Extremozymes and their Industrial Applications

Extremophiles belong to members of all three domains of life, i.e., bacteria, archaea, and eukarya. However, a high proportion of extremophiles are archaea and bacteria. These microbes live under chemical and physical extremes that are usually lethal to cellular molecules, yet they not only manage to survive but even thrive in such conditions. Extremophiles have important practical and industrial uses. They are a valuable source of industrially important enzymes also known as extremozymes. Recent research has revealed that extremozymes have unique structural features essential for biocatalysis under extreme conditions. Extremozymes have great commercial values and are known for their potential use in biotechnology, biomining, and bioremediation.

Extremozymes and their Industrial Applications highlights the current and topical areas of research in this rapidly growing field of extremophiles and their applications. Expert researchers from around the globe are trying to uncover the underlying mechanisms responsible for their specific adaptations under extreme environments. The topics covered include the ability of acidophiles to maintain a neutral intracellular pH, the way psychrophiles "loosen up" their proteins at low temperatures, and other equally ingenious adaptations and metabolic strategies that extremophiles use to survive and flourish under extreme conditions.

Extremozymes and their Industrial Applications also covers the established biotechnological uses of extremophiles and the most recent and novel applications, including their exploitation for enzyme production. Potential use of extremophiles and their enzymes in the generation of sustainable energy, biomass conversion, agro-waste processing, and biocontrol of phytopathogens is also covered. The book will be very useful for researchers and students working in the area of industrial microbiology and biotechnology, and microbial ecologists. It is also recommended reference text for those interested in the biochemistry and microbiology of extremophiles, as well as for those interested in bioprospecting, biomining, biofuels, and biodegradation.

• Presents information exclusively based on extremozymes and their application in industries
• Chapters have been collected from various experts and deals with contemporary issues related to extremozymes and their usability in various industries
• Enriched with suitable illustrations that assist in increasing readership and broaden the reach of the book amongst scholars and academicians

• Language: English
• Publisher: Academic Press
• Release date: Jun 15, 2022
• ISBN: 9780323904230

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Chapter 1

Extremozymes and their applications

Dipayan Samanta¹, ³, Tanvi Govil¹, ², Priya Saxena¹, Payal Thakur¹, Adhithya Narayanan⁴ and Rajesh K. Sani¹, ², ³,    ¹Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, Rapid City, SD, United States,    ²Composite and Nanocomposite Advanced Manufacturing Center—Biomaterials (CNAM-Bio Center), Rapid City, SD, United States,    ³BuG ReMeDEE Consortium, South Dakota School of Mines and Technology, Rapid City, SD, United States,    ⁴School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, India

Abstract

The constant motivation for the synthesis of green products has attracted the attention of researchers toward extremophiles and extremozymes. These naturally-derived, extreme catalysts are fast, efficient, selective, specific, consistent, and stable under conditions that can denature their nonextreme counterparts. The additional advantages of using thermozymes in biotechnology include the reduced risk of microbial contamination, improved transfer rates, and improved solubility of substrates under the operating conditions. This powerful development in genetic engineering field is transforming the way these extremozymes are seen and applied. As a result, the demand of extremozymes as hosts for a number of value-added product manufacturing such as biofuels, biopolymers, cyclodextrin, biosensors, keratin-based products, and the design of chiral drugs is on a rise. This book chapter summarizes the application of thermozymes in industrial and biotechnological fields. The chapter also details the list of recombinant extremozymes (e.g., amylases, lipases, laccases, chitinases, prolidases, and esterases) and their tunable catalytic nature during product manufacturing processes.

Keywords

Biofuels; extremozymes; recombinant enzymes; thermophiles; Value-added products

1.1 Introduction

The relentless increase in global pollution level due to the processes implemented by industries to manufacture chemical products and the direct disposal of these recalcitrant compounds has directed many to the constant search for green synthetic products over the past several decades [1–3]. The use of enzymes as catalysts to obtain value-added products has paved the way toward an ecofriendly synthetic era [4]. The chemical reactions governing the synthesis of most industrial products are carried out at high temperatures and pressures, extreme pH, and in a nonaqueous environment [5,6]. In such unusual extreme conditions, where mesophilic enzymes denature, extremozymes are found promising to withstand and catalyze biochemical reactions [7]. The sources of these extremozymes are the extremophiles, which can grow in environments that are inhospitable to support life [8]. The extreme growth environments may include extreme temperatures (>55°C and <4°C) and pH (pH>8 and pH<4), high pressure (>500 atm), highly toxic metallic concentrations (arsenic, cadmium, copper, and zinc), high radiation (>5000 Gy γ radiation and >400 J/m² UV), high salinity (2–5M NaCl or KCl) and low oxygen tension (<10%) [9,10], to name a few.

While a high proportion of extremophiles are archaea, some extremophiles belong to the domain bacteria [11]. Nevertheless, in both the domains, extremophiles can be classified into acidophile, hyper-thermophile, thermophile, psychrophile, alkaliphile, endolith, hypolith, oligotroph, piezophile, radioresistant, metallotolerant, toxitolerant, microaerophile and xerophile [12]. Owing to the optimal activity and excellent stability of extremozymes under extreme conditions, they have found vital applications in various biotechnological industries [13]. The extremozymes are advantageous toward enacting of ecofriendly processes and producing biodegradable products, hence, reducing toxic waste and product size [14]. Moreover, one of the limitations in chemical synthesis is the nontunable physiochemical behavior of the desired product, which can easily be bypassed through the use of extremozymes [15,16]. The enzymatic propriety such as enantioselectivity, chemo-selectivity, regioselectivity, stereoselectivity, and choroselectivity has made biocatalysis promising for the production of perfectly structure-controlled materials [17,18]. Despite the fact that the yield of extremozymes is lower than mesophilic enzymes, the extremozymes’ intrinsic properties such as consistency, reproducibility, economic feasibility, and high purity during production and extraction play a significant role while harnessing an enzyme for industrial applications. This innovative research has made feasible a substantial increase in demand for extremozymes to attain a biobased economy.

Within extremozymes, the heat-active thermozymes have drawn greater attention and promising applications in academia and industry [19]. Thermozymes are resistant to irreversible inactivation at elevated temperatures, that is >55°C, and this property allows them to act as novel catalysts with high research interest in otherwise biologically challenging environments.

This chapter focuses on thermozymes, that is cyclodextrin glucanotransferases, β-Glucosidase, chitinases, prolidases, cellulases, and keratinases, and describe their usage in biotechnological fields. This chapter also details some of the genetically engineered extreme enzymes (e.g., halophilic amylases, thermophilic pullulanase) and their tunable catalytic nature during the product manufacturing process.

1.2 Thermozymes

Even though enzymes have been used to produce various food and alcoholic beverages since the beginning of the 17th century, the significant understanding, commercialization, and active applications of enzymes witnessed a breakthrough with the discovery of enzyme diastase, known as amylase, in 1833 [20–22]. In 1914 the isolation of trypsin from the animal pancreas marked the first enzyme preparation process development [23]. The lone step toward the commercial-scale production of enzyme was made by Novozymes in the 1960s, with the mass production of proteases from Bacillus spp., which was used in detergents [24,25]. In 1974 Taq polymerase, extracted from a thermophilic eubacterial microbe Thermus aquaticus (discovered by T. D. Brock and colleagues), was identified as an enzyme that can withstand the protein-denaturing conditions (high temperature) required during PCR [26]. Since then, many thermophilic, psychrophilic, halophilic, and acidophilic enzymes have been studied, characterized, and used for various industrial applications. Hence, while the journey from chemical catalysts to enzymes is quite promising with an aim to mitigate the adverse consequences on the environment [10,27], the innovative exploration of extremozymes is intriguing due to the fact that extremozymes can cope with extreme industrial conditions. An overview of the applications of various extremozymes and their characteristics are listed in Table 1.1. Amongst all extremozymes, thermostable enzymes are gaining much interest in various industrial applications, which are described below.

Table 1.1

NA, Not Applicable. The information in table was compiled using references [28–34].

Thermophilic bacteria and archaea have drawn the attention of several researchers because of the wide range of industrial applications of their thermozymes for example, proteases, lipases, and polymer degrading enzymes such as cellulases, chitinases, prolidases, and amylases [35,36]. Thermophilic microorganisms grow at temperature between 55°C and 80°C, while those that grow ≥80°C are hyperthermophiles [29]. Most of the thermophilic bacterial and archaeal organisms are widely distributed among the genera: Bacillus, Thermoanaerobacter, Clostridium, Fervidobacterium, Thermus, Rhodothermus, Aquifex, and Thermotoga [37,38]. In contrast, most of the hyperthermophiles belong to the archaea, which fall under the genera- Sulfolobus, Acidanus, Pyrodictium, Pyrolobus, Pyrobaculum, Desulfurococcus, Thermoproteus, Thermofilum and Staphylothermus [39,40]. All of these archaea genera descended from the phyla, Crenarchaeota, are very efficient in producing esterases, mannanases, pullulanases, phytases, ferritins, etc. [41,42]. Three other archaeal phyla—Eurachaeota, Korarchaeota, and Nanoarchaeota, also belong to the hyperthermophilic groups but are less exploited [43]. Table 1.2 lists the thermophilic and hyperthermophilic extremozymes, their physiochemical properties, and respective applications. The overview of the most common thermozymes along with their enzyme classes and contribution in various commercial fields are shown in Fig. 1.1.

Table 1.2

NA, Not available.

Figure 1.1 Overview of the enzyme classes of thermozymes and their contributions to various commercial fields.

Out of all the extremozymes, thermophilic enzymes have found the most commercial use to date mainly due to their characteristic inherent stability and optimal activity at elevated temperatures. Their thermal stability is not owed to any precise or unique characteristic but results from an additive action of specific structural, and dynamic attributes that dispense these thermophilic proteins an utmost stability, compared with mesophilic-derived enzyme-analog [72]. Several in-depth studies concerning the three-dimensional structures of the thermophilic enzymes have fascinatingly covered this fact [73,74]. The unique surface charge property, protein core hydrophobicity, exposed thermolabile amino acids, and increased flexibility due to shorter loops, are majorly responsible for the stability [75,76]. Moreover, the more significant number of disulfide bonds between two cysteine residues within these enzymes provides strong resistance to unfolding at high temperatures and maintain their structural rigidness and integrity [77,78]. Also, high temperatures favor the substantial increase in solubility of many reagents, particularly polymeric substrates, and reduce the risk of mesophilic contamination, which might result in unfavorable complications. The basic three-dimensional structures of each of the selected thermozymes were extracted from Protein Data Bank [79], and are shown in Fig. 1.2. Selected thermophilic enzymes are discussed below.

Figure 1.2 Three-dimensional structures of the selected thermozymes along with their functions- (A) cyclodextrin glucanotransferase [80], (B) β-Glucosidase [81], (C) chitinase [82], (D) prolidase [55], (E) cellulase [83], and (F) keratinase [84].

1.2.1 Cyclodextrin glucanotransferase

(CGTase, EC 2.4.1.19). This enzyme, consists of CBM20 domain, belongs to the carbohydrate binding module family 20. It is an important hydrolytic enzyme within the starch utilization pathway of several bacteria for example, Bacillus and Geobacillus, and regulates various glucan transfer reactions with starch [85]. The biocatalysis of polysaccharides by CGTase initiates with the cleavage of one α-1, 4-linkage within the glucan molecule, followed by the transfer of the newly formed reducing end of the substrate to either its own nonreducing end or to the nonreducing end of a separate linear acceptor molecule [86,87]. Three transferase reaction strategies that include coupling, cyclization, and hydrolysis have been reported to be governed by CGTases. With the aid of these transglycosylation reactions biocatalyzed by CGTases during the degradation of starch, alpha-cyclodextrins and beta-cyclodextrins are produced. The intra-ring conversion of cyclodextrins from alpha to beta and vice-versa has also been made possible using CGTases.

Cyclodextrin has found applications in the food processing industry for the preparation of reduced-cholesterol products and escalating the bioavailability of desired molecules [88,89]. Cyclodextrins facilitate hydrophobic-hydrophilic interactions within protein-protein and other molecules [90,91]. Due to their specific cavity structures, cyclodextrins are in use as an encapsulation additive in the food industry for numerous purposes, such as (1) preventing the degradation of lipophilic food and enhancing its shelf life, (2) solubilizing food pigments and vitamins, (3) controlled release of specific food ingredients, (4) suppressing unwanted odors/tastes and reducing unwanted components, such as trans-fats, allergens, mycotoxins, acrylamides, bitter compounds, (5) removing cholesterol in food and thus controlling the body weight and blood lipid profile, (6) prebiotics to improve the lower gut microflora by the selective proliferation of bifidobacterial components, and (7) dietary fibers that can reduce the glycemic index of food [92–95]. Today, cyclodextrins have been posed as a widely used food supplement in baked goods, cereals, dairy products, and transparent soft drinks, among others, for their antiobesity and antidiabetic effects [96].

Several thermophilic microorganisms that secret CGTases mainly belong to the Bacillus and Geobacillus genera, such as Bacillus alcalophilus, Bacillus circulans, Bacillus lehensis, and Geobacillus stearothermophilus [97,98]. Other microorganisms such as Pyrococcus furiosus, Thermoanaerobacter spp, Anaerobranca gottschalkii, Thermotoga maritima, Thermus thermophilus, Aquifex aeolicus, and Rhodothermus marinus are also used to extract CGTases, though not commonly but promisingly [99,100]. Although CGTases have been reported from mesophilic microorganisms too, CGTases from the thermophilic microorganisms have advantages over mesophilic microorganisms due to the faster kinetic rates. For instance, the CGTase from mesophilic bacterium Paenibacillus macerans (km value of the soluble enzyme with starch as the substrate is 2.5 mM) loses 80% of its enzymatic activity at 60°C [101]. In contrast to CGTase thermophilic bacterium B. circulans can withstand a broad range of temperatures (40°C–70°C with a km value of 0.83 mM using starch as the substrate) [102]. Therefore, the lower km value for the thermophilic bacteria suggests a higher kinetic rate and higher productivity than the km values for the mesophilic bacteria [103]. Fu et al. reported the advantages of thermophilic CGTases over mesophilic CGTases while studying the molecular dynamics of the wild-type CGTase and thermostable mutants of CGTase from a mesophilic bacteria Bacillus macerans. The study described that CGTase mutants with added salt bridges witnessed an enhancement in the protein’s electrostatic interactions and had higher thermostability and better kinetics than the CGTase from mesophiles [104]. Such improved thermostability of CGTases allows their application at elevated temperatures for liquefication of starch, with reduced problems of microbial contamination. Furthermore, the use of thermophilic CGTases for the production of cyclodextrins from the starch allow for a shortened production time for achieving reasonable yields. Industrially, liquefaction of starch is accomplished in a jet cooker at 105°C (primary treatment) followed by a hold at 90°C (secondary treatment) [103]. Thus the use of thermostable CGTases will be advantageous, as they can be easily integrated into the process without extra cost. Thus, because of the defining properties of CGTases to form cyclodextrins from starch, which typically need harsh conditions (80°C–90°C, >8 pH) [105], CGTases isolated from thermophiles have attracted undivided attention.

1.2.2 β-Glucosidase

(EC 3.2.1.21). This enzyme mediates the hydrolyzation of a carbohydrate substrate's glycosidic bond to liberate nonreducing terminal glycosyl residues from the β-D-glycosides (β-D-galactosides, α-L-arabinosides, β-D-xylosides, β-D-fucosides), and oligosaccharides [106,107]. β-glucosidases function in cyanoamino acid metabolism, phenylpropanoid biosynthesis, and starch and sucrose metabolism pathways within bacteria and archaea [108]. Some thermophilic microorganisms such as Thermotoga naphthophila [109], Thermofilum pendens [110], Thermus thermophilum [111], P. furiosus [112], Pyrococcus horikoshii [113], Caldicellulosiruptor bescii [114], and T. maritima, are prominent sources of β-glucosidase. Thermostable β-glucosidases are promising for applications in industries due to their high substrate affinity (lower km values) and retention of enzyme activity for example, about 80% of its enzyme activity is at optimum temperature. For instance, the β-glucosidase from the hyperthermophilic bacterium T. thermophilum has the km value of 4 mM with cellobiose as the substrate [115]. In contrast the β-glucosidase from the mesophilic bacterium Agrobacterium tumefaciens has the km value of 8.3 mM with the same substrate [116]. Moreover, the β-glucosidase from the thermophilic and acidophilic archaebacterium T. pendens has the km value of 0.149 mM using cellobiose as substrate and can withstand a temperature range above 80°C, without losing a significant fraction of its enzyme activity [117]. These examples prove that thermophilic enzymes have are significantly valuable in maintaining the enzyme’s stability at much higher temperatures (above 80°C) without compromising their km values. In comparison, the enzyme activity of mesophilic β-glucosidase decreases to a significant fraction (approximately by 90%) at high temperatures (above 55°C) [118].

In terms of the application, β-glucosidase is a highly versatile enzyme with a favorable industrial prospect for the degradation of lignocellulosic biomass [117,119,120]; hydrolysis of isoflavones [121]; production of biofuels from cellulose rich wastes; [122]; hydrolysis of resentful compounds during juice extraction and release of aroma from wine grapes [123–125]. The glucose production using P. furiosus, along with the endocellulase treatment (from P. horikoshii), has also found remarkable attention [112]. Furthermore, β-glucosidases are the crucial enzymes in producing aromatic compounds from glycosidic precursors present in fruits and fermenting products in the flavor industry [126,127]. β-Glucosidases can improve the organoleptic properties of citrus fruits and juices in which bitterness is in part due to a glucosidic compound, naringin, whose hydrolysis requires β-glucosidase, followed by α-rhamnosidase [128]. β-Glucosidase has a broad range of applications in food processing industries and, hence known as flavor enzyme [129]. β-Glucosidases hydrolyze almost all anthocyanin products and help in effortless separation because anthocyanidins and sugar aglycones are less soluble than anthocyanins, possess little color, and tend to precipitate [130].

1.2.3 Chitinase

(3.2.1.14). This enzyme belongs to the glycoside hydrolase family 19, plays a role in the random endo-hydrolysis of N-acetyl-β-D-glucosaminide (1–4)-β-linkages in chitin and chitodextrins. Chitins are the second most abundant polysaccharide in the ecosystem after cellulose. They are major components of the structural framework of fungal cell walls, the exoskeleton and gut lining of insects, and the shells of crustaceans [131,132]. Chitinases show endo- and exo-hydrolytic activity using a double displacement reaction mechanism. Based on the above two activities, chitinases have been divided into two main categories- endochitinases and exochitinases. The exo-chitinases are further classified into two subcategories: chitobiosidases and 1–4-β-glucosaminidases. Although the property of transglycosylation is expected within the enzymes from the transferase class, the transglycosylation reactions using chitinases are also reported. Chitinases contain a catalytic domain, a fibronectin type III domain, and a chitin binding domain [133]. The catalytic domain with one amino acid in the C-terminal region is sufficient for chitinase activity. Therefore chitinases are vital in a wide range of applications with critical roles in the degradation of crystalline polysaccharides [134].

Chitinase-producing microorganisms are diversely found in the ecosystem, mostly in the form of archaea such as Thermococcus chitonophagus, and Thermococcus kodakarensis [135]. A minimal group of bacteria that also produce chitinases are Bacillus licheniformis and Streptomyces thermoviolaceus OPC-520. With a motive to differentiate between the benefits of the thermophilic chitinases over the mesophilic chitinases, chitinases from T. chitonophagus (hyperthermophilic archaeon) have been observed to obtain the km value of 0.14 mM using chitin as a substrate [135], whereas the mesophilic bacterial genus Streptomyces have shown the km value of 20 mM with the same substrate [136]. The higher viscosity and solubility of the substrates and the lower viscosity of the derived products while using the thermophilic chitinases, also add to the advantages besides, their higher substrate specificity (as observed from the lower km values of the thermophilic chitinases). Moreover, the pH of most thermophilic chitinases is within the acidophilic range (below 4.5), which can be considered as the most useful parameter to use these chitinases in harsh industrial conditions.

Chitinases possess fascinating hydrophobic features that render them appealing for protein engineering studies and increase researchers' escalating attention [137,138]. Chitinases are useful in forming chito-oligosaccharides, chitosan, and other chitin derivatives by the degradation of chitin from biomass [139]. Chitooligosachharides are known to possess antitumor, antimicrobial, immunomodulatory, antioxidant, and antiinflammatory properties and find applications in the food and drug industries [140]. Chitinases from thermophiles such as R. marinus, B. licheniformis, Cohnella sp., Myceliophthora thermophila C1, Thermomyces lanuginosus SSBP, have been reported to be efficient for the generation of chito-oligosaccharides by hydrolyzing chitin at elevated temperatures [141].

Chitinases are indeed quite diverse in their functionality and find their suitability even for field applications such as in the biocontrol of fungal phytopathogens and invasive pests affecting crops [142,143], as well as for the bioremediation in the aquatic ecosystem. Marine wastes constitute several billions of tons of chitin deposits (for example, shellfish wastes generated in the food industry) [144]. These marine wastes can be managed by an efficient enzymatic system of chitin degradation and bioconversion into soluble monomers and chitin oligosaccharides and chitosan oligosaccharides for use in the food and chemical industries [145]. For the marine chitin wastes bioremediation, high temperature, and 40% sodium hydroxide (NaOH) are employed for their demineralization and deproteination, generating low-quality heterogeneous mixtures [141]. The use of NaOH is harmful to the water bodies. Hence, the application of thermophilic chitinases that can hydrolyze the crystalline and insoluble chitin raw materials to chito-oligosaccharides can be a valuable eco-friendly addition to the current thermochemical processes and can bring efficiency to the process along with significant cost reduction [141]. Further, thermophilic chitinases can be used against invasive pests that can withstand arid and high temperature conditions. In the health care sector, thermophilic chitinases with longer shelf life are formulated in lotions and creams to control antifungal infections and preserve the drug’s integrity for an elongated period [146].

1.2.4 Prolidase

(EC 3.4.13.9). It is a multifunctional enzyme that belongs to the peptidase family M24 (methionyl aminopeptidase family). Prolidases are also known as proline dipeptidases and can degrade dipeptides using the proline or hydroxyproline residue located at the C-terminal position [147]. Prolidase is an essential enzyme in the collagen metabolism pathway. It releases a free proline for collagen recycling and, therefore acts as a step-limiting factor in regulating collagen biosynthesis. Thermophilic archaea such as P. horikoshii and P. furiosus have drawn greater attention as the source of thermophilic prolidases with a lack of microorganisms from the bacterial domain. The km values of prolidases from P. horikoshii and P. furiosus using L-Leu-L-Pro as a substrate are 8 and 1.8 mM, respectively [148]. Further, the prolidase enzyme from the hyperthermophilic archaea P. horikoshii can withstand a temperature of 114°C without compromising with the enzyme activity [149].

The crystallographic structure of thermophilic prolidase depicted the homodimeric quaternary structure. In contrast the quaternary structure of prolidase from the mesophilic microorganisms contains a monomeric arrangement [150]. Thus concerning the faster kinetics and enzyme stability at higher temperatures, thermophilic prolidases are significantly valuable over the mesophilic prolidases, with high turnover frequency due to the presence of homodimeric units.

Prolidases from P. furiosus and P. horikoshii have been used in extensive applications such as in component of biodecontamination cocktail, hydrolyzes organophosphorus compounds, thereby reducing their harmful nature, as a biosensor for fluorine-containing organophosphorus compounds, and prevents effects of toxic organophosphorus exposure [149,151]. Prolidases from Alteromonas sp. can be used as biodecontamination foams due to their high activity against G-type nerve agents, such as soman and sarin. In the food industry, prolidases are used as a proteolytic enhancer and therefore, are employed in the cheese-ripening process to improve cheese taste and texture [152].

1.2.5 Cellulase

(EC 3.2.1.4). Cellulases are glycosyl hydrolases that cleave cellulose (an unbranched glucose polymer composed of β-glucose units), lichenin, and cereal β-D-glucans by hydrolyzing β(1→4)-d-glucoside linkages [153,154]. Cellulase has a vital role to play as it degrades and recycles cellulosein the environment. Likewise, their application in industries for the degradation of lignocellulosic is an emerging research area, with broader potential in the paper, pulp, textile, and biofuel industries.

In nature, aerobic and anaerobic thermophilic fungi (Trichoderma reesei, Orpinomyces sp., Humicola insolens, Penicillium decumbens, Neosartorya fischeri), bacteria (Clostridium thermocellum, Clostridium cellulovorans, Ruminococcus flavefaciens, C. bescii, Thermotoga petrophila, R. marinus, Caldibacillus cellulovorans, Acidothermus cellulolytic, Bacillus amyloliquefaciens, Bacillus subtilis, Xylanimicrobium pachnodae, Lachnoclostridium phytofermentans, Acquifex geolicus, Paenibacillus lautus, and Peudomonas campinasensis BL11, Herbinix hemicellulosilytica gen, Herbivorax saccincola gen.), archaea (P. furiosus), and actinomycetes (Thermobifida fusca, Thermobifida halotolerans) have been documented to be efficient cellulase enzyme sources [155–158]. Industrially, for agro-biomass conversion, thermophilic cellulases have garnered interest for their high thermostability, higher specific activity, shorter hydrolysis time, multifunctionality, and broader substrate utilization. For instance, cellulase from a moderately thermophilic soil bacterium, T. fusca, has a km value of 0.197 mM with the β-D-cellobioside as a substrate [74]. On the other hand, using the same substrate, the cellulase producing mesophilic uncultured bacteria grown at 37°C showed km values from 20 to 151 mM with cellobioside, β-glucan and hydroxyethylcellulose, which is much higher as compared to the thermophilic cellulases. Therefore the mesophilic organism limits the faster kinetics of the reaction while using cellulase as the biocatalyst. Besides, thermostable cellulases are promising toward the pretreatment of the biomass, thus reducing the energy cost of the process by 20%–40%, improving the substrate's solubility, and reducing its viscosity.

The last decades have depicted the immense increase in cellulases’ applications in biofuel and the pulp and paper industries [159,160]. The pulping processes that include mechanical refining and grinding of the woody raw material result in high fines, bulk, and stiffness of the cellulosic pulp [161,162]. While with the help of biomechanical pulping method using cellulases, an energy savings of nearly 20%–40% have been reported during refining and modifications in tunable strength properties [162,163]. Improvements in drainage and beat-ability in the paper mills before and after pulping have been observed using mixtures of cellulases (endoglucanases I and II) and hemicellulases [164]. Endoglucanases can reduce the pulp viscosity with a lower degree of hydrolysis. Simultaneously cellulases enhance the bleachability of kraft pulp producing a final brightness score as compared to that of xylanase treatment [165]. On a similar line, deinking in textile industries is carried out using cellulases alone, or in combination with xylanases [166,167]. Most of the applications that have been reported so far for the release of ink from the pulp surface by partial hydrolysis of carbohydrate molecules use cellulases and hemicellulases [168,169]. Due to the strong resistance to anionic surfactants and oxidizing agents, thermostability, pH-stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases, cellulases can be promising to be used as laundry detergent too [24]. Further, in bioremediation, cellulases can be potentially utilized for the treatment of hypersaline wastewater to remove cellulose [170]. As per the global cellulase market research report 2018, the demand and application of cellulase in textile, food, beverages, paper and pulp industry will reach approximately USD 2.3×10⁸, with a compound annual growth rate (CAGR) of 5.5% [171]. These data, studies, and reports suggest that the demand of cellulases for waste management and hydrolysis of plant biomass, has expanded impressively in the last decade or so.

1.2.6 Keratinase

It is the exclusive group of proteases that facilitates the complete degradation of complex and obstinate proteins to valuable amino acids in an eco-friendly way [172,173]. The enzyme was previously assigned with the EC number 3.4.4.25. However, due to ambiguous functionality and a greater number of proteases, the enzyme has been limitedly assigned only to the enzyme class and subclass. The in-vivo role of keratinases is to break down keratin and recycle the amino acids for further synthesis of proteins. Some of the most prominent sources of thermophilic keratinases are Thermoactinomyces candidus, Meiothermus sp. 140, Actinomadura keratinilytica, Caldicoprobacter algeriensis, and Thermoactinomyces sp. YT06.

The thermophilic keratinases differ from mesophilic keratinases based on their specificity toward the formation of products. Beta keratinases obtained from Thermobacter keratinaphilus can break both the alpha and beta polypeptide chains of keratin [172]. In contrast keratinases (30°C) from B. subtilis can only degrade alpha keratin found in hair and wools.

The ability of keratinases to bind to complex and insoluble substrates (e.g., feathers, wool, silk, collagen, elastin, horns, hair, azokeratin, and nails) made their characteristics distinguishably unique from others [174]. Based on the sulfide content, or cysteine content, classification of keratin-rich biomass into soft and hard keratin has been proposed. The textile industry uses keratinases for processing wool fibers [175]. Wool is implicated of structural proteins with high degree of cross-linked disulfide bridges that give fibers tensile strength and offer deep resistance to degradation [176]. The use of keratinases is also marked in the cosmetic industry for the treatment of acne, calluses, keratinized and dry skin removal, and treatment of psoriasis [177,178]. On the other hand, keratinases may be very effective in loosening the nail plates [179].

1.3 Recombinant thermozymes

The product manufacturing units in industries rely primarily on the techno-economic aspects. Their main objective is to (1) enhance the yield during production with less utilization of raw materials, thereby increasing the efficiency of conversion, (2) lessen the overall energy consumption, thereby circumventing the energy-intensive process as in chemical catalysis [180,181]. Over the past decades, the enrichment of recombinant DNA (rDNA) technology methods and protocols has paved the way toward the genetic modifications in enzymes [182,183]. This genetically tunable nature of the enzymes has drawn much attention in recent years and has mainly been applied toward the production of industrially valuable products [184,185]. Therefore, biocatalysis using recombinant enzymes has been promising to encompass these fundamental two points, with more focus on the increased conversion and lowering the activation energy. Nowadays, many extremozymes are modified using genetic tools to tune the yield and physicochemical properties of the products. The recombinant extremozymes and their physical properties with applications are listed in Table 1.3.

Table 1.3

NA, Not available.

1.3.1 Thermophilic pullulanase

Pullulanase is generally used as a starch-debranching enzyme with extensive application in food, chemical, and pharmaceutical industries. The thermostability of the pullulanase enzyme is highly desirable since the starch-debranching process requires a high temperature. Pang et al. chose the surficial residue Lys419 from wild-type pullulanase (type II) of Anoxybacillus sp. WB42, and replaced it with arginine using the strategy of surficial residue replacement and disulfide bond introduction, thereby introduced two disulfide bonds between Thr245 and Ala326 and Trp651 and Val707. As a result, in comparison to the wild type, a 1.5 fold increase in the specific activity (98.20 U/mg) and kcat/km value (12.22 mL/mg s) of the mutated pullulanase was observed [187]. Kim et al. [199], performed site-directed mutagenesis and obtained seven individual mutants from Thermus sp. strain IM6501, and confirmed the optimal temperature at 75°C, which was 15°C higher than the wild type. Higher optimum temperature and thermostability of pullulanase improves the hydrolytic efficiency in the starch-debranching process [199]. Hence, investigation of genetic engineering to create thermostable pullulanase is a subject of tremendous interest for increased applications in starch-based industries, principally those directed for glucose production.

1.3.2 Thermostable lipase

Lipases are an essential group of enzymes with promising applications in the detergent, biosurfactant, cosmetics, and pharmaceutical industries. Thermostable lipases that resist denaturation conditions in industrial processes that use high temperatures and/or organic solvents are a practical necessity. Moderately actively thermophiles have been reported from isolates belonging to the genus Bacillus [202]. Nevertheless, stability of lipases at high temperatures is a requirement for industrial processes. Chopra et al. [188], constructed the Pro247-Ser variant of Bacillus lipase using homology modeling, and rational designing. The mutated enzyme showed an increase in the thermostability of the enzyme by 60-fold at 60°C. A decline in km value was observed with evidence toward a 12-fold enhancement in the catalytic activity. The mutant's hydrolytic activity was also investigated, and it was found that the conversion of the methyl oleate was increased by twofold, compared to the wild type. This study shows that a single amino acid substitution can alter the thermostability and enzyme activity of lipases toward the organic solvents, and pose them as promising biocatalysts for commercialization [188].

1.3.3 Thermostable β-cyclodextrin glucanotransferase

Lee et al., reported the mutations of β-cyclodextrin glucanotransferase from Bacillus firmus var. alkalophilus. H59Q, and Y96M mutants were constructed and β-cyclodextrin glucanotransferase was produced using a recombinant E. coli with a secretive expression system extracellularly. The Y96M mutant showed that the conversion into β-cyclodextrin has been increased from 28.6% to 39% without any significant changes in the β-cyclodextrin polymer ratio. The advantage sought was the reduction of linear combinations of malt oligosaccharides during the production. Li et al. showed that Asp577 mutations of β-cyclodextrin glucanotransferase from B. circulans, is important to increase the catalytic activity of the enzyme. The mutants D577G and D577A, increased the cyclization activity of the enzyme. The km values of D577G and D577A were 36.1% and 18.0% lesser, respectively, thereby increasing the kcat/km values by 43.9% and 23.0% than that of the wild-type enzyme, respectively. These mutations increased not only the affinity of CGTase for maltodextrin but also the catalytic efficiency of the cyclization reaction