Microbial Enzymes for Pollutants Degradation

Biocatalysis offers the green and clean solution to chemical processes and it is emerging as a sustainable alternative to chemical technology. A variety of chemical processes are now carried out by biocatalysts (enzymes) (Gautam et al., 2017; Prakash et al., 2013). Enzymes are biocatalysts that facilitate the conversion of substrates into products by lowering their required activation energy. Recent developments in technology showed that biocatalysis through isolated enzyme is more economical compared to whole-cell utilization. Enzymes are not restrained by inhibitors of microbial metabolism and are also applicable in extreme conditions, limiting microbial activity. Moreover, enzymes are more effective at low pollutant concentrations and can work in the presence of microbial predators or antagonists. The small size of enzymes makes them more mobile compared to microorganisms. All of these properties render enzymes very useful for bioremediation (Rao et al., 2010; Saxena et al., 2020). Bioremediation is carried out by the enzymes that mainly belong to oxidoreductases, hydrolases, and transferases. The main producers of these enzymes are bacteria, fungi (especially white-rot fungi), plants, and microbe-plant association (Rao et al., 2010).

Microbial Oxidoreductases

The oxidoreductases perform humification of different phenolic compounds that are generated from the decomposition of lignin in the soil environment. Similarly, oxidoreductases can also detoxify toxic xenobiotic substances such as phenolic or anilinic compounds polymerization, copolymerization with other substrates, or binding to humic substances (Karigar and Rao, 2011; Park et al., 2006). Oxygenases are the oxidoreductases that perform the oxidation of reduced substances by utilizing oxygen from molecular oxygen and FAD/NADH, NADPH as a co-substrate. Oxygenases are categorized into two groups (monooxygenases and dioxygenases) based on the number of oxygen atoms used for oxygenation (Arora et al., 2009; Karigar and Rao, 2011). Mono-oxygenases incorporate one atom of oxygen into the substrate; whereas, dioxygenases add both atoms of oxygen to a substrate. Mono-oxygenases are grouped into flavin-dependent mono-oxygenases and P450 mono-oxygenases, based on the co-factor requirement (Arora et al., 2010) (Table 1.2). Dioxygenases


Microbial Enzymes for Pollutants Degradation


Enzyme Action


Microbial oxidoreductases


  • • Desulfurization, dehalogination, denitrification, biodegradation, amminification, hydroxylation and biotranformation of aromatic and aliphatic compounds
  • • Methane monooxygenases performs the degradation of methanes, alkanes, cycloalkanes, alkenes, haloalkenes, ethers, and aromatic and heterocyclic hydrocarbons

Arora et al.

  • (2010) ; Grosse et al, (1999); Karigar and Rao
  • (2011)


• Degrade aromatic compounds into aliphatic products

Krzmarzick et al. (2018); Sharma et al. (2019)


  • • Laccases catalyze the oxidation of phenolic compounds such as polyphenols, amino phenols, and methoxy phenols
  • • Diamines, aromatic amines and related substances, N-heterocyles, phenothiazines, thio groups, etc. are oxidized

Barrios-Estrada et al. (2018); Yadav et al. (2018)

Microbial peroxidases



  • • Non-phenolic lignin derivatives are degraded to homologous ketones or aldehydes
  • • Involves in hydroxylation of benzylic methylene groups of aromatic ring cleavages
  • • Catalyzes oxidation of phenolic compounds such as vanillyl alcohol, catechol, acetosyringone, syringic acid, and guaicol etc. preferentially at a much faster rate compared to non-phenolic compounds

Ikehata et al. (2004); Kumar and Chandra (2020); Wong (2009)



  • • It oxidizes both phenolic and non-phenolic compounds
  • • Phenolic compounds such as phenol containing dyes, amines, and lignin derivatives

Kumar and Chandra (2020)



  • • Are able to oxidize phenolic, non-phenolic and lignin derivatives
  • • They do not require any mediator for oxidation of compounds

Knop et al. (2016); Kumar and Chandra (2020)

Hydrolytic enzymes


  • • Catalyze the hydrolysis and synthesis of long chain acylglycerols
  • • Perform hydrolysis of triglycerides and reverse reaction (interesterification and esterification)

Hassan et al. (2018); Lajis (2018)


• Conduct proteolysis by hydrolysis of peptide bonds that link amino acids together in polypeptide chain forming the protein

Wanyonyi and Mulaa (2019)

Cellulases and hemicellulases

  • • Cellulases catalyze the hydrolysis of cellulose into simple glucose units
  • • Hemicellulases perform the hydrolysis of hemicelluloses to release simple fermentable sugars

Kumar et al.


are a multicomponent enzyme system that incorporates oxygen into the substrate. Dioxygenases primarily oxidize aromatic compounds and therefore are applicable in environmental remediation (Karigar and Rao, 2011).

Laccases are oxidoreductases that contain a copper active center and are widely used for environmental protection. Laccases are broadly practiced for the treatment of effluents from dye, textile, leather, pulp and paper, and petrochemical industries. The efficiency of enzymes can be improved by using it in combination with redox mediator molecules. Laccases are involved in dye degradation, especially azo dyes, that are very difficult to degrade as they contain polyphenolic components (Abadulla et al., 2000; Kanagaraj et ah, 2015; Le et ah, 2016). Lignin peroxidase (LiP) belongs to the class oxidoreductase that degrades lignin and its derivatives in the presence of H202. These are heme-containing enzymes that are mainly produced by fungi and bacteria and degrade the polymer via the oxidative process (Kumar and Chandra, 2020). Manganese peroxidase (MnP) is also a heme-containing enzyme that is crucial for lignin degradation. It is a glycoprotein dependent on H202 that requires Mn++ ion for the oxidation of monoaromatic phenols (Kumar et ah, 2017; Kumar and Chandra, 2020). The processing of olive oil, distillery, and pulp and paper industry produces several billion liters of colored, toxic, and harmful wastewater over the world annually. Pulp and paper effluents have been treated with several fungi such as Ceriporiapsis subvermispora, Phenerocheate chrysosporium, Trametes versicolor, Rhizopus oryzae, etc. (Yadav and Yadav, 2015). Versatile peroxidases (VP) are known as hybrid enzymes that belong to oxidoreductase family. They have combined catalytic activities of both LiP and MnP, hence designated as hybrid enzymes. VPs are able to oxidize the compounds with low to high redox potential due to two additional active sites via a mechanism similar to that described for LiPs (Knop et ah, 2016; Kumar and Chandra, 2020; Ravichandran et al.„ 2019).

Hydrolytic Enzymes

Hydrolytic enzymes such as lipase, protease, cellulases, xylanase, and amylase play an important role in the waste treatment and sustainable industrial processing. Lipases are the ubiquitous enzymes that act at the interface between hydro- phobic lipid substrates and hydrophilic aqueous medium to catalyze the hydrolysis of ester bonds in triglycerides. Lipases act on a broad range of substrates in harsh reaction conditions without any requirement of expensive co-factors (Gupta et ah, 2004; Wang et ah, 2019). Lipases are explored as the promising alternatives to assist the degradation of effluents rich in lipids, especially in milk and meat industries. The lipase pretreatment improves the hydrolysis and dissolution of fats available in the effluent from milk processing and reduces the time consumed in later treatments (Adulkar and Rathod, 2014; Golunski et ah, 2017). Lipase assists the degradation of slop oil, a by-product of oil refining, and also generates during cleanup of oil tanks and filters. The high content of hydrocarbons (C3-C40) and other organic compounds make this waste difficult to eliminate from the environment. The combination of Bacillus cereus EN18 and lipase degraded slop oil efficiently (Marchut- Mikolajczyk et ah. 2020).

Proteases are enzymes of ubiquitous nature that catalyze the breakdown of proteins into peptides and amino acids. Based on catalytic action, proteases are divided into exopeptidases and endopeptidases. Proteases are exploited in food, pharmaceutical, leather, and detergent industry (Sharma et ah, 2017). Proteases catalyze the breakdown of proteinaceous substances that enter the environment due to the shedding and moulting of appendages and animal carcasses. Industries such as poultry, fishery, and leather also generate proteinaceous pollutants. Proteases hydrolyze the peptide bond in an aqueous environment (Karigar and Rao, 2011). Proteases can increase the degradation rated of biodegradable substances such as activated sludge, allowing more efficient treatment processes (Kara and Kumar, 2015). Proteases efficiently removes the vanes from the shaft of the detached bird feather and dehairs the goat skin. Protease treatment also reduces the BOD, COD, and pH of tannery waste effluent (Majumder et ah, 2015).

A huge quantity of lignocellulosic biomass is generated through forestry, agricultural practices, timber, pulp and paper, and many agro-industries that create an environmental pollution problem. This large amount of residual plant biomass considered as ‘waste’, can be converted into value-added products such as bioethanol, biomethane, biohydrogen, biobutanol, organic acids, microbial polysaccharides, xylitol, etc. (Kumar et ah, 2016b). The bioconversion of lignocellulosic wastes into valuable products is completed in three steps: pretreatment, hydrolysis of carbohydrates, and fermentation of monomer sugars. The hydrolysis of cellulose and hemicelluloses into fermentable sugars is carried out by cellulases and xylanases. Cellulases, hemicellu- lases, and pectinases are the hydrolytic enzymes that decompose the cellulose, hemicelluloses, and pectin, respectively. Cellulases are a multi-component enzymatic system that consists of three major groups of enzymes: endo-p-1, 4-glucanases, exo- [3-1, 4-glucanase, and (3-glucosidases. Endo-(3-l, 4-glucanases randomly cleave (3-1, 4-glycosidic linkages in amorphous part of cellulose away from chain ends. Exo-(3-l, 4-glucanase, or cellobiohydrolase produces cellobiose by attacking cellulose from reducing and non-reducing chain ends. (3-glucosidases that converts cellobiose and soluble oligosaccharides into glucose (Bansal et ah, 2009; de Castro and de Castro, 2012; Kumar et ah, 2019b). The enzymes that break down the hemicelluloses are collectively called hemicellulases. Endoxylanases randomly cleave the backbone of the xylan chain, producing a mixture of xylooligosaccharide (Kumar et ah, 2019b).

Phytoremediation for Pollutants Degradation and Heavy Metals Removal

Phytoremediation is the in-situ technology that exploits plants and their rhizosphere to remove the contaminants or lower their bioavailability in soil and water. It results in the ecological rehabilitation of contaminated sites (DalCorso et ah, 2019). Various organic pollutants such as polynuclear aromatic hydrocarbons, PCBs, and pesticides are degraded by phytoremediation. It is also able to remove inorganic pollutants like heavy metals (Rezania et ah, 2015). Phytoremediation is considered more suitable when pollutants cover a wide area and are present within the root zone of the plants (Chibuike and Obiora, 2014). Plants and rhizosphere, i.e. soil and microorganisms associated with roots, perform the mechanisms that altogether are responsible for the rehabilitation of contaminated soil. The phytoremediation system includes phytodegradation, phytoextraction, phytovolatilization, phyto(rhizo)satabilization, and phyto(rhizo)filtration (DalCorso et al., 2019). Phytoextraction is the process of absorption of contaminants and their translocation to aerial parts of plants, mainly shoots and sometimes leaves. The removal of heavy metals and other pollutants from contaminated water by plant roots is known as rhizofiltration (Khan and Faisal, 2018; Martmez-Alcala et al., 2012). A wide variety of plants have been tested for phytoremediation purposes. The plants belonging to multiple species of families such as Brassicaceae, Poaceae, Fabaceae, Asteraceae, Salicaceae, Chenopodiaceae, and Careophylaceae have shown phytoremediation potential. Some individual species from families including Cyperaceae, Amaranthaceae, Cannabaceae, Cannaceae, Typhaceae, and Pontederiaceae have also been used for phytoremediation. Every plant shows certain advantages and suffers from some limitations for phytoremediation applications (Gawronski and Gawronska, 2007).

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