Nature’s Green Catalyst for Environmental Remediation, Clean Energy Production, and Sustainable Development

BENNY THOMAS1’, DIVYA MATHEW2, and K. S. DEVAKY’

'Assistant Professor, Department of Chemistry, St. Berchmans College, Changanassery, Kerala, India

  • 2FDP Substitute, Department of Chemistry, St. Berchmans College, Changanassery, Kerala, India
  • 1Professor■, School of Chemical Sciences, Mahatma Gandhi University, Kottayam, Kerala, India

'Corresponding author. E-mail: This email address is being protected from spam bots, you need Javascript enabled to view it

ABSTRACT

Green chemistry is the design and application of chemical processes to reduce the practice and generation of materials hazardous to human health and the environment. The exclusion of widely dispersed anthropogenic pollutants is one of the main concents for a sustainable improvement for our planet. Comparing to traditional physicochemical methods, bioremediation is the safest, least troublesome, and most economic treatment. Enzymatic bioremediation has risen as an attractive alternative to traditional methods. Nowadays exciting new opportunities for biocatalysis toward the production of renewable and clean energy sources are rapidly emerging. Based on the premise that these alternatives can contribute to a cleaner environment, especially when using renewable agricultural products, the demand for these energies is increasing.

AN INTRODUCTION TO BIOREMEDIATION

The overall quality of the environment inextricably determines the quality of life on earth. Collective awareness of the environment where we live will lead to an intensive search for designing greener technologies. Biotechnology can offer new platforms for providing proper awareness of the environment to the mankind. Further, biotechnology provides opportunities for the transformation of the pollutants into benign substances, generation of biodegradable materials from renewable sources, and emerging ecologically safe industrial and discarding procedures for the welfare of the population. Bioremediation technology is an effective eco-friendly approach for removing toxic pollutants from the soil and aquatic environment through appropriate organization, conservation, and renovation of the environment.1 hi this process, organic wastes are biologically degraded under controlled maimer to a harmless state below established concentration limits.

PERSPECTIVE FOR ENZYMATIC BIOREMEDIATION

The prime goal of green chemistiy is the reduction or elimination of the practice and generation of hazardous materials through the design, fabrication, and application of chemical procedures and products. Biocatalysts— enzymes and whole cells—offer a greener alternative to conventional organic synthesis under mild reaction conditions in the framework of low energy necessities and minimal problems of isomerization and rearrangement. In addition, biocatalysts are biodegradable. They may exhibit chemo-, regio-, and stereoselectivity ensuring huge reduction in the formation of by-products and evading the necessity for functional group activation, protection, or deprotection.2

ENVIRONMENTAL APPLICATIONS AND BENEFITS OF DIFFERENT ENZYMES

Removal of widely dispersed anthropogenic organic pollutants is one of the main challenges for the sustainable improvement for our planet and the survival of mankind.3 Compared to the conventional physicochemical methods, bioremediation offers enzymatic bioremediation green, minimum troublesome, and most cost-effective treatment using whole microorganisms, either naturally occurring or introduced or isolated enzymes. They degrade the persistent hazardous contaminants into nontoxic or less toxic compounds. Furthermore, enzymatic bioremediation offers more simple systems than whole organisms. Most xenobiotics—the chemical compounds like drags, pesticides, or carcinogens that are foreign to a living organism—can be submitted to enzymatic bioremediation. For instance, polycyclic aromatic hydrocarbons (PAHs), polynitrated aromatic compounds, pesticides such as organochlorine insecticides, bleach plant effluents, synthetic dyes, polymers, and wood preservatives like creosote and pentachlorophenol can be succumbed to enzymatic bioremediation. New developments in the design and application of enzymatic “cocktails” for biotreatment of wastewaters have recently emerged. From a green environmental point of view, the application of enzymes instead of chemicals or microorganisms undoubtedly has advantages like lack of toxic side-products, enhanced bioavailability, and higher scale production.4 For enzymatic bioremediation, the enzyme should be kept at optimal reaction conditions to display high substrate affinity with Km in the micromolar range, supporting thousands of product turnovers. Altogether, the enzymes should exhibit high robustness under the selection of external factors and low dependency on expensive redox cofactors like NAD(P)H, which would be prohibitive in a commercial setting. The disposal and management of the sludge produced from biomass during living cell mediated bioremediation is a problematic concent in developing countries where the demand for water treatment is high. Depending on the choice, enzymes can either function at mild conditions replacing harsh conditions and harsh chemicals or work in extreme conditions and hence save energy and prevent pollution. The high specificity of enzymes results in fewer unwanted side effects and by-products. Besides, enzymes can readily be absorbed back into nature owing to their biodegradability. In short, for a sustainable future, enzymatic bioremediation will definitely open an eco-friendly strategy in a wide range of industries. In literature, there are lots of reports on enzymatic environmental remediation satisfying the principles of green chemistry.

5.3.1 OXIDOREDUCTASES

Oxidoreductases are a class of enzymes used to detoxify hazardous compounds by oxidative coupling.5 The oxidation is carried out through the transfer of electrons from reductants to oxidants and results in the release of chloride ions, CO,, and methanol. These enzymes cleave the chemical bonds of organic pollutants yielding energy and transferring electron from the reduced organic matter (donor) to another compound (acceptor). During the redox reactions, the contaminants are finally oxidized to harmless compounds. Various species of bacteria, fungi, and higher plants are responsible for the production and secretion of oxidoreductases. The energy generated by oxidoreductase during degradation and oxidation of pollutants to harmless compounds is utilized by microorganisms for their metabolic process. Oxygenases, monooxygenases, and dioxygenases are the main members of the oxidoreductase enzyme family.4 Oxidoreductases have been used in the degradation of many natural and man-made pollutants, especially from textile industries, for example, phenolics, anilinics, and dyes.6 The Gram-positive bacteria Bacillus safensis produces oxidoreductase to degrade the petroleum compounds. Chlorinated phenolic compounds are the major harmful component in the effuents of paper and pulp industry. Many fungal species are rich in extracellular oxidoreductase enzymes and are well-thought-out to be apt for the exclusion of these chlorinated phenolic compounds. The filamentous structure of fungi helps them to reach the soil pollutants more effectively than bacteria. White-rot fungi, Panus tigrinus, and its extracellular oxidoreductase can remove the phenols, color, and organic load released from olive mill wastewater. Petroleum-based hydrocarbons and chlorinated compounds like DDT, BHC, and so on can be effectively degraded by the oxidoreductases secreted from the plant families of Fabaceae, Gramineae, and Solanaceae. Further, chlorinated solvents, explosives, and petroleum hydrocarbons can be degraded by phytoremediation.7 Oxidoreductase enzymes released by bacterial species are helpful for the reduction of radioactive metals. The electrons released by the oxidation of organic pollutants are used for the reduction of radioactive elements.8

5.3.1.1 OXYGENASES

Enzyme oxygenase belongs to the family of oxidoreductases. They transfer oxygen from molecular oxygen utilizing FAD (flavin adenine dinucleotide)/ NADH (nicotinamide adenine dinucleotide)/NADPH (nicotinamide adenine dinucleotide phosphate) as a co-substrate. Oxygenases are responsible for the aerobic degr adation of aromatic compounds by increasing their reactivity or water solubility or bringing about cleavage of the aromatic ring.910 The addition of molecular oxygen is an essential step for their degr adation. Thus, they catalyze the cleavage of the aromatic ring by adding one or two molecules of oxygen. Additionally, halogenated organic compounds especially herbicides, insecticides, fungicides, plasticizers, and intermediates for chemical synthesis can be degraded by specific oxygenases. Furthermore, they assist dehalogenation reactions of halogenated methanes, ethanes, and ethylenes in association with multifunctional enzymes.411 On the basis of the number of O, molecules involved, oxygenases are classified into two subclasses.

5.3.1.2 MONOOXYCENASES

Monooxygenases catalyze the oxidation of simple alkanes, complex steroids, and fatty acids.12 Monooxygenases act as biocatalysts in bioremediation process and synthetic chemistry due to their highly region selectivity and stereoselectivity on a wide range of substrates.4 These enzymes require only molecular oxygen for their activities and utilize the substrate as reducing agent.13 They utilize the substrate as reducing agent and necessitate only molecular oxygen for then activities. The main reactions catalyzed by monooxygenases include desulfurization, dehalogenation, denitrification, ammonification, hydroxylation, biotransfonnation, and biodegradation of various aromatic and aliphatic compounds.14 Majority of monooxygenases comprise cofactors,4 for example, flavin-dependent monooxygenases and P450 monooxygenases. Methane monooxygenase is the best one for the degradation of substituted aliphatic, aromatic, and heterocyclic hydrocarbons. Monooxygenase catalyzes oxidative dehalogenation reactions under oxygen-rich conditions whereas they consequence reductive dechlorination affording the formation of labile products under low oxygen levels. Methane monooxygenases are found in cytoplasmic membrane and cytoplasm.

5.3.1.3 DIOXYCENASES

Dioxygenases are multicomponent enzyme systems responsible for the degradation of aromatic pollutants into nontoxic materials. On the basis of their mode of action, aromatic dioxygenases have the element of both aromatic ring hydroxylation and aromatic ring cleavage. Aromatic ring hydroxylation dioxygenases degrade the chemical compounds by the addition of two molecules of oxygen into the ring while aromatic ring cleavage dioxygenases cleave the aromatic rings of compounds.4 They have Rieske (2Fe-2S) cluster and mononuclear iron in their alpha subunit, and hence, they thought to belong to the family of Rieske nonheme iron oxygenases.15

Aromatic hydrocarbon dioxygenase includes toluene dioxygenase, catechol dioxygenase, and so on. They introduce molecular oxygen into their substrate.16 They are effectual for environmental remediation owing to their enantiospecific oxygenation property of a wide range of substrates

5.3.2 LACCASES

Laccases are an interesting group of ubiquitous, oxidoreductase enzymes offering great potential for bioremediation applications.17 They are produced by certain plants, fungi, insects, and bacteria. They are multi-copper oxidases that catalyze the oxidation of phenolic and aromatic compounds present in the soil and water. They are always produced in the cell, but can be secreted extracellularly. They are able to degrade the ortho- and paradiphenols, aminophenols, polyphenols, polyamines, lignins, and aiyl diamines as well as some inorganic ions into less hazardous or nontoxic materials.18-20 In addition, they can oxidize, decarboxylate, and demethylate the methoxy substituted phenolic acids.

Depolymerization of lignin is catalyzed by these enzymes resulting in a variety of phenols. The generated phenol derivatives can be utilized as nutrients for microorganisms or they can be repolymerized to humic materials by laccase itself.21 Laccases can effectively decolorize azo dyes by oxidizing their bonds and finally transform them into less harmful substances. The decolorization activity is mainly due to two laccase isozymes purified from fungus Trametes hispida.22 Laccase immobilization on solid support increases their stability, half-life, and resistance to protease enzymes. Laccase (isolated from fungus Trametes versicolor) immobilized on porous glass beads offers higher thermal, pH, and storage stabilities in the remediation platform.23 Laccase can reduce the dioxygen molecules of pollutants into water by removal of electrons from the organic substrate.24 Synthetic laccase is also reported in paper and pulp industry to boost bleaching of pulp and textiles.4 The substrate specificity of laccases displays strong dependence on pH, and their specificity can be subdued by azides, cyanides, and halides excluding iodide.25 Different laccases display different tolerance power toward the inhibition by halides. The nitrogen concentration in fungi is responsible for laccase production and shows a direct proportionality. Both homologous and heterologous methods can be adopted for the production of recombinant laccases.

5.3.3 PEROXIDASES

Peroxidases are universal enzymes useful for the oxidation of lignin and other phenolic compounds. Hydrogen peroxide and mediators are essential for then oxidation and degr adation activities.4 For instance, phenolic radicals produced by oxidation of phenolic compounds, aggr egates and become less soluble and hence precipitated quickly. These peroxidases may be haem and non-haem proteins.26 In mammals, they strengthen the immune system and assist hormone regulation. Auxin metabolism, lignin and suberin formation, cross-linking of cell wall components, defense against pathogens, or cell elongation are their major functions in plants.26,27 The haem peroxidases are of two types. They are found in animals, plants, fungi, and prokaryotes. In mammals, they strengthen the immune system or help in hormone regulation. In plants, they assist auxin metabolism, lignin and suberin formation, and defense against pathogens or cell elongation. Non-haem peroxidases comprise thiol peroxidase, alkylhydroperoxidase, haloperoxidase, manganese catalase, and NADH peroxidase. The thiol peroxidase is the largest one and comprises glutathione peroxidases and peroxy redoxins as subfamilies.26 The lignin peroxidase (LiP) and manganese peroxidase (MnP) are the most studied enzymes because of their greater potential for the degradation of toxic substances. Horseradish peroxidases can be effectively immobilized as cross-linked enzyme aggregates (HRP-CLEAs) using ethylene glycol-bis [succinic acid N-hydroxysuccinimide] as the cross-linker. HRP-CLEAs are found to be effective in the oxidative para-dechlorination of toxic contaminants and carcinogens like 2,4,6-trichlorophenol with high efficiency.28 Soybean peroxidase (SBP) and chloroperoxidase are well studied for the degradation of thiazole compounds. Peroxidases are further classified into LiP, MnP, and versatile peroxidase (VP).4,29

5.3.3.1 LIPS

LiPs are monomeric haem-containing proteins and secondary metabolites of white-rot fungus.4,30 In LiPs, Fe (III) is penta-coordinated with histidine residue and four tetrapyrrole nitrogens. They necessitate hydrogen peroxide as co-substrate and veratryl alcohol as mediator for catalyzing the oxidation of toxic pollutants.31,32 The degr adation of pollutants proceeds through two- electron oxidation of the native ferric enzyme by H,Ov33 Additionally, LiP degrades lignin and other phenolic compounds. The presence of H,0, and veratryl alcohol (mediator) is essential for the degr adation activity of LiP.

During the course of the reaction, HnO, gets reduced to H,0, and the enzyme LiP gets oxidized. The oxidized LiP fUrther gains an electron from veratryl alcohol and returns to its native reduced state forming veratryl aldehyde. But veratryl aldehyde gets reduced back to veratryl alcohol by taking an electron from the substrate. LiPs display a great potential for the treatment of wastewater in the field of bioremediation. Lignin degradation by bacterial peroxidases is more efficient as compared to fungal peroxidases regarding their specificity and thermostability. LiPs generally degrade the plant cell wall constituent lignin. For LiP-catalyzed degr adation, the aromatic contaminants should have a minimum redox potential higher than 1.4 V.34

5.3.3.2 MnPs

MnPs are haem-containing extracellular enzymes. They are produced by lignin-degrading basidiomycetes fungi. They can effectively oxidize Mn2+ into Mn3+ by a sequence of reactions.4,32 Further, they catalyze the degr adation of several phenols, amine-containing aromatic compounds, and dyes.31 Several acidic amino acid residues and one haem group containing manganese binding site are present in enzyme MnP. Mn2+ ion can stimulate the production of the enzyme MnP and serve as the substrate for the enzyme MnP. The Millions generated act as a mediator for the oxidation of phenolics and afford Mn3+-chelates. Xenobiotic pollutants may be buried deep within the soil, and hence, they are not primarily accessible to the enzymes. But the Mn3+-chelates are small enough to diffuse into areas inaccessible even to the enzyme.31 For instance, MnP immobilized on chitosan beads activated by glutaraldehyde shows a greater potential for decolorization of dye effluent fr om the textile industry, hr addition, the stability and half-life of MnP can be enhanced by immobilization.

5.3.3.3 MICROBIAL VPs

VPs are capable of oxidizing Mn2+, methoxybenzenes, and phenolic aromatic compounds directly, hr comparison with peroxidases, VP exhibits extraordinary substrate specificity to oxidize the phenolic and nonphenolics even in the absence of manganese.35 Consequently, a highly efficient VP overproduction is essential for the bioremediation of recalcitrant pollutants.36,37

5.3.4 HYDROLYTIC ENZYMES

The soil and water pollution by industrial effluents is a serious problem of the modem world.4 Bioremediation offers a safe and economic alternative to commonly used remedies. Extracellular hydrolases secreted by microbes play a key role in degradation of organic polymers and toxic compounds with molecular weights less than 600 Da that can diffuse through the pores of the cell.38 Hydrolytic enzymes are most commonly used for the bioremediation of pesticides and insecticides by disrupting chemical bonds like ester, peptide, and carbon-halide in the toxic molecules. Extracellular hydrolytic enzymes like amylases, proteases, lipases, DNases, pullulanases, and xyla- nases have diverse potential applications in the areas of food industry, feed additive, biomedical sciences, and chemical industries.39 The hemicellulase, cellulase, and glycosidase are of much application in biomass degr adation.40

5.3.4.1 LIPASES

Lipases are specific for the breakdown of lipids.41 They are produced by bacteria, plants, actinomycetes, and animal cells. They are helpful in the drastic reduction of total hydrocarbon content hi the contaminated soil through processes like hydrolysis, interesterification, alcoholics, and aminolysis. They are beneficial for the hydrolysis of triglyceride, the main component of natural oil or fat, into diacylglycerol, monoacylglycerol, glycerol, and fatty acids.4 Lipolytic reactions occur at the lipid-water interface, where lipolytic substrates usually form equilibrium between monomeric, micellar, and emulsified states, hi the biphasic oil-water system, the enzyme lipase gets adsorbed on to the oil-water interface in the bulk of the v'ater phase and then breaks the ester bonds of triolein to diolein, monoolein, and glycerol, and oleic acid is formed at each consecutive reaction stage. The glycerol formed is hydrophilic in nature and gets dissolved into the v'ater phase. Monoacylglycerol is effectively used as an emulsifying agent in the food, cosmetic, and pharmaceutical industries. Lipase activity is one of the most usefiil parameters for testing the degradation of hydrocarbon in soil.42

5.3.4.2 CELLULASES

Cellulases are the key enzymes for the degradation of cellulose, the most abundant biopolymer found on the earth.43 Cellulases are generally produced by microorganisms.44 They may be cell-bound, associated with cell envelope, and extracellular. Usually, cellulases are composed of a mixture of several enzymes such as endoglucanase and exoglucanase or cellobiohydrolase.45 The endoglucanase attacks regions of low crystallinity in the cellulose fiber and creates free chain ends. But the exoglucanase removes cellobiose units from the free chain ends of cellulose and degrades it. The enzyme action of p-glucosidase is helpfi.il for the hydrolysis of cellobiose to glucose units. Cellulases are useful in the detergent and washing powders manufacturing industries, where cellulose microfibrils produced during processes are removed by these enzymes.46 Alkaline cellulases can be employed for the bioremediation of ink in paper and pulp industry during the recycling of paper and waste management.47-48 Cellulase can adapt to harsh environmental conditions like extreme pH and temperature.

5.3.4.3 PhlOSPHOTRI ESTERASES

Phosphodiesterases have application in the degradation of chemical wastes released from industrial fields and pesticides (e.g., parathion, malathion) used in crop fields.49 Parathion is an organophosphatic component in herbicides and insecticides.50 Organophosphate is an ester of phosphoric acid and is degraded by phosphodiesterases. There are aryldialkylphosphatase, organophosphorus hydrolase, and recombinant thermostable phosphodiesterases too.

5.3.4.4 HALOALKANE DEHALOCENASES

Halogenated compounds produced by both natural activities and manmade efforts are present everywhere in the soil. These compounds may be hazardous, toxic, mutagenic, or carcinogenic. Haloalkane dehalogenases are useful for the hydrolysis of carbon halogen bonds present in the various halogens containing contaminants and produce alcohol and halides.51 The active site of haloalkane dehalogenase is present between the main domains of an eight-stranded p-sheet helices. First haloalkane dehalogenase discovered from the bacterium Xanthobacter autotrophicus has the ability to degrade 1, 2-dichloroethane. Several dehalogenases have been cloned and characterized from Gram-positive and Gram-negative haloalkane degrading bacteria.

5.3.4.5 PROTEASES

Proteases are class of enzymes responsible for the hydrolysis and reverse synthesis of peptide bonds in aqueous environment and nonaqueous environment, respectively.52 They hydrolyze the proteinaceous substance produced by shedding and molting of appendages, death of animals, and as the by-product of poultry, fishery, leather, detergent, and pharmaceutical industries.53 Based on the mode of catalysis of peptide chain, there are endopepti- dase like serine endopeptidase, cysteine peptidase, aspartic endopeptidases, metallopeptidases, and so on, and exopeptidases such as aminopeptidase, carboxypeptidase, and so on. The endopeptidase acts on the iimer regions of peptide chain whereas the exopeptidases are sensitive to the terminal amino or carboxylic position of chain only.54 Proteases are beneficial for the manufacture of cheese, detergent, non-calorific artificial sweetener, and effective therapeutic agents. The alkaline proteases are usefi.il for removing hairs in the leather industry. Subtilisin in combination with antibiotics is helpful in the treatment of bums and wounds.55

 
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