Polysaccharides of Starchy and Lignocellulose Materials and their Use in Ethanol Production: Enzymes and other Factors Affecting the Production Process
Applied Food Biotechnology,
Vol. 9 No. 2 (2022),
Background and Objective: Nowadays, production of ethanol involves many kinds of plant based materials, from conventionally used starchy materials such as rye, wheat, corn and barley to lignocellulose materials serving in second-generation bioethanol production. While raw materials containing simple sugars do not require such complex mashing processes, starchy and lignocellulose materials need significant processing. This review provides an in-depth description of the structures of starchy raw materials commonly used in production of first-generation ethanol. Furthermore, the review describes the structure of lignocellulose biomasses used for second-generation bioethanol production.
Results and Conclusion: Methods commonly used in distilleries to release starch from plant raw materials belong to pressure-thermal pretreatments known as steaming or pressureless liberation of starch methods. Literature shows that amylolysis is strongly determined by the morphology of starch granules. The larger the speciﬁc surface area of granules, the greater their susceptibility to amylolysis. The key stage in preparation of starch raw materials for fermentation is starch hydrolysis, which consists of two steps of liquefaction and saccharification. Several species of bacteria (e.g. Bacillus licheniformis) and fungi (e.g. Aspergillus niger) are available that are capable of producing enzymes necessary for starch hydrolysis. Enzymes needed for starch hydrolysis are divided into 1) liquefying enzymes such as α-amylase produced by Aspergillus niger and Bacillus licheniformis or can be found in malt and 2) saccharifying enzymes such as glucoamylase, β-amylase and maltogenic α-amylase of fungal, bacterial and malt origins. Proteases and phytases are used to support mashing process hydrolases of non-starch polysaccharides (xylanase and pullulanase).
Conflict of interest: The authors declare no conflict of interest.
- ▪ Amylases ▪ Cellulose ▪ Ethanol ▪ Polysaccharides ▪ Starch ▪ Supportive enzymes
How to Cite
Aehle W. Enzymes in Industry, Production and Application. WILEY-VCH, The Netherlands, 2007.
Balcerek M, Pielech-Przybylska K. Effect of supportive enzymes on chemical composition and viscosity of rye mashes obtained by the pressureless liberation of starch method and efficiency of their fermentation. Eur Food Res Technol. 2009; 229: 141-151.
Raigond P, Ezekiel R, Raigond B. Resistant starch in food: A review. J Sci Food Agric. 2015; 95: 1968-1978.
Rocha TS, Cunha VAG, Jane J, Franco CML,Structural Characterization of Peruvian Carrot (Arracacia xanthorrhiza) Starch and the Effect of Annealing on Its Semicrystalline Structure. J. Agric. Food Chem. 2011; 59(8): 4208-4216.
Strąk‐Graczyk E, Balcerek M, Pielech-Przybylska K, Zyzelewicz D. Simultaneous saccharification and fermentation of native rye, wheat and triticale starch. J Sci Food Agric. 2019; 99: 4904-4912.
Blazek J, Gilbert EP. Effect of enzymatic hydrolysis on native starch granule structure. Biomacromolecules 2010; 11: 3275-3289.
Sujka M, Jamroz J, Starch granule porosity and its changes by means of amylolysis. Int Agrophys. 2007; 21: 107-113.
Qi X, Tester RF. Effect of native starch granule size on susceptibility to amylasehydrolysis. Starch/Starke 2016; 68: 807-810.
Langenaeken NA, De Schepper FC, De Schutter DP, Courtin CM. Different gelatinization characteristics of small and large barley starch granules impact their enzymatic hydrolysis and sugar production during mashing, Food Chem. 2019; 295: 138-146. https://doi.org/10.1016/j.foodchem.2019.05.045
Dhital S, Butardo VM, Jobling SA, Gidley MJ. Rice starch granule amylolysis-Differentiating effects of particle size, morphology, thermal properties and crystalline polymorph. Carbohydr Polym. 2015; 115: 305-316.
Pielech-Przybylska K, Balcerek M, Nowak A, Wojtczak M, Czyzowska A, Dziekonska-Kubczyk U, Patelski P. The effect of different starch liberation and saccharification methods on the microbial contaminations of distillery mashes, fermentation efficiency and spirits quality.
Molecules 2017; 22: 1-20.
Hourston JE, Ignatz M, Leubner‐Metzger G, Reith M, Steinbrecher T. Biomechanical properties of wheat grains: the implications on milling. J R Soc Interface. 2017; 14:1-12.
Li E, Dhital S, Hasjim J. Effects of grain milling on starch structures and flour/starch properties. Starch/Staerke 2014; 66: 15-27.
Moran ET. Starch: Granule, amylose-amylopectin, feed Preparation and recovery by the fowl's gastrointestinal tract. J Appl Poultry Res, 2019; 28(3)566-586.
Hoover R. The Impact of heat-moisture treatment on molecular structures and properties of starches isolated from different botanical sources. Crit Rev Food Sci Nutr. 2010; 50(9): 835-847.
Burgess SA, Lindsay D, Flint SH. Thermophilic bacilli and their importance in dairy processing. Int J Food Microbiol. 2010; 144(2): 215-225.
Srichuwong S, Fujiwara M, Wang X, Seyama T, Shiroma R, Arakane M. Simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash for the production of ethanol. Biomass Bioenerg. 2009; 33: 890-898. https://doi.org/10.1016/j.biombioe.2009.01.012
Saha BC, Nichols NN, Qureshi N, Kennedy Gj, Iten LB, Cotta MA. Pilot scale conversion of wheat straw to ethanol via simultaneous saccharification and fermentation. Bioresource Technol. 2015; 175: 17-22.
Naguleswaran S, Li J, Vasanthan T. Amylolysis of large and small granules of native triticale, wheat and corn starches using a mixture of α‐amylase and glucoamylase. Carbohydr Polym. 2012; 88: 864-874.
Shariff YN, Uthumporn U, Karim AA. Hydrolysis of native and annealed tapioca and sweet potato starches at subgelatinization temperature using a mixture of amylolytic enzymes. Int Food Res J. 2017; 24(5): 1925-1933.
Strąk‐Graczyk E, Balcerek M. Effect of pre-hydrolysis on simultaneous saccharification and fermentation of native rye starch. Food Bioprocess Technol. 2020; 13: 923-936.
Liu C, Li K, Li X, Zhang M, Li J. Formation and structural evolution of starch nanocrystals from waxy maize starch and waxy potato starch. Int J Biol Macromol. 2021; 180: 625-632.
Xu QS, Yan YS, Feng JX. Efficient hydrolysis of raw starch and ethanol fermentation: a novel raw starch-digesting glucoamylase from Penicillium oxalicum. Biotechnol Biofuels. 2016;9(1):1-18.
Szymanowska-Powałowska D, Lewandowicz G, Błaszczak W, Szwengiel A. Structural changes of corn starch during fuel ethanol production from corn flour. J Biotechnol Computat Biol Bionanotechnol. 2012;9(3):333-341.
Uthumporn U, Shariffa YN, Karim A. Hydrolysis of native and heat-treated starches at sub-gelatinization temperature using granular starch hydrolyzing enzyme. Appl Biochem Biotechnol. 2012;166(5):1167-1182.
DuPont TM. Granular starch hydrolyzing enzyme for ethanol production. https://www.dupont.com/content/dam/dupont/products-and-services/industrial-biotechnology/documents/DuPont-STARGEN002-WB-web-EN.pdf. (Accessed 30 Aug 2020).
Balcerek M, Pielech-Przybylska K. Effect of simultaneous saccharification and fermentation conditions of native triticale starch on the dynamics and efficiency of process and composition of the distillates obtained. J Chem Technol Biotechnol. 2013;88(4):615-622.
Strąk‐Graczyk E, Balcerek M. Effect of pre-hydrolysis on simultaneous saccharification and fermentation of native rye starch. Food Bioprocess Technol. 2020; 13: 923-936.
Cheng X,Zhang M, Adhikari B, Islam MN. Effect of power ultrasound and pulsed vacuum treatments on the dehydration kinetics, distribution and status of water in osmotically dehydrated strawberry: a combined NMR and DSC study. Food Bioprocess Technol. 2014;7(10):2782–2792.
Pietrzak W, Kawa-Rygielska J. Ethanol fermentation of waste bread using granular starch hydrolyzing enzyme: effect of raw material pretreatment. Fuel 2014; 134: 250-256.
Abedi E, Pourmohammadi K, Jahromi M, Niakousari M, Torri L. The effect of ultrasonic probe size for effective ultrasound-assisted pregelatinized starch. Food Bioprocess Technol. 2019; 2: 1852-1862.
Tse TJ, Wiens DJ, Reaney MJT. Production of Bioethanol-A Review of Factors Affecting Ethanol Yield. Fermentation 2021; 7(4): 268.
Absar N, Zaidul ISM, Takigawa S, Hashimoto N, Matsuura-Endo C, Yamauchi H, Noda T. Enzymatic hydrolysis of potato starches containing different amounts of phosphorus. Food Chem. 2009; 112(1): 57-62.
Cornaggia C, Evans DE, Draga A, Mangan D, McCleary BV. Prediction of potential malt extract and beer filterability using conventional and novel malt assays. J Institute brew. 2019; 125(3): 294-309.
Dong L, Piao Y, Zhang X, Zhao C, Hou Y, Shi Z. Analysis of volatile compounds from a malting process using headspace solid-phase micro-extraction and GC–MS. Food Res Int. 2013;51:783-789.
Souza PMD. Application of microbial α-amylase in industry-a review. Braz J microbial. 2010; 41(4): 850-861.
De Souza PM. De Oliveira MP. Application of microbial α-amylase in industry-a review. Braz J Microbiol. 2010; 41(4): 850-861.
Avwioroko OK, Anigbor AA, Unachukwu NN, Tonukari NJ. Isolation, identification and in silico analysis of α-α amylase gene of Aspergillus niger strain CSA35 obtained from cassava undergoing spoilage. Biochem Biophys Rep. 2018; 14: 35-42.
Delkash-Roudsari S, Zibaee A, Abbasi Mozhdehi MR. Digestive α-α-amylase of bacterocera oleae gmelin (diptera: tephritidae): biochemical characterization and effect of proteinaceous inhibitor. J King Saud Univ Sci. 2014;26(1):53-58. https://doi.org/10.1016/j.jksus.2013.05.003
Inam-ul-Haq A, Hussain I. Mutational effects of thermo-stable a-amylase producing Bacillus Species. Schol Adv Animal Vet res. 2015; 3(1): 35-41.
Gupta A, Gautam N, Modi DR. Optimization of α-α amylase production from free and immobilized cells of Aspergillus niger. J Biotechnol Pharm Res. 2010;1(1):1-8.
Sundarram A, Murthy TP. α-amylase production and applications: a review. J Appl Environ Microbiol. 2014;2(4):166-175.
Sudan SK, Kumar N, Kaur I, Sahni G. Production, purification and characterization of raw starch hydrolyzing thermostable acidic α-α-amylase from hot springs. India. Int J Biol Macromol. 2018; 117: 831-839.
Far BE, Ahmadi Y, Khosroushahi AY, Dilmaghani A. Microbial alpha-amylase production: Progress, challenges and perspectives. Adv Pharm Bull. 2020; 10(3): 350-358.
Wang X, Kan G, Ren X, Yu G, Shi C, Xie Q. Molecular cloning and characterization of a novel α-α-amylase from antarctic sea ice bacterium Pseudoalteromonas sp. M175 and its primary application in detergent. Biomed Res Int. 2018; 1-16.
Dou S, Chi N, Zhou X, Zhang Q, Pang F, Xiu Z. Molecular cloning, expression and biochemical characterization of a novel cold-active α-α-amylase from Bacillus sp. dsh19-1. Extremophiles 2018; 22(5): 739-749.
Sharma A, Satyanarayana T. Microbial acid-stable α-amylases: characteristics, genetic engineering and applications. Process Biochem. 2013; 48(2): 201-211.
Tarhriz V, Mohammadzadeh F, Hejazi MS, Nematzadeh G, Rahimi E. Isolation and characterization of some aquatic bacteria from Qurugol Lake in Azerbaijan under aerobic conditions. Adv Environ Biol. 2011; 5(10): 3173-3178.
Oboh G. Isolation and characterization of amylase from fermented cassava (Manihot esculenta Crantz) wastewater. Afr J Biotechnol. 2005; 4(10): 1117-1123.
Abou-Elela G, El-Sersy NA, Wefky SH. Statistical optimization of cold adapted α-α-amylase production by free and immobilized cells of Nocardiopsis aegyptia. J Appl Sci Res. 2009; 5(3): 286-292.
Ozcan D, Sipahioglu HM. Simultaneous production of α-α and beta amylase enzymes using separate gene bearing recombinant vectors in the same Escherichia coli cells. Turk J Biol. 2020; 44: 201-207
De Schepper CF, Michiels P, Buve C, Van Loey AM, Courtin CM. Starch hydrolysis during mashing: a study of the activity and thermal inactivation kinetics of barley malt α-amylase and β-amylase, Carbohydr Polym. 2021; 255: 117494, https://doi.org/10.1016/j.carbpol.2020.117494.
Rama Mohan Reddy P, Ramesh B, Mrudula Gopal Reddy S, Seenayya G. Production of thermostable β-amylase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: Optimization of nutrient levels using response surface methodology. Process Biochem. 2003; 39 (3): 267-278.
Kumar P, Satyanarayana T. Microbial glucoamylases: characteristics and applications. Crit Rev Biotechnol. 2009;29(3):225-255. https://doi.org/10.1080/07388550903136076
Carrasco M, Alcaino J, Cifuentes V, Baeza M. Purification and characterization of novel cold adapted fungal glucoamylase. Microb Cell Fact. 2017; 16: 75.
Lam WC, Pleissner D, Lin CSK. Production of fungal glucoamylase for glucose production from food waste. Biomolecules 2013;3(3):651-661.
Monma M, Mikuni K, Ishigami H, Kainuma K. Purification of the glucoamylase components of Chalara paradoxa by affinity chromatography and chromatofocusing. Carbohydr Res. 1987; 159: 255-261.
Nagasaka Y, Muraki , Kimura A, Suto M, Yokota A, Tomita F. Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 1995; 44: 451-458.
Kataria A, Sharma R, Sharma S, Singh B, Kaur G, Yakubu M. Recent applications of bio-engineering principles to modulate the functionality of proteins in food systems. Trends Food Sci Technol. 2021; 113: 54-65.
Liu HL, Wang WC. The predicted unfolding order of the beta-strands in the starch binding domain from Aspergillus niger glucoamylase. Chem Phys Lett. 2002; 366: 284-290.
Marulanda VA, Botero Gutierrez CD, Cardona Alzate CA. Chapter 4 - Thermochemical, Biological, Biochemical and Hybrid Conversion Methods of Bio-derived Molecules into Renewable Fuels. In: Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals and Bioproducts. Editor(s): Hosseini M., Woodhead Publishing, 2019; 59-81.
Motta FL andrade CCP, Santana MHA. A Review of Xylanase Production by the Fermentation of Xylan: Classification, Characterization and Applications. Sustainable Degradation of Lignocellulosic Biomass-Techniques, Applications and Commercialization, 2013: 10:251-276
Collins T, Gerday C, Feller G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev. 2005; 29: 3-23.
Biely P, Markovic O, Mislovicova D. Sensitive detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in gels. Anal Biochem. 1985; 144: 147-151
Shi ZJ, Xiao LP, Deng J, Xu F, Sun RC. Isolation and characterization of soluble polysaccharides of Dendrocalamus brandisii: a high-yielding bamboo species. Bioresources 2011; 6(4): 5151-5166
Pauly M, Keegstra K. Plant cell wall polymers as precursors for biofuels. Curr Opin Plant Biol. 2010; 13(3): 304-311. https://doi.org/10.1016/j.pbi.2009.12.009
Czarnecki Z, Nowak J. Effects of rye pretreatment and enrichment with hemicellulolytic enzymes on ethanol fermentation efficiency. Electron J Pol Agric Univ Topic. Food Sci Technol. 2001; 4(2): 12.
Scheffler A, Bamforth CW. Exogenous β‐glucanases and pentosanases and their impact on mashing. Enzyme Microbiol Technol. 2005; 36: 813-817.
Balcerek M, Pielech-Przybylska K. Effect of supportive enzymes on chemical composition and viscosity of rye mashes obtained by the pressureless liberation of starch method and efficiency of their fermentation. Eur Food Res Technol. 2009; 229: 141-151. https://doi.org/10.1007/s00217-009-1035-y
Adler-Nissen J. Enzymic Hydrolysis of Food Proteins, Elsevier, 1986.
Lantero OJ, Fish JJ. Solvay Enzymes, Inc., assignee. 1993. Process for Producing Ethanol. US Patent no. 5,231,017.
Gohel V, Duan G, Maisuria VB. Impact of an acid fungal protease in high gravity fermentation for ethanol production using indian sorghum as a feedstock. Biotechnol Progress. 2013; 29(2): 329-336.
Cheng KC, Demirci A, Catchmark JM. Pullulan: Biosynthesis, production and applications. Appl Microbiol Biotechnol. 2011; 92: 29-44.
Bertoldo C, Antranikian G. Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr Opin Chem Biol. 2002;6(2):151-160.
Hi, SL, Tan JS, Ling TC, Ariff AB. Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res. 2012.
Roy A, Messaoud EB, Bejar S. Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp. US149. Enzyme Microb Technol. 2003; 33(5): 720-724.
Duffner F, Bertoldo C andersen JT, Wagner K, Antranikian G. A new thermoactive pullulanase from Desulfurococcus mucosus: Cloning, sequencing, purification and characterization of the recombinant enzyme after expression in Bacillus subtilis. J Bacteriol. 2000; 182(22): 6331-6338.
Leveque E, Janecek S, Haye B, Belarbi A. Thermophilic archaeal amylolytic enzymes. Enzyme Microb Technol. 2000; 26(1): 3-14.
Kunamneni A, Singh S. Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7. Enzyme Microb Technol. 2006; 39(7): 1399-1404. https://doi.org/10.1016/j.enzmictec.2006.03.023
Jensen BD, Norman BE. Bacillus acidopullyticus pullulanase: applications and regulatory aspects for use in food industry. Process Biochem. 1984; 1: 397-400.
Poliakoff M, Licence P. Sustainable technology: Green chemistry. Nature. 2007; 450(7171): 810-812.
Lei XG, Porres JM. Phytase enzymology, applications and biotechnology. Biotechnol Lett. 2003; 25: 1787-1794. https://doi.org/10.1023/a:1026224101580
Greiner R, Konietzny U. Phytase for food application. Food Technol Biotechnol. 2006; 44(2): 125-140.
Singh B, Satyanarayana T. Microbial phytases in phosphorus acquisition and plant growth promotion. Physiol Mol Biol Plants. 2011; 17: 93-103.
Sparvoli F, Cominelli E. Seed biofortification and phytic acid reduction: A conflict ofInterest for the Plant? Plants 2015; 4(4): 728-755.
Mikulski D, Klosowski G. Phytic acid concentration in selected raw materials and analysis of its hydrolysis rate with the use of microbial phytases during the mashing process. J Inst Brew. 2015; 121(2): 213-218.
Polyakov VA, Serba EM, Overchenko MB, Ignatova NI, Rimareva LV. The effect of a complex phytase-containing enzyme preparation on the process of rye wort fermentation. Foods Raw Mat. 2019; 7(2): 221-228.
Stedile T, Ender L, Meier HF, Simionatto EL, Wiggers VR. Comparison between physical properties and chemical composition of bio-oils derived from lignocellulose and triglyceride sources. Renew Sustain Energy Rev. 2015; 50: 92-108.
Karp SG, Woiciechowski AL, Soccol VT, Soccol CR. Pretreatment strategies for delignification of sugarcane bagasse: a review. Braz archives biol and technol. 2013; 56(4): 679-689.
Perez-Carrillo E, Serna-Saldivar SO, Chuck-Hernandez C, Cortes-Callejas ML. Addition of protease during starch liquefaction affects free amino nitrogen, fusel alcohols and ethanol production of fermented maize and whole and decorticated sorghum mashes. Biochem Eng J. 2012; 67: 1-9.
Vanholme R, Morreel K, Ralph J, Boerjan W. Lignin engineering. Curr Opin Plant Biol. 2008; 11(3): 278-285. https://doi.org/10.1016/j.pbi.2008.03.005
Zhang Y, Li Q, Su J, Lin Y, Huang Z, Lu Y, Sun G, Yang M, Huang A, Hu H, Zhu Y. A green and efficient technology for the degradation of cellulosic materials: Structure changes and enhanced enzymatic hydrolysis of natural cellulose pretreated by synergistic interaction of mechanical activation and metal salt. Biores Technol. 2015; 177: 176-181.
Pandey A. Handbook of Plant-Based Biofuels. New York: CRC Press, 2009.
Yu Z, Zhang B, Yu F, Xu G, Song A. A real explosion: the requirement of steam explosion pretreatment. Biores Technol. 2012; 121: 335-341.
Cardona CA, Quintero JA, Paz IC. Production of bioethanol from sugarcane bagasse: Status and perspectives. Biores Technol. 2009; 101(13): 4754-4766.
Sarkar N, Ghosh SK, Bannerjee S, Aikat K. Bioethanol production from agricultural wastes: A overview. Renew Energy. 2012; 37(1): 19-2
Kim JS, Lee YY, Kim TH. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Biores Technol. 2016; 199: 42-48.
Maki M, Tin Leung K, Qin W. The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci. 2009; 5(5): 500-516.
Wei Y, Das S, Berke-Schlessel D, Ji H, McDonough J, Feng L, Zhang X, Zhai W, Cao Y. Synthesis of a re-usable cellobiase enzyme catalyst through in situ encapsulation in nonsurfactant templated sol-gel mesoporous silica. Top Catal. 2012; 55: 1247-1253.
Lai C, Zeng GM, Huang DL, Zhao MH, Wei Z, Huang C, Xu P, Li NJ, Zhang C, Chen M, Li X, Lai M, He Y. Synthesis of gold-cellobiose nanocomposites for colorimetric measurement of cellobiase activity. Spectrochimica Acta Part A. 2014; 132: 369-374. https://doi.org/10.1016/j.saa.2014.04.091.
Cannella D, Jorgensen H. Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production?. Biotechnol Bioeng. 2014; 111(1): 59-68.
Pielech-Przybylska K, Balcerek M, Patelski P, Dziekonska-Kubczak U. Solutions for improvement of saccharification and fermentation of high gravity rye mashes. Int Agrophys. 2019; 33: 1-10.
Liang Y, Yesuf J, Schmitt S, Bender K, Bozzola J. Study of cellulases from a newly isolated thermophilic and cellulolytic Brevibacillus sp. strain JXL. J Ind Microbiol Biotechnol. 2009;36:961-970.
Yanase S, Yamada R, Kaneko S, Noda H, Hasunuma T, Tanaka T, Ogino C, Fukuda H, Kondo A. Ethanol production from cellulosic materials using cellulase expressing yeast. Biotechnol J. 2010; 5: 449-455. https://doi.org/10.1002/biot.200900291
Lopez-Linares CJ, Garcia-Cubero MT, Lucas S, Gonzalez-Benito G, Coca M. Microwave assisted hydrothermal as greener pretreatment of brewer’s spent grains for biobutanol production. Chem Eng J. 2019; 368: 1045-1055.
del Rio PG, Gomes-Dias JS, Rocha CMR, Romani A, Garrote G, Domingues L. Recent trends on seaweed fractionation for liquid biofuels production. Bioresource Technolog. 2020; 299: 122613.
Lugowoj S, Balcerek M, Pielech-Przybylska K. Buckwheat as an interesting raw material for agricultural distillate production. Pol J Food Nutr Sci. 2020; 70(2): 125-137.
Chen A, Xu T, Ge Y, Wang L, Tang W, Li S. Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase. Enzyme Microbiol Technol. 2019; 124; 79-83.
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