Biotechnology of Microbial Enzymes

Dr. Goutam Brahmachari is a full professor of organic chemistry at Visva-Bharati University, Santiniketan, India. His research focuses on developing practical synthetic methods for carbon–carbon and carbon–heteroatom bonds using C–H functionalization, cascade, and cross-coupling techniques, with applications in medicinal heterocyclic scaffolds. He has also studied the chemistry and biology of traditionally used Indian medicinal plants, identifying novel phytochemicals of interest.He is the founding Series Editor of Elsevier’s Natural Product Drug Discovery book series and an elected fellow of the Royal Society of Chemistry. His awards include the Acharya P. C. Ray Memorial Lecture Award (2025) from the Indian Science News Association (ISNA), Kolkata, India; the Dr. Satyajit Chakraborti Memorial Award (2025) from IEM, Kolkata, India; the Chemical Research Society of India Bronze Medal (2021); the Dr. Basudev Banerjee Memorial Award (2021) from the Indian Chemical Society; the Indian National Science Academy Teachers Award (2019); the Dr. Kalam Best Teaching Faculty Award (2017); and the Academic Brilliance Award (2015).

• 1. Biotechnology of microbial enzymes: production, biocatalysis, and industrial applications—an overviewGoutam Brahmachari1.1 Introduction 1.2 An overview of the book1.2.1 Chapter 2 1.2.2 Chapter 3 1.2.3 Chapter 4 1.2.4 Chapter 5 1.2.5 Chapter 6 1.2.6 Chapter 7 1.2.7 Chapter 8 1.2.8 Chapter 9 1.2.9 Chapter 10 1.2.10 Chapter 11 1.2.11 Chapter 12 1.2.12 Chapter 13 1.2.13 Chapter 14 1.2.14 Chapter 15 1.2.15 Chapter 16 1.2.16 Chapter 17 1.2.17 Chapter 18 1.2.18 Chapter 19 1.2.19 Chapter 20 1.2.20 Chapter 21 1.2.21 Chapter 22 1.2.22 Chapter 23 1.2.23 Chapter 24 1.2.24 Chapter 25 1.2.25 Chapter 261.3 Concluding remarks 2. Useful microbial enzymes—an introductionBeatriz Ruiz-Villafa´n, Romina Rodrı´guez-Sanoja and Sergio Sa´nchez2.1 The enzymes: a class of useful biomolecules 2.2 Microbial enzymes for industry 2.3 Improvement of enzymes 2.4 Discovery of new enzymes 2.5 Concluding remarks Acknowledgments Abbreviations References 3. Production, purification, and application of microbial enzymes Anil Kumar Patel, Cheng-Di Dong, Chiu-Wen Chen, Ashok Pandey and Reeta Rani Singhania3.1 Introduction 3.2 Production of microbial enzymes 3.2.1 Enzyme production in industries 3.2.2 Industrial enzyme production technology 3.3 Strain improvements 3.3.1 Mutation 3.3.2 Recombinant DNA technology 3.3.3 Clustered regularly interspaced short palindromic repeats-Cas9 technology 3.3.4 Protein engineering 3.4 Downstream processing/enzyme purification 3.5 Product formulations 3.6 Global enzyme market scenarios 3.7 Industrial applications of enzymes 3.7.1 Food industry 3.7.2 Textile industry 3.7.3 Detergent industry 3.7.4 Pulp and paper industry 3.7.5 Animal feed industry 3.7.6 Leather industry 3.7.7 Biofuel from biomass 3.7.8 Enzyme applications in the chemistry and pharma sectors 3.8 Concluding remarks Abbreviations References 4. Solid-state fermentation for the production of microbial cellulasesSudhanshu S. Behera, Ankush Kerketta and Ramesh C. Ray4.1 Introduction 4.2 Solid-state fermentation 4.2.1 Comparative aspects of solid-state and submerged fermentations 4.2.2 Cellulase-producing microorganisms in solid-state fermentation 4.2.3 Extraction of microbial cellulase in solid-state fermentation 4.2.4 Measurement of cellulase activity in solid-state fermentation 4.3 Lignocellulosic residues/wastes as solid substrates in solid-state fermentation 4.4 Pretreatment of agricultural residues 4.4.1 Physical pretreatments 4.4.2 Physiochemical pretreatment 4.4.3 Chemical pretreatments 4.4.4 Biological pretreatment 4.5 Environmental factors affecting microbial cellulase production in solid-state fermentation 4.5.1 Water activity/moisture content 4.5.2 Temperature 4.5.3 Mass transfer processes: aeration and nutrient diffusion 4.5.4 Substrate particle size 4.5.5 Other factors 4.6 Strategies to improve production of microbial cellulase 4.6.1 Metabolic engineering and strain improvement 4.6.2 Recombinant strategy (heterologous cellulase expression) 4.6.3 Mixed-culture (coculture) systems 4.7 Fermenter (bioreactor) design for cellulase production in solid-state fermentation 4.7.1 Tray bioreactor 4.7.2 Packed bed reactor 4.7.3 Rotary drum bioreactor 4.7.4 Fluidized bed reactor 4.8 Biomass conversions and application of microbial cellulase4.8.1 Textile industry4.8.2 Laundry and detergent 4.8.3 Paper and pulp industry 4.8.4 Bioethanol and biofuel production 4.8.5 Food industry 4.8.6 Agriculture 4.9 Concluding remarksAbbreviationsReferences5. Hyperthermophilic subtilisin-like proteases from Thermococcus kodakarensisRyo Uehara, Hiroshi Amesaka, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya and Shun-ichi Tanaka5.1 Introduction 5.2 Two Subtilisin-like proteases from Thermococcus Kodakarensis KOD1 5.3 TK-subtilisin 5.3.1 Ca21-dependent maturation of Tk-subtilisin 5.3.2 Crystal structures of Tk-subtilisin 5.3.3 Requirement of Ca21-binding loop for folding 5.3.4 Ca21 ion requirements for hyperstability 5.3.5 Role of Tkpro 5.3.6 Role of the insertion sequences 5.3.7 Cold-adapted maturation through Tkpro engineering 5.3.8 Degradation of PrPSc by Tk-subtilisin5.3.9 Tk-subtilisin pulse proteolysis experiments5.4 Tk-SP5.4.1 Maturation of Pro-Tk-SP5.4.2 Crystal structure of Pro-S359A5.4.3 Role of proN5.4.4 Role of the C-domain5.4.5 PrPSc degradation by Tk-SP5.5 Concluding remarksAcknowledgmentsAbbreviationsReferences6. Enzymes from basidiomycetes—peculiar and efficient tools for biotechnologyThaı´s Marques Uber, Emanueli Backes, Vinı´cius Mateus Salvatore Saute, Bruna Polacchine da Silva, Rubia Carvalho Gomes Correˆ a, Camila Gabriel Kato, Fla´vio Augusto Vicente Seixas, Adelar Bracht and Rosane Marina Peralta6.1 Introduction6.2 Brown- and white-rot fungi6.3 Isolation and laboratory maintenance of wood-rot basidiomycetes6.4 Basidiomycetes as producers of enzymes involved in the degradation of lignocellulose biomass6.4.1 Enzymes involved in the degradation of cellulose and hemicelluloses6.4.2 Enzymes involved in lignin degradation 6.5 Production of ligninolytic enzymes by basidiomycetes: screening and production in laboratory scale6.6 General characteristics of the main ligninolytic enzymes with potential biotechnological applications6.6.1 Laccases6.6.2 Peroxidases6.7 Industrial and biotechnological applications of ligninolytic enzymes from basidiomycetes6.7.1 Application of ligninolytic enzymes in delignification of vegetal biomass and biological detoxification for biofuel production6.7.2 Application of ligninolytic enzymes in the degradation of xenobiotic compounds6.7.3 Application of ligninolytic enzymes in the degradation of textile dyes6.7.4 Application of ligninolytic enzymes in pulp and paper industry6.8 Concluding remarksAcknowledgmentsAbbreviationsReferences7. Metagenomics and new enzymes for the bioeconomy to 2030Patricia Molina-Espeja, Cristina Coscolı´n, Peter N. Golyshin and Manuel Ferrer7.1 Introduction7.2 Metagenomics7.3 Activity-based methods for enzyme search in metagenomes7.4 Computers applied to metagenomic enzyme search7.5 Concluding remarksAcknowledgmentsReferences8. Enzymatic biosynthesis of β-lactam antibioticsSwati Srivastava, Reeta Bhati and Rajni Singh8.1 Introduction8.2 Enzymes involved in the biosynthesis of β-lactam antibiotics8.2.1 Isopenicillin N synthase8.2.2 β-Lactam synthetase8.2.3 Carbapenam synthetase (Cps)8.2.4 Tabtoxinine β-lactam synthetase (Tbl S)8.2.5 Deacetoxycephalosporin C synthase and deacetylcephalosporin C synthase8.2.6 Clavaminic acid synthase8.2.7 Nonribosomal peptide synthetases8.3 Semisynthetic β-lactam derivatives8.4 Concluding remarksAbbreviationsReferences9. Insights into the molecular mechanisms of β-lactam antibiotic synthesizing and modifying enzymes in fungiJuan F. Martı´n, Carlos Garcı´a-Estrada and Paloma Liras9.1 Introduction 9.1.1 Penicillin and cephalosporin biosynthesis: a brief overview 9.1.2 Genes involved in penicillin and cephalosporin biosynthesis 9.2 ACV synthetase 9.2.1 The ACV assembly line 9.2.2 The cleavage function of the integrated thioesterase domain 9.3 Isopenicillin N synthase 9.3.1 Binding and lack of cyclization of the LLL-ACV 9.3.2 The iron-containing active center 9.3.3 The crystal structure of isopenicillin N synthase 9.3.4 Recent advances in the cyclization mechanism 9.4 Acyl-CoA ligases: a wealth of acyl-CoA ligases activate penicillin side-chain precursors 9.5 Isopenicillin N acyltransferase (IAT) 9.5.1 Posttranslational maturation of the IAT 9.5.2 The IPN/6-APA/PenG substrate-binding pocket 9.5.3 A transient acyl-IAT intermediate 9.5.4 The origin of IAT: an homologous AT in many fungal genomes 9.6 Transport of intermediates and penicillin secretion 9.6.1 Transport of isopenicillin N into peroxisomes 9.6.2 IAT is easily accessible to external 6-APA 9.6.3 Intracellular traffic of intermediates and secretion of penicillins 9.7 Production of semisynthetic penicillins by penicillin acylases 9.7.1 Molecular mechanisms of penicillin acylases 9.7.2 Novel developments in industrial applications of penicillin acylases 9.8 Concluding remarks Abbreviations References 10. Role of glycosyltransferases in the biosynthesis of antibioticsPankaj Kumar, Sanju Singh, Vishal A. Ghadge, Harshal Sahastrabudhe, Meena R. Rathod and Pramod B. Shinde 10.1 Introduction 10.2 Classification and structural insights of glycosyltransferases10.3 Role of glycosylation in enhancing bioactivity10.3.1 Vancomycin10.3.2 Tiacumicin B10.3.3 Amycolatopsins10.3.4 Digitoxin10.3.5 Aminoglycosides10.4 Engineering biosynthetic pathway of antibiotics by altering glycosyltransferases10.4.1 Combinatorial biosynthesis10.4.2 Glycorandomization10.5 Identification of glycosyltransferases and glycosylated molecules using bioinformatics10.6 Concluding remarksAbbreviationsReferences11. Relevance of microbial glucokinasesBeatriz Ruiz-Villafa´n, Diana Rocha, Alba Romero and Sergio Sa´nchez11.1 Introduction11.2 Synthesis, biochemical properties, and regulation11.3 Structure11.4 Catalytic mechanism11.5 Production11.6 Potential applications in industrial processes 11.7 Concluding remarksAcknowledgmentsReferences12. Myctobacterium tuberculosis DapA as a target for antitubercular drug designAyushi Sharma, Ashok Kumar Nadda and Rahul Shrivastava12.1 Introduction12.1.1 Tuberculosis: global epidemiology12.2 Challenges encountered by the scientific communities12.3 MTB cell wall: a source of drug targets12.3.1 Targeting MTB cell wall enzymes12.4 The diaminopimelate (DAP) pathway (lysine synthesis pathway)12.5 Dihydrodipicolinate synthase (DapA)12.5.1 Structure of MTB DapA12.5.2 Action mechanism of MTB DapA12.5.3 Active site of MTB DapA12.5.4 Kinetic parameters of MTB DapA12.5.5 Regulation of MTB DapA activity12.5.6 Inhibitors against MTB DapA12.6 Previous experiments targeting MTB Dap pathway enzymes12.7 Significance of inhibitors against MTB Dap pathway enzymes12.8 Concluding remarksAcknowledgmentAbbreviationsReferences13. Lipase-catalyzed organic transformations: a recent update Goutam Brahmachari13.1 Introduction 13.2 Chemoenzymatic applications of lipases in organic transformations: a recent update13.3 Concluding remarks References14. Tyrosinase and Oxygenases: Fundamentals and Applications Shagun Sharma, Kanishk Bhatt, Rahul Shrivastava and Ashok Kumar Nadda14.1 Introduction14.2 Origin and Sources14.2.1 Tyrosinase14.2.2 Oxygenase14.3 Molecular Structure of Tyrosinase and Oxygenase14.3.1 Molecular structure of Tyrosinase14.3.2 Oxygenase14.4 Mechanism of Catalytic Action14.4.1 Tyrosinase: mechanism of the reaction14.4.2 Oxygenase14.5 Applications of Tyrosinase and Oxygenase14.5.1 Biological applications14.5.2 Applications in food industry 14.5.3 Applications in bioremediation14.5.4 Medicinal applications14.5.5 Industrial applications14.6 Concluding RemarksAcknowledgementAbbreviationsReferences15. Application of microbial enzymes as drugs in human therapy and healthcareMiguel Arroyo, Isabel de la Mata, Carlos Barreiro, Jose´ Luis Garcı´a and Jose´ Luis Barredo15.1 Introduction15.2 Manufacture of therapeutic enzymes15.2.1 Production and purification15.2.2 Preparation of “single-enzyme nanoparticles”: SENization15.2.3 Oral enzyme therapy15.3 Examples of microbial enzymes aimed at human therapy and healthcare15.3.1 “Clot buster” microbial enzymes15.3.2 Microbial enzymes as digestive aids15.3.3 Microbial enzymes for the treatment of congenital diseases15.3.4 Microbial enzymes for the treatment of infectious diseases: enzybiotics15.3.5 Microbial enzymes for burn debridement and fibroproliferative diseases: collagenase15.3.6 Enzymes for the treatment of cancer15.3.7 Other enzymes for the treatment of other health disorders15.4 Concluding remarksAbbreviationsReferences16. Microbial enzymes in pharmaceutical industryNidhi Y. Patel, Dhritiksha M. Baria, Dimple S. Pardhi, Shivani M. Yagnik, Rakeshkumar R. Panchal, Kiransinh N. Rajput and Vikram H. Raval16.1 Introduction16.2 Cataloging of hydrolases used in pharmaceutical industry16.3 Microbial enzymes in pharmaceutical processes16.3.1 Therapeutics16.3.2 Antiinflammatory16.3.3 Enzybiotics16.4 Concluding remarksAbbreviationsReferences17. Microbial enzymes of use in industryXiangyang Liu and Chandrakant Kokare17.1 Introduction17.2 Classification and chemical nature of microbial enzymes17.2.1 Amylases17.2.2 Catalases17.2.3 Cellulases17.2.4 Lipases17.2.5 Pectinases17.2.6 Proteases17.2.7 Xylanases17.2.8 Other enzymes17.3 Production of microbial enzymes 17.3.1 Fermentation methods 17.3.2 Purification methods 17.4 Applications of microbial enzymes 17.4.1 Plastic/polymer biodegradation 17.4.2 Food and beverage17.4.3 Detergents 17.4.4 Removal of pollutants 17.4.5 Textiles 17.4.6 Animal feed 17.4.7 Ethanol production 17.4.8 Other applications 17.5 Future of microbial enzymes 17.6 Concluding remarks References 18. Microbial enzymes used in food industry Pedro Fernandes and Filipe Carvalho18.1 Introduction 18.1.1 A global perspective on the use of enzymes in the food industry18.1.2 Identification/improvement of the right biocatalyst 18.1.3 Enzyme sources and safety issues 18.2 Microbial enzymes in food industry 18.2.1 Production of enzymes for food processing 18.2.2 Formulation of enzymes for use in food processing 18.2.3 Granulation of enzymes 18.2.4 Tablets18.2.5 Immobilization18.2.6 Applications in food industries18.3 Concluding remarks AbbreviationsReferences19. Carbohydrases: a class of all-pervasive industrial biocatalystsArchana S. Rao, Ajay Nair, Hima A. Salu, K.R. Pooja, Nandini Amrutha Nandyal, Venkatesh S. Joshi, Veena S. More, Niyonzima Francois, K.S. Anantharaju and Sunil S. More19.1 Introduction 19.2 Classification of carbohydrases 19.2.1 Glycosidases 19.2.2 Glycosyltransferase 19.2.3 Glycosyl phosphorylases19.2.4 Polysaccharide lyases19.2.5 Carbohydrate esterases19.3 Sources19.3.1 Marine microorganisms19.3.2 Rumen bacteria 19.3.3 Genetically modified organisms19.3.4 Fungi and yeasts 19.4 Industrial production of carbohydrase 19.4.1 Enzyme immobilization19.5 Industrial applications of carbohydrases 19.5.1 Enzymes involved in the production of beverages19.5.2 Enzymes involved in the production of prebiotics19.5.3 Enzymes involved in syrup and isomaltulose production19.5.4 Enzymes in dairy industry19.5.5 Carbohydrases in animal feed production 19.5.6 Carbohydrase application in pharmaceutical industries19.5.7 Carbohydrases involved in detergent19.5.8 Carbohydrases in wastewater treatment19.5.9 Agriculture19.5.10 Enzymes in textile industry19.5.11 Carbohydrases involved in biofuel production19.5.12 Carbohydrases involved in paper industry19.6 Concluding remarksAbbreviationsReferences20. Role of microbial enzymes in agricultural industryPrashant S. Arya, Shivani M. Yagnik and Vikram H. Raval20.1 Introduction20.2 Soil and soil bacteria for agriculture20.3 Microbial enzymes20.3.1 Nitro-reductase20.3.2 Hydrolases20.3.3 1-Aminocyclopropane-1-carboxylic acid deaminase20.3.4 Phosphate-solubilizing enzymes20.3.5 Sulfur-oxidizing and reducing enzymes20.3.6 Oxidoreductases20.3.7 Zinc-solubilizing enzymes20.4 Microbial enzymes for crop health, soil fertility, and allied agro-industries20.4.1 Crop health (assessment via biocontrol agents)20.4.2 Soil fertility (indicator enzymes)20.4.3 Allied agro-industrial applications20.5 Agricultural enzyme market20.6 Concluding remarks AbbreviationsReferences21. Opportunities and challenges for the production of fuels and chemicals: materials and processes for biorefineriesCarolina Reis Guimara˜ es, Ayla Sant’Ana da Silva, Daniel Oluwagbotemi Fasheun, Denise M.G. Freire, Elba P.S. Bon, Erika Cristina G. Aguieiras, Jaqueline Greco Duarte, Marcella Fernandes de Souza, Mariana de Oliveira Faber, Marina Cristina Tomasini, Roberta Pereira Espinheira, Ronaldo Rodrigues de Sousa, Ricardo Sposina Sobral Teixeira and Viridiana S. Ferreira-Leita˜o21.1 Introduction21.2 Brazilian current production and processing of lignocellulosic sugarcane biomass21.2.1 Cellulosic ethanol: worldwide production and feedstock description 21.2.2 Lignocellulosic biomass components and biomass-degrading enzymes21.2.3 Perspectives and difficulties of cellulosic ethanol production 21.2.4 Enzyme-based initiatives for ethanol production at commercial scale 21.2.5 Perspectives on the use of microalgae as sources of fermentable sugars 21.3 Technical and economic prospects of using lipases in biodiesel production21.3.1 Current biodiesel production and perspectives21.3.2 Biocatalytic production of biodiesel21.3.3 Feedstocks used for biodiesel production21.3.4 Enzymatic routes for biodiesel production21.3.5 Enzymatic biodiesel: state of the art21.3.6 Perspectives for enzymatic biodiesel production21.4 Perspectives on biomass processing for composites and chemicals production21.5 Biogas/biomethane production21.5.1 Enzymes applied to improve anaerobic digestion21.5.2 Generation and use of biogas/biomethane in Brazil 21.5.3 Hydrogen production 21.5.4 Sequential production of hydrogen and methane21.6 Concluding remarksAbbreviationsReferences22. Use of lipases for the production of biofuelsThais de Andrade Silva, Julio Pansiere Zavarise, Igor Carvalho Fontes Sampaio, Laura Marina Pinotti, Servio Tulio Alves Cassini and Jairo Pinto de Oliveira22.1 Introduction22.2 Lipases22.2.1 Immobilization of lipases22.2.2 Immobilization methods and supports22.3 Feedstocks22.3.1 Vegetable oils22.3.2 Animal fats22.3.3 Oily waste22.3.4 Microalgae oil and biomass22.4 Catalytic process22.4.1 Effect of temperature22.4.2 Effect of water content22.4.3 Effect of acyl acceptor22.4.4 Effect of solvent22.4.5 Effect of molar ratio22.5 Reactors and industrial processes22.6 Concluding remarksReferences23. Microbial enzymes used in textile industryFrancois N. Niyonzima, Veena S. More, Florien Nsanganwimana, Archana S. Rao, Ajay Nair, K.S. Anantharaju and Sunil S. More23.1 Introduction23.2 Isolation and identification of microorganism-producing textile enzymes23.3 Production of textile enzymes by bacteria and fungi23.4 Process aspect optimization for producing microbial textile enzymes23.4.1 Effect of initial pH medium for the secretion of textile enzymes by microorganisms 23.4.2 Influence of incubation temperature on the production of textile enzymes by microorganisms23.4.3 Effect of agitation on the secretion of textile enzymes by microorganisms23.4.4 Influence of inoculum concentration on the production of textile enzymes by microorganisms23.4.5 Effect of initial time on the secretion of textile enzymes by microorganisms23.4.6 Influence of carbon sources on the production of textile enzymes by microorganisms 23.4.7 Effect of nitrogen sources on the production of textile enzymes by microorganisms23.5 Purification strategies of textile enzymes23.6 Microbial enzymes used in the textile industry23.6.1 Biodesizing by α-amylases23.6.2 Bioscouring by pectinases aided by proteases, cutinases, and lipases23.6.3 Biostone-washing by neutral cellulases23.6.4 Biobleaching by laccases, catalases, and peroxidases 23.6.5 Biodyeing and printing by pectinases and peroxidases23.6.6 Biopolishing/biofinishing by acid cellulases23.6.7 Use of the mixture of microbial enzymes in textile fabric material processing23.7 Immobilization of textile enzymes23.8 Genetic engineering of bacteria- and fungi-producing textile enzymes23.9 Manufacturers of some commercial textile enzymes23.10 Textile industry effluents’ treatment23.11 Concluding remarksReferences 24. Microbial enzymes in bioremediationShivani M. Yagnik, Prashant S. Arya and Vikram H. Raval24.1 Introduction24.2 Robust microbes/superbugs in bioremediation 24.2.1 Xenobiotic and persistent compounds24.2.2 Robust microbes and their application in bioremediation24.2.3 Metabolic pathway engineering for high-speed bioremediation24.3 Role of microbial enzymes24.3.1 Dye degradation24.3.2 Remediation of hydrocarbon and benzene, toluene, ethylbenzene, and xylene compounds24.3.3 Heavy metal remediation 24.3.4 Pesticide degradation24.4 Remedial applications for industries24.4.1 Designing and developing environmental biosensor24.4.2 Immobilization and bioengineering24.4.3 Biotransformation and bioleaching24.5 Concluding remarksAbbreviationsReferences25. The role of microbes and enzymes for bioelectricity generation: a belief toward global sustainability Lakshana Nair G, Komal Agrawal and Pradeep Verma25.1 Introduction25.2 Bioresources: biorefinery25.3 Hydrolytic enzymes and their applications in various sectors 25.3.1 Ligninolytic enzymes25.3.2 Laccases25.3.3 Cellulases25.3.4 Xylanases25.3.5 Amylases25.3.6 Pectinases25.3.7 Lytic polysaccharide monooxygenases25.3.8 Lipases25.4 Bioelectricity and microbial electrochemical system25.4.1 Working of the microbial fuel cell25.4.2 Use of wastes for electricity generation25.4.3 Hydrolytic enzymes in microbial fuel cell25.5 Limitations and their possible solutions in biorefinery and bioelectricity generation25.6 Prospects25.7 Concluding remarksAbbreviationsReferences26. Discovery of untapped nonculturable microbes for exploring novel industrial enzymes based on advanced next-generation metagenomic approachShivangi Mudaliar, Bikash Kumar, Komal Agrawal and Pradeep Verma26.1 Introduction 26.2 Need for nonculturable microbe study 26.3 Problems associated with nonculturable microbial studies 26.3.1 Relationship with coexisting microbes 26.4 Culture-independent molecular-based methods 26.4.1 Isolation of sample DNA 26.4.2 Metagenomic library construction 26.4.3 Metagenomics 26.4.4 Metatranscriptomics 26.4.5 Metaproteomic 26.5 Different approaches for metagenomic analysis of unculturable microbes 26.5.1 Sequence-based screening 26.5.2 Function-based screening 26.6 Next-generation sequencing and metagenomics 26.6.1 Benefits of metagenomic next-generation sequencing 26.7 Application of unculturable microbes and significance of next-generation metagenomic approaches26.7.1 Agricultural applications 26.7.2 Clinical diagnosis 26.7.3 Xenobiotic degradation 26.7.4 Industrial applications 26.7.5 Bioeconomy 26.8 Concluding remarks Conflict of interest Abbreviations References Index