyphenol using are toxic, mutagenic and potential endocrine disruptors ( laccase. Another example of a biotransformation by laccases, not only Some Bacillus species have the ability to grow on DMP as a sole leading to polymerization products, was reported by source of carbon. Isoesterases present in the cell-free extract of a Bacillus Here, the laccase-catalyzed formation of a lactone from the sp. culture are responsible for the initial de-esteriﬁcation ( polycyclic musk fragrance galaxolide (HHCB) was shown.
while a cocktail of enzymes containing proteases, esterases, The peroxidase-mediated degradation of BPA was studied and amylases and tyrosinases are collectively involved in the degradation optimized with respect to critical reaction parameters such as pathway of DMP by growing bacteria When puriﬁed temperature, pH, protective agents and peroxide concentrations by cutinase and puriﬁed yeast esterase were compared regarding their In addition, thirteen other BPA derivatives, also efﬁciency of dipentyl phthalate degradation, cutinase exhibited a extensively used in plastic synthesis, were nearly completely removed remarkable activity The degradation products were under the same conditions. Resulting oligomer precipitates were mainly 1,3-isobenzofurandione when using cutinase and a mixture of 1,3- easily removed from the aqueous solution by ﬁltration. In another isobensofurandione with pentyl methyl phthalate (PeMP) when using the recent study, observed a reduced esterase. Toxicity monitoring showed that PeMP caused inhibition of genotoxicity of a 0.5 mM BPA solution after its continuous treatment bacterial growth and cellular stress. evaluated cutinase in a lab-scale ﬁxed-bed reactor using bitter gourd peroxidase and yeast esterase for the degradation of di-(2-ethylhexyl)-phthalate immobilized on ﬂy ash together with guaiacol as a mediator (DEHP). Consistent with the above mentioned results, fungal cutinase was (0.3 mM). At a ﬂow rate of 20 mL h−1, 76% removal was retained highly efﬁcient in the degradation of DEHP and esterase treatment led to after 30 days. In an attempt to overcome the inherent drawbacks of toxic degradation products.
peroxidases in wastewater processing, i.e. their cost and stability, Polyurethanes are widely used synthetic polymers with poor commercially available Hb from bovine blood was assessed for the biodegradability. These polymers result from condensation of removal of BPA in the presence of electrochemically generated H2O2 polyisocyanates and polyalcohols. They are used in medical, automo- Hb was immobilized on carbon ﬁbers, which tive and industrial applications. Esterases which hydrolyze ester bonds served as a cathode, and immersed in a jacketed beaker (35 mL) along of polyester polyurethanes are putative key enzymes in their microbial with the stainless steel anode. Under optimized conditions, 51% of 100 mg L−1 BPA was removed after 2 h, as compared to a biochemical Cyanides, inorganic compounds with a ―C N group, are potent removal of 35% (Hb + H2O2 supplemented), and an electrochemical respiratory inhibitors. An estimated 3 million tons of cyanide per year removal of less than 5%.
are used in industrial processes including the production of chemical Haloacetic acids (HAA), especially chloroacetic acids, are toxic intermediates, synthetic ﬁbers, rubber and pharmaceuticals, as well as byproducts formed upon disinfection by addition of chlorine to ore leaching, coal processing and metal plating ).
drinking and other process waters. As an alternative to activated Cyanides are produced by certain bacteria, fungi, algae and plants only carbon, electroenzymatic degradation of trichloroacetic acid and in small concentrations; therefore their presence in the environment bromoacetic acid was proposed using Hb immobilized on carbon is mainly attributed to human activities ). Cyanide nanotubes The results proved that reduced waste treatment, if done, is usually a two-stage alkaline chlorination– heme can be effectively regenerated by the electrode and that Hb oxidation process, resulting in problematic sludge with high chlorine exhibits high and stable activity. Tri-, di-, and monochloroacetic acids content. Biological degradation of cyanide has been suggested as an were converted sequentially and could be completely dechlorinated environmentally friendly and inexpensive alternative to conventional at a redox potential of −200 mV. The concept was ﬁnally illustrated in processes ). used an electroenzymatic packed-bed reactor where the average current immobilized S. loti, a cyanide hydratase producer, to degrade cyanide efﬁciency was almost 100%, i.e. HAA were the only electron acceptors wastes to formamide in a continuous reactor system. Up to 100 mM of cyanide was degraded in 2 h and the immobilized mycelia were more Adsorption capacity of papain instead of its catalytic properties stable than the free mycelia retaining about 55% and 15% activity after 3 was exploited for mercury abatement in water. Papain, immobilized on and 6 days at 24 °C respectively. High level of cyanide hydratase activity activated charcoal and alginate beads, was used in a ﬁnishing step to was also reported in Gloeocercospora sorghi and Helminthosporium remove low ppm to ppb levels of mercury remaining after turcicum. Immobilized G. sorghi completely converted cyanide conventional bulk unit operations (i.e. ﬁltration or precipitation) (70 mmol added continuously at 7.5 mL h−1) into formamide for However, this process 30 days and the enzyme stability was dramatically enhanced by the is not based on enzymatic catalysis, as papain is characterized by a addition of glucose in the feed ). strong tendency to bind metals due to the presence of four sulfhydryl designed a packed-bed reactor based on immobilized Fusarium groups in its active center. With both supports, results were oxysporum (cyanide hydratase producer) and Methylobacterium sp. for satisfactory since 99% of the mercury could be removed from the integrated degradation of cyanide and formamide (refer to Section solutions and the adsorption was characterized by fast kinetics.
Nitriles are organic cyanide (R―C N) compounds which are 2.5. Chemical industry present in the environment due to either natural or industrialsyntheses. Xenobiotic nitrile compounds are used as solvents, The chemical industry is a pillar of the modern economy, converting pharmaceuticals, intermediates in organic synthesis, pesticides, etc.
raw materials (oil, natural gas, air, water, metals, and minerals) into tens ). Most of them are toxic, carcinogenic and of thousands of different products. Chemicals are used in the mutagenic () and thus their release into the manufacturing of a wide variety of consumer goods and other products environment needs to be limited. Researchers have described various that are essential for agriculture and industry. However, a long history of organisms, which possess different pathways for cyanide and nitrile Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011), P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx soil and sediments which contribute to their low biodegradability and persistence in the environment. They represent a potential health A mixed culture of bacteria producing risk due to their mutagenicity and carcinogenicity. Bioremediation different nitrile hydrolyzing enzymes (including NHase, nitrilase and technology using microorganisms has been extensively evaluated for amidase) was grown in batch and continuous culture and metabolized PAH-contaminated soils ().
acrylonitrile, fumaronitrile, succinonitrile, etc. ( showed that the laccase from Trametes Using this consortium, the authors reported the remediation of versicolor could oxidize 11 PAHs (acenaphthylene, anthracene, benzo[a] an efﬂuent from acrylonitrile manufacturing industries with a 75% pyrene, acenaphthene, ﬂuoranthene, pyrene, benzo[a]anthracene, reduction in COD and 99% reduction in toxicity. chrysene, benzo[b]ﬂuoranthene, benzo[k]ﬂuoranthene) to corre- have also reported that acrylonitrile decontamination of polymer sponding quinones, plus 3 others (naphthalene, ﬂuorene, and emulsions (aqueous latexes) can be efﬁciently achieved by NHase phenanthrene) when HBT was added (refer to The treatment. used a solvent-tolerant nitrile hydratase same laccase was immobilized on kaolinite and tested for the removal for the biotransformation of nitriles under mild conditions. A of anthracene and benzo[a]pyrene in water in presence of 2,2′-azino- combination of NHase and amidase-producing microorganisms bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) resulted in an efﬁcient conversion (N90%) of 6 M acetonitrile to acetic ). used free fungal laccases for the remediation acid in 10 h. This procedure could be suitable for the treatment of of a PAH contaminated soil. Reasonable transformation of PAH was acetonitrile containing wastes on site or in treatment plants achieved without addition of mediators. The authors hypothesized that (Nitrilases are easily inactivated and their phenol-like compounds in the soil may enhance the enzymatic non-covalent binding onto carriers (hydrophobic, ionic interactions) transformation of PAHs. A priori, the treatment of hydrophobic is more promising than other immobilization methods due to the pollutants adsorbed on organic matters in soil by enzymes in aqueous mildness of this technique solution seems paradoxical. However the addition of miscible organic Emissions of volatile halogenated organic compounds threaten solvents or surfactants in aqueous reaction media (to desorb PAHs) environmental integrity. demonstrated the ability coupled to stabilized enzymatic catalysis in such matrices could solve of lyophilized R. erythropolis cells to continuously transform 1- the problem. Recently, designed a spiral-bed chlorobutane through haloalkane dehalogenase (DHA) activity in a reactor using bitter gourd peroxidase immobilized on the surface of solid–gas bioﬁlter. Solid–gas systems have some advantages over calcium-alginate beads for the continuous treatment of an anthracene- liquid–solid systems, e.g. no solubility and diffusion problems, no need containing model efﬂuent. The reactor was operated over a month (40% for additional solvents and simpliﬁed biocatalyst recovery. Cells treated retained removal efﬁciency), for the treatment of 0.5 mM anthracene in with lysozyme were more active (higher initial rate of degradation) but organic mixture (acetone/N-N dimethylformamide, 35% v/v in water) DHA activity declined with time due to a pH decrease caused by HCl supplemented with 0.1 mM guaiacol as mediator. Further development generation. The addition of volatile triethylamine (Lewis base) was and scale-up of enzymatic remediation of PAHs are clearly restricted at highly beneﬁcial in maintaining the activity. Later, the moment by the use of solvents and mediators as well as the cost of pretreated the biocatalysts with ammonia vapor to further stabilize the DHA (75% residual activity after 60 h) and extended degradation studiesto C5 and C6 halogenated compounds (chloro- and bromo-substituted).
3. Research directions in enzyme technology A DHA from Bradyrhizobium japonicum was cloned and heterologouslyexpressed in Escherichia coli (The recombinant Enzyme catalysis has been exploited in food and beverage processing enzyme displayed broad substrate speciﬁcity toward haloalkanes and for centuries. Nowadays scientists and engineers are aware of the was able to hydrolyze longer compounds than DHA from Rhodococcus.
enormous potential and versatility of enzymatic reactions and are facing For detailed information on aerobic pathways of the bacterial the challenge of their exploitation. This challenge is signiﬁcant as the degradation of halogenated compounds and enzymes involved, the cheap supply of highly active and stable biocatalysts has yet to become a reader can refer to .
reality. Very recently, tools to improve existing biocatalysts, whole cells Recently, the Defense Treaty Reduction Agency has published a call or enzymes, used in established processes have been partially reviewed for new biocatalysts to ﬁnd an environmentally friendly way to remove (). Here, we present examples for the use of these OPs OPs present in some pesticides (e.g.
tools in connection with above-mentioned enzymes which also have parathion, coumaphos, paraoxon) and nerve agents (e.g. sarin, VX, sulfur potential environmental applications ).
mustard) display neurotoxicity in insects and mammals because of theirability to inactivate esterases in the central nervous system, leading tothe accumulation of neurotransmitters and receptor overstimulation.
3.1. Discovery of new enzymes Severe poisonings may cause respiratory failure, convulsions and death(). OPs leaking from crop ﬁelds and warfare Better enzymes are needed to meet the requirements of process agent depots accumulate in ground- and municipal waters. In a engineering and to enable sustainable bioprocesses to compete comparative study, fungal cutinase and yeast esterase were evaluated equitably with "older" processes. The search for new enzyme activities for the degradation of malathion . Cutinase was more among known or previously unknown organisms is called bioprospect- suitable than esterase and had the advantage of transforming malathion ing. The classic culture-dependent way of screening organisms for into the non-toxic malathion diacid. Indeed, production of monoacid interesting metabolic capabilities or ﬁnding them "accidentally" and malathion by esterase appeared to severely inhibit protein synthesis in subsequently identifying, isolating and characterizing responsible bacterial cells. An esterase from a metagenomic library of bovine rumen enzymes is time-consuming, laborious and seldom systematic, yet it bacteria was successfully expressed in Pichia pastoris at a high expression represents an established and proven methodology. Even in the era of "- level of 4.0 g L−1 The results revealed a good omics", this methodology has its value in prospecting enzymes and OP hydrolytic activity since cadusafos, coumaphos, diazinon, dyfonate, remains important in combination with molecular biological methods, ethoprophos, fenamiphos, parathion and methylparathion could be like cloning, heterologous expression, etc. Examples of its persisting relevance include the discovery of novel secreted fungal peroxidases, Polycyclic aromatic hydrocarbons (PAH) are ubiquitous pollutants like aromatic peroxygenases and dye-decolorizing peroxidases, exhibit- mainly resulting from incomplete combustion of organic matter. Because ing surprising catalytic properties as reviewed by of their hydrophobicity, they are easily adsorbed onto organic matter, Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi: P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx For several decades, extremophiles have more than excited the approach does not have a high success rate (i.e. percentage of mutants interest of researchers. The ﬁrst commercialized enzyme from such with desired properties) since it introduces random point mutations organisms was the DNA-polymerase of Thermus aquaticus ( while substantial changes in enzyme functions may require changes Nevertheless, key drawbacks associated with the culture of of not only single amino acids but modiﬁcations of larger portions of extremophiles are the harsh conditions imposed on the equipment the amino acid sequence (insertion, deletion, inversion, etc.), during fermentations and downstream processing, e.g. high salt occurring naturally during recombination events. Nevertheless, concentrations (halophiles), high temperatures (thermophiles) or directed evolution represents an approach yielding impressive pressures (piezophiles) results, especially when mutations affect the active site Advances in molecular biology and analytical techniques coupled ). This was shown for one variant of OPH which exerted a 25-fold with automation and computerized data processing enable a modern higher activity against methyl parathion compared to the native lab to have a high-throughput approach in enzyme prospection.
enzyme ). In a review, also Genome sequence databases, or even more powerful metagenome reported that directed evolution using DNA shufﬂing had produced databases, allow an in silico screening of the whole diversity of enzymes, bacterial mutants expressing OPH with a 725-fold higher hydrolytic not limited by their expression or the culturing of the respective activity against the pesticide chlorpyrifos. Stabilizing mutations often affect the protein surface by adding electrostatic and hydrogen bonds Whereas genome-mining is suitable for prokaryotic enzymes, introns in ). stated that mutations at eukaryotic genes currently hamper a broad exploitation of fungal, the surface rather than in the active site of laccase improved its animal and plant genomes, thus calling for the application of (meta) stability in solvents. The laccase mutant they obtained after ﬁve transcriptomic approaches. This is illustrated by rounds of directed evolution exhibited 19.2-fold higher stability than who combined metatranscriptomics with a functional screening to the native enzyme in 50% (v/v) water–ethanol mixtures.
arrive at expressed, functional fungal phosphatases. The next major The choice between rational design and directed evolution breakthrough can be expected, as proteomics is starting to provide depends on the level of knowledge – including mechanistic comprehensive libraries. Thus, in combination with the above men- understanding – and on the existence of a selection scheme for a tioned methods, proteomics offers the possibility to directly derive given enzyme For instance, directed evolution applied speciﬁc enzymes based on metabolic capabilities of microbial commu- to cellulase is hampered by the development of an efﬁcient selection nities. Furthermore, it is able to overcome limitations of nucleic acid or screening method for mutant cellulases due to the insoluble and based approaches like post-translational modiﬁcations or alternative heterogeneous nature of the cellulose substrate ).
splicing which can alter catalytic properties signiﬁcantly On the other hand, proposed that directed evolution would be particularly attractive for the improvement ofcytochromes P450, as one-step detoxifying enzymes. Mutants 3.2. Designer enzymes producing active, detoxifying P450-enzymes would be capable ofgrowing with high concentrations of substrates toxic to the host cells Industrial applications of enzymes often require conditions (pH, and may be easily selected. This is especially interesting within the ionic strength, organic solvents) that are not congruent with optimal scope of the present review, as the selection of enzymes for activities and stability as suggested by an enzyme's physiological environmental applications is often based on their detoxiﬁcation context. To fulﬁll the requirements of industry, protein engineers can abilities and single enzyme-systems rather than degradation path- use directed evolution and rational design to tailor catalytic efﬁciency ways are preferred, thus allowing for the construction of such (Km, Kcat), extend stability (pH, T, solvents) and even modify the selection systems.
reaction mechanisms (). The impact of Both approaches have already successfully produced stable and these two methodologies on enzyme technology and bioprocess highly active biocatalysts. Following a directed evolution methodology, engineering is growing in importance. Rational design introduces reported the heterologous expression of laccase deﬁned changes in the amino acid sequence using site-directed which exhibited a 22-fold higher Kcat for ABTS, a common substrate for mutagenesis based on the knowledge of three-dimensional structure laccases. obtained a mutant of CYP450 from of enzymes, functions and mechanisms. Molecular modeling predicts Pseudomonas putida with an enhanced activity against PAHs (phenan- how mutations would affect enzyme properties such as selectivity, threne, ﬂuoranthene, pyrene and benzo[a]pyrene) after two directed activity and stability, using databases and structures of homologous mutations in the active site. The reader may also refer to enzymes as a model if the structure of the studied protein is unknown for successful constructions of other heme enzymes such as ). , for example, were able to increase nitrile hydratase activity of papain by 4 orders of Directed evolution and rational design appear to be complemen- magnitude. highlighted new computational tools for tary and their combination increases the chance of obtaining protein design dedicated to small libraries built on knowledge of advanced biocatalysts (). In a two-step optimiza- protein sequence, structure and function. Rational design might still tion, computer modeling is used to identify key amino acids be in its infancy, but an increasing understanding of structure– responsible of enzyme activity and directed evolution is subsequent- function relationships and post-translational processing of proteins ly applied for mutagenesis of the DNA sequence of the "hot spot" alongside with steadily increasing computational power for simula- residues and selection of mutants. The process may be iterative and tions and modeling, will soon make this methodology one of the most after selection of evolved mutants in a ﬁrst round, another rational valuable tools for protein engineers.
design step may be carried out. followed a In contrast, a popular method mimicking natural evolution called combined approach in an attempt to enhance stability and activity of directed evolution requires much less information. Indeed using a fungal peroxidase for its use as dye-transfer inhibitor in laundry random mutagenesis, it is possible to improve catalytic properties detergents. They obtained a mutant enzyme with 174 times the stability without information about protein spatial structure. However, for and 100 times the oxidative activity of the wild-type enzyme. And directed evolution one still needs to have the gene coding for the recently, the total laccase activity (including secretion by Saccharomyces enzyme of interest cloned and ready for expression. In earlier cerevisiae and kinetics) was enhanced 34,000 times after eight rounds of approaches mutations were introduced in whole bacterial genomes.
molecular evolution (). This is one tangible demon- In addition, suitable methods for the screening of the obtained stration of the possibilities offered by the above tools and concepts of mutants for desired properties must be at hand. By its nature, this protein engineering toward tailoring laccases (and, by extension, other Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),

P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx Fig. 4. Overview of research directions in enzyme technology aiming at an efﬁcient bioprocess.
industrially relevant enzymes) to different applications ( expression of native P450 sequence, slight changes in the sequence led to a high level of expression. In addition, an approach to overcome The drop in stability caused by the accumulation of mutations was the lack of P450 reductase activity would be the co-expression or the identiﬁed and redressed by rational design. construction of P450:P450 reductase fusion proteins ( followed an unusual path to improve a haloalkane dehalogenase from Rhodococcus rhodochrous. They have redesigned residues in the A recent thoughtful analysis of the combined role of structural channel connecting the bulk and the buried active site instead of biology and genetic engineering interventions for heterologous concentrating on amino acids from the active site or its vicinity. After expression shows that novel, ﬂexible and highly efﬁcient fungal directed evolution, they obtained a mutant enzyme with a 32-fold enzymes like laccases are possible Among other enhanced activity toward 1,2,3-trichloropropane.
recommendations, the authors underline the possibility of exploiting Heterologous enzyme expression has several advantages over less studied aspects such as the enzyme's glycosylation status: the cultivation of wild-type strains for enzyme production. Microbial structure of glycans could have a decisive impact not only on catalytic fermentations performed with improved strains are usually short, efﬁciency (including redox potential range) but also on stability in economical and allow the overproduction of tightly regulated harsh environments ().
enzymes. Conversely, exploitation of e.g. wild-type white rot fungifor laccase production in bioreactors is still limited for two main 3.3. Insoluble biocatalysts reasons: ﬁrst, an advanced downstream processing is required torecover the target enzymes; second, fermentations of wild-type The development of insoluble biocatalysts is beneﬁcial for all kinds of strains are inherently linked to secondary metabolism and associated enzyme applications but decisively important in a low added value sector process instability, due to protease activity, polysaccharide produc- of the bioeconomy such as environmental services. Bioremediation/bio- tion, uncontrolled growth, etc. ().
degradation with free enzymes is hardly feasible as long as the enzymes have listed 49 laccases heterologously are the major cost-determining factor of the treatment. Heterogeneous expressed with varying degrees of success.
biocatalysis allows biocatalyst recycling in addition to a better process CYP450 from mammalian cells may offer some advantages over control, in turn leading to reduced treatment costs.
their bacterial counterparts and their catalytic properties are better Immobilized enzymes consist of at least two elements; (a) the non- investigated. They have been expressed in yeast ( catalytic part which aims at aiding the separation of the biocatalyst from but expression in bacteria offered other advantages. Although it its surroundings and (b) the catalytic function The dilution was hampered by proteolysis of the NADPH-P450 reductase and of speciﬁc enzyme activity (U enzyme g−1 support) and additional Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi: P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx diffusion constraints are drawbacks of carrier-bound immobilization.
the reaction process could be followed by colorimetry and the shells' Carrier-free immobilization approaches like the crosslinking of enzyme magnetic properties allowed an easy separation. Using a different layer- aggregates (CLEA®, enzyme precipitation followed by chemical reticu- by-layer approach, spherical biopolymer particles made of 80% HRP lation) are possible alternatives, e.g. laccase-CLEA® were fabricated by co-precipitating calcium carbonate and the enzyme and peroxidase-CLEA® ). However, the pure- followed by cross-linking and dissolution of the inorganic matrix protein nature of the biocatalysts thus obtained limits the choice of ). Up to three enzymes – β-glucosidase, reactor types, e.g. an application in packed bed reactors is not readily glucose oxidase and HRP – were co-immobilized in separate and deﬁned feasible until problems of fragility and deformability of the aggregates compartments, still expressing their respective activities. Multicom- are overcome.
partment systems may be desirable in drug delivery, bioreactors and Reversible soluble–insoluble supports have been developed to solve sensors when more than one function is required in a controlled diffusion constraints encountered in heterogeneous biocatalysis.
Covalent immobilization of chitinase and protease on reversiblysoluble polymeric supports (hydroxypropyl methylcellulose acetate 3.4. Additives: redox mediators, cosubstrates and protecting agents succinate) was demonstrated ).
These biocatalysts were water-soluble at pH above 5.5 and insoluble Some oxidoreductases such as laccase and peroxidase have a broad below pH 4.5. Chitin hydrolysis was exempliﬁed in repeated batch substrate spectrum. The range of applications, however, can be further operations with pH cycles which facilitated the enzyme recovery.
expanded to complex or high redox-potential molecules with redox- Recently, developed a protein engineering approach mediators. These are chemicals which act as "intermediate substrates" consisting in genetically fusing amino acids (α-helical leucine zipper for enzymes, because their oxidized radical forms react with the target domain, hexa-histidine tags, etc.) with OPH to produce OPH mutants compounds They not only broaden the enzyme spectrum able to self-assemble in hydrogels beyond a given concentration.
but commonly enhance the degradation rate. Typical redox mediators Nanobiocatalysis is a growing research area which may reconcile for laccases are HBT, ABTS, guaiacol and syringaldazine. The laccase high speciﬁc activity and rigidity of biocatalysts. Advances in nanotech- mediated oxidation of the PAHs anthracene and benzo[a]pyrene using nology have opened new possibilities in catalysis for environmental ABTS and HBT was reported ().
applications. Nanoporous/nanostructured matrices, carbon nanotubes, achieved a degradation of naproxen, an anti- nanoﬁbers and nanoparticles are novel supports which allow the inﬂammatory drug, by including HBT in the reaction medium. Although manipulation of an enzyme environment at the nm-scale mediators broaden the substrate range and expand the applicability of Above all, the high speciﬁc surface area of these materials allows laccases, their use is hampered by limitations such as high/extra costs superior enzyme payloads, leading to high volumetric activity and and additional pollution generated by the mediator itself. Their eventually intensiﬁed processes. For instance, laccase was immobilized utilization at industrial or ﬁeld scale will probably stay limited until an on silica nanoparticles produced with the Stöber method effective technique allows their recovery or alternative mediators, ). A loss of 60% of laccase activity occurred following the cheap and eco-friendly, are available e.g. from lignocellulose residues immobilization but the laccase-nanoparticle conjugates could still In the ﬁeld of wood pulp bleaching, efﬁciently degrade BPA. OPH-carbon nanotubes conjugates were progress is made in the research of natural mediators and syringalde- conceived for decontamination of nerve agents. OPH was irreversibly hyde seems to be a promising alternative as delignifying agent regarding immobilized onto carbon nanotubes, then incorporated in commercial its toxicity and efﬁcacy ( water-based paint and air-dried to obtain a coating of 450 μm Some enzymes need cosubstrates (e.g. P450: NADP(H), MnP: thickness ). The biocatalytic composites, containing H2O2/Mn2+) for their catalytic cycle. One major drawback in the only 0.07% (w/w) OPH, were more stable than native free enzyme and exploitation of peroxidases is their need for peroxides, which have to could decontaminate N99% of 10 g m−2 of paraoxon within 30 min and be delivered in a controlled manner, keeping the concentration high more than 95% of diisopropylﬂuorophosphate in 45 min.
enough to avoid rate limitations and low enough to keep suicide- Biomimetic mineralization has opened a new range of possibilities to inactivation of the enzymes at a minimum. To overcome this problem, entrap enzymes under mild conditions co-immobilized MnP and glucose oxidase on ). Biomineralization is the formation of silica beads to create a catalytic cascade where H2O2 is produced by hierarchically structured biological organic–inorganic materials such glucose oxidase and readily consumed by MnP. Further investigations as bones, shells and teeth. The ﬁeld was expanded to the production of on enzyme loading and kinetic constants would allow a ﬁne-tuned synthetic materials and called bioinspired/biomimetic material synthe- system with a stoichiometric delivery of H2O2. Another option is the sis (Papain was entrapped by biomimetic silication which combination of enzymatic catalysis with in situ electrochemical is the condensation of orthosilicates catalyzed by cationic polymers generation of H2O2, which is exempliﬁed in ). The immobilized enzymes exhibit enhanced pH and For cosubstrates that need to be regenerated, such as NAD(H), the thermal stability compared to free enzymes. have constraints outlined above for mediators also apply. Immobilization of reported the silica deposition onto a gold surface mediated by lysozyme cosubstrates can prevent a washing out from a continuously operated with concomitant immobilization of active OPH. This enzyme could reactor. reported the immobilization of NAD(H) onto degrade paraoxon over 2 days, but its activity decreased over time along nanoparticles. They mixed glutamate dehydrogenase, lactate dehydro- with the loss of silica. have described the genase and NAD(H), all immobilized separately on silica particles, and entrapment of horseradish peroxidase using biosilication methodology demonstrated a successful production of α-ketoglutarate and lactate for an amperometric sensing application. The bioactive silica matrix was with a cyclic regeneration of NAD(H).
formed by the condensation of tetramethyl orthosilicate precursors in Numerous studies reported the use of additives, e.g. synthetic the presence of polyethyleneimine and peroxidases. With entrapment, polymers, salts or sugars as protecting agents for various enzymes ( more than one enzyme or functionality can be simultaneously Enzyme activation and stabilization using immobilized Multifunctional capsules were produced by synthetic polymers like polyethylene glycol (PEG) provide a stable and and applied to BPA and phenol degradation. Based on ﬁxed enzyme conformation with high activity compared to their free Tyr entrapped in a chitosan core, these workers produced a stable form without polymer addition Likewise, millimetric platform including various functions incorporated through a stabilization as CLEA®s partly results from a stable enzyme conforma- layer-by-layer technique. Beside the main oxidative activity of Tyr, the tion obtained after addition of a precipitant (e.g. PEG) and locked in capsules also bound oxidized products (quinones) on the core material, place by the crosslinking. Such additives can be applied for the storage of Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011), P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx biocatalysts but pose problems in processes because high concentra- have combined an electro-enzymatic (peroxidase tions are generally needed to obtain a substantial stabilization effect.
immobilized on Celite and electrogenerated H2O2) and electrochemical(electrogenerated OCl−) process for the remediation of petrochemical 3.5. Reactor and process design wastewater at pilot scale (40 L). The reactor was equally divided intotwo sections allowing enzymatic and chemical reactions in series. The An efﬁcient kinetic modeling is mandatory to operate and control prototype exhibited impressive results with a removal efﬁciency of 85 to enzyme reactors. The most signiﬁcant factors affecting a continuous 93% over a 30-day period. In comparison with solely the electrochemical enzymatic treatment of aqueous efﬂuents are the residence time (RT), process (OCl−), the COD removal efﬁciency of the integrated system the biocatalyst loading (amount of enzymes) and the environmental was two-fold higher, with a power consumption reduced by 30% conditions (temperature, pH, composition of aqueous phase). The (1.99 W g−1 COD). The complex aromatic compounds (methylethyl choice of RT is related to the desired conversion yield and therefore benzene, α-methyl styrene, acetophenone, benzaldehyde, etc.) in the directly linked to pollutant load. The catalyst loading is calculated efﬂuent were transformed mainly into aliphatic compounds (dodecene, with respect to conversion yield (and thus RT), and the catalyst hexadecene, heptadecanal, etc.) by ring cleavage.
recycling rate is linked to the enzyme inactivation rate, dependent on Enzymes are putative tools for the treatment of micropollutants environmental conditions (). Complexity can arise (BPA, NP, hormones, etc.). Substrate afﬁnity of the enzyme, charac- from multi-substrate reactions and substrate/product inhibitions. The terized by Km, is a crucial factor since micropollutant concentrations reactor design is linked to the biocatalyst formulation and vice-versa, are typically much lower than the Km values (up to 6 orders of e.g. suspended solids in water may hamper biocatalyst recovery if its magnitude), limiting the reaction rate. Non-isothermal conditions formulation is inadequate. Also, diffusion-limited catalysis generally might represent a potential tool to increase the efﬁciency of occurs with enzymes immobilized on porous materials with pore sizes enzymatic treatments against pollutants at low concentrations. below 8 to 10 times the enzyme diameter (). In this case, a assayed tyrosinases and diffusion term may be included in the kinetic modeling of the process.
laccases immobilized on nylon and modiﬁed polypropylene mem- Low energy and space demanding processes such as ﬁxed or branes in a thermodialysis process for the degradation of BPA and ﬂuidized bed reactors and membrane bioreactors are favored. In phenol derivatives. Thermodialysis is the selective transport across a domestic wastewaters (i.e. complex efﬂuents), enzymatic treatment is hydrophobic membrane separating two solutions at different tem- generally foreseen as a polishing step (or tertiary treatment), while it peratures. Compared to the same system operated under isothermal is often carried out as pretreatment in more deﬁned industrial conditions, a higher increase of enzymatic activity was shown for the efﬂuents. Nowadays, there is a trend toward integration of physical/- lowest concentrations tested (50–1000 μM) and for substrates having chemical treatments with enzyme reactors such as CSTR plus (ultra) the lowest degradation rate in isothermal conditions. Overall, an ﬁltration, immobilized enzymes on membranes, CSTR plus electro- increase in the enzyme activity was found, proportional to the applied chemistry, etc.
transmembrane temperature difference. In a similar device, have reported the have also observed a lower apparent Km of immobilized lipase use of membrane reactors (ultraﬁltration, 30 kDa) for the oxidative for dimethyl phthalate. The percentage of increase of the reaction rate treatment of phenol-containing synthetic wastewater with free for an actual temperature difference of 1 °C between the membrane tyrosinase and peroxidase, respectively. The enzymes were main- sides rises when DMP concentration decreases (up to 24% °C−1 of tained in the reactor by recycling of the retentate, and the polymer increase). Future studies applying substrate concentrations in the products were gradually adsorbed on the membrane. Under real submicromolar range should validate if non-isothermal conditions are conditions, rather fast clogging of the membrane may occur in such a promising approach to enhance the degradation rate of systems. On the other hand, the use of immobilized tyrosinase on siliceous supports or cross-linkedtyrosinase aggregates () would allow the use of membranes with a higher cut-off (microﬁltration) and would reduceclogging and fouling. In addition, superior stability achieved through A number of waste/pollutant treatments where the use of enzymes immobilization reduces the overall cost of operation. For further might be beneﬁcial have been identiﬁed. Biological wastes were process intensiﬁcation, tyrosinase can be efﬁciently immobilized onto preferentially handled by hydrolases while oxidoreductases and the membrane itself Instead of a lyases are used to tackle various chemical pollutants. Hydrolases membrane technology to recover the biocatalyst, render wastes more amenable to conventional biological treatments covalently immobilized HRP on magnetic beads (79% (i.e. activated sludge) and to bioconversions to value-added sugars, immobilization yield) and applied them in a lab-scale magnetically proteins and lipids which have various outlets such as animal feed, stabilized ﬂuidized bed reactor for continuous transformation of 4- culture media and biodiesel. Oxidoreductases are valuable for the chlorophenol (4-CP). For the treatment of the 4-CP, detoxiﬁcation of textile efﬂuent streams and waters containing have compared soybean peroxidase and UV treatments. Better phenols, drugs and hormones, often in association with ﬁltration results in terms of removal efﬁciency for concentrated solutions (up to 0.5 g L−1 4-CP) were obtained with the UV treatment at high intensity The need for environmental sustainability is the key driving force for (10 J cm−2), but the generation of hazardous soluble benzoquinone the development of enzyme technology for environmental stewardship.
and hydroquinone was mentioned. While peroxidase treatment This review shows that equivalent outcomes have been obtained with required high enzyme concentration to be effective, it produced enzyme technology and with existing technologies, and that better insoluble and thus easily removable polymers. The design of a novel results can be achieved when enzyme-based and classical technologies photobioreactor where photodegradation would be followed by are combined. However, the limited number of ﬁeld applications enzymatic polymerization was proposed. indicates a gap between academia and industry. As the use of enzymes combined ultrasound and enzymatic treatment, and showed an in environmental applications often yields only limited added value, this enhanced removal of 2-chlorophenol compared to the single collaboration is crucial. An integrated approach is needed to efﬁciently treatments. It was alleged that sonication can act physically through transfer scientiﬁc ﬁndings to the market and make it a reality within the accelerated diffusion and enzyme structure modiﬁcations and emerging bioeconomy. The development of a biocatalytic process and chemically through elimination of inhibiting intermediates via the path to its application need input from diverse disciplines. A hydroxyl radical production.
comprehensive identiﬁcation of the target environmental application Please cite this article as: Demarche P, et al, Harnessing the power of enzymes for environmental stewardship, Biotechnol Adv (2011),doi: P. Demarche et al. / Biotechnology Advances xxx (2011) xxx–xxx Fig. 5. Integrated development of an enzyme-based process in environmental technology.
involves not only physicochemical data but current and expected Arica MY, Bayramoglu G. Reversible immobilization of tyrosinase onto polyethyleneimine- grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes. J Mol Catal B: legislation along with evaluation of potential markets for the technology to be developed. Together with life cycle assessment and proﬁtability Arica MY, Altintas B, Bayramoglu G. Immobilization of laccase onto spacer-arm attached studies, this should lead to a holistic decision making process to pursue non-porous poly(GMA/EGDMA) beads: application for textile dye degradation.
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IN THE HIGH COURT OF DELHI AT NEW DELHI Ex.P. No. 81/2005 11th August, 2015 LILLY ICOS LLC. Mr. Chander M. Lall, Ms. Nancy Ray and Mr. Anuj Nain, Advs. AJANTA PHARMA LIMITED Mr. Rajiv Nayar, Sr. Adv. with Mr. Yogender Nath Bhardwaj, Adv. HON'BLE MR. JUSTICE VALMIKI J.MEHTA To be referred to the Reporter or not?

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Im Auftrag der Stadtgemeinde Saalfelden seit 1996 Kinder & Jugendzentrum Saalfelden TREFFPUNKT · Berglandstraße 28 Gefördert durch die Stadtgemeinde Saalfelden und Mitteln des Land Salzburg Im Auftrag der Stadtgemeinde Saalfelden seit 1996 Im Wandel der Zeit! Das Kinder und Jugendzentrum Saalfelden „Treffpunkt", befi ndet sich ständig im Wandel. Viele der „älteren" Jugendlichen in der Altersgruppe von 15 bis 18 Jah-ren, haben nicht nur neue Interessen, sondern stehen auch vor neuen Herausfor-derungen wie dem Schulabschluss, den Beginn einer Lehre oder weiterführende Schule, der Führerscheinprüfung, dem Bundesheer oder Zivildienst und so wei-ter. Sie treffen sich im Jugendzentrum um sich diesbezüglich bei gleichaltrigen Alexander Houtman BEdauszutauschen und auch um Erfahrungswerte von BetreuerInnen anzuhören. Leiter des Kinder & Auffallend sind dabei die „kurzen" Besuche im Jugendzentrum - die Gespräche Jugendzentrum Saalfeldenmit den BetreuerInnen sind dafür aber intensiver.