Abstract
α-Amylase is the major biocatalyst involved in saccharification process of starch-based biofuel production. Most of the α-amylases reported in literature are not stable and catalytically active in presence of metabolites (biosolvents, and bioacids), and other media components such as salts and reducing agents, hence starch-based biofuels are produced by two-step process of separate hydrolysis by α-amylase, and later the fermentation of biofuel production. Finding new α-amylase with special properties can assist to integrate the process of saccharification and biofuel fermentation. We report a new α-amylase produced from wild type Bacillus strain IBT108. This α-amylase (10.5 U/ml) can be produced from wheat bran (5%) based medium. It can be purified by diethylaminoethyl weak anion exchange-based fast protein liquid chromatography. Purified α-amylase has molecular mass of 68 kDa with a high specific activity (734.8 U/mg), and significant Vmax (1428.6 U/mg) and Km (2.8 mg/ml). Protein fingerprinting analysis reveals that it is a unique member of super-families of AmyAc and MaltAmyC which can act on both α-1,4 and α-1,6-glycosidic linkages of starch. It shows optimal activity at pH 7 and 70 °C, retains 72–92% of its activity in acidic pH range between 4 and 6.5, and stable at 60 °C for 2 h. Notably, it is resistant to 5 mM concentration of urea, dithiothreitol, β-mercaptoethanol, surfactants and EDTA, also shows > 70% of its activity in presence of 1 M solvents. Consequently, adding this α-amylase (2.5–3 U/ml) assists Clostridia to effectively utilize 375 g/l waste potato, and produce high concentration of butanol (20.41–23.74 g/l) and hydrogen (3.20–4.38 l/l).
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Enzymes, B.: Global Biofuel Enzymes Industry. https://prnewswire.com/news-releases/global-biofuel-enzymes-industry-300671711 (2018). Accessed 20 May 2019
Novozymes: Liquozyme®: Fewer chemicals. More yield. https://novozymes.com/en/advance-your-business/bioenergy/Liquozyme (2019). Accessed 10 Aug 2019
Enzymes, N.: Biofuel and bioenergy, thermostable alpha amylase. https://enzyme-india.com/amylase-enzymes-biofuel (2015). Accessed 20 May 2019
Virunanon, C., Ouephanit, C., Burapatana, V., Chulalaksananukul, W.: Cassava pulp enzymatic hydrolysis process as a preliminary step in bio-alcohols production from waste starchy resources. J. Clean. Prod. 39, 273–279 (2013)
Li, H.G., Ma, X.X., Zhang, Q.H., Luo, W., Wu, Y.Q., Li, X.H.: Enhanced butanol production by solvent tolerance Clostridium acetobutylicum SE25 from cassava flour in a fibrous bed bioreactor. Bioresour. Technol. 221, 412–418 (2016)
Li, X., Li, Z., Zheng, J., Shi, Z., Li, L.: Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824 using cassava as substrate. Bioresour. Technol. 125, 43–51 (2012)
Sharma, A., Satyanarayana, T.: Microbial acid-stable α-amylases: characteristics, genetic engineering and applications. Process Biochem. 48(2), 201–211 (2013)
Van der Maarel, M.J.E.C., van der Veen, B., Uitdehaag, J.C.M., Leemhuis, H., Dijkhuizen, L.: Properties and applications of starch-converting enzymes of the α-amylase family. J. Biotechnol. 94(2), 137–155 (2002)
Liu, Y.H., Lu, F.P., Li, Y., Yin, X.B., Wang, Y., Gao, C.: Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600. Appl. Microbiol. Biotechnol. 78(1), 85–94 (2008)
Arikan, B.: Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15. Bioresour. Technol. 99(8), 3071–3076 (2008)
Asoodeh, A., Chamani, J., Lagzian, M.: A novel thermostable, acidophilic α-amylase from a new thermophilic “Bacillus sp. Ferdowsicous” isolated from Ferdows hot mineral spring in Iran: purification and biochemical characterization. Int. J. Biol. Macromol. 46(3), 289–297 (2010)
Miller, G.L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31(3), 426–428 (1959)
Rajagopalan, G., He, J., Yang, K.L.: Production, purification, and characterization of α-amylase from solventogenic Clostridium sp. BOH3. Bioenergy Res. 7(1), 132–141 (2014)
Rajagopalan, G., He, J., Yang, K.L.: A highly efficient NADH-dependent butanol dehydrogenase from high-butanol-producing Clostridium sp. BOH3. Bioenergy Res. 6(1), 240–251 (2013)
Paquet, V., Croux, C., Goma, G., Soucaille, P.: Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 57(1), 212–218 (1991)
Abernathy, D.G., Spedding, G., Starcher, B.: Analysis of protein and total usable nitrogen in beer and wine using a microwell ninhydrin assay. J. Inst. Brew. 115(2), 122–127 (2009)
Shirazi, O.U., Khattak, M.M.A.K., Shukri, N.A.: Quantitative analysis of minerals in the selected formulations of spices and herbs using ICP-MS. Prog. Nutr. 18, 161–165 (2016)
Rajagopalan, G., He, J., Yang, K.L.: Direct fermentation of xylan by Clostridium strain BOH3 for the production of butanol and hydrogen using optimized culture medium. Bioresour. Technol. 154, 38–43 (2014)
Burton, K.: A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62(2), 315–323 (1956)
Rajagopalan, G., Krishnan, C.: Optimization of agro-residual medium for α-amylase production from a hyper-producing Bacillus subtilis KCC103 in submerged fermentation. J. Chem. Technol. Biotechnol. 84(4), 618–625 (2009)
Tiwari, S., Shukla, N., Mishra, P., Gaur, R.: Enhanced production and characterization of a solvent stable amylase from solvent tolerant Bacillus tequilensis RG-01: thermostable and surfactant resistant. Sci. World J. 2014, 972763 (2014)
Burhan, A., Nisa, U., Gökhan, C., Ömer, C., Ashabil, A., Osman, G.: Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6. Process Biochem. 38(10), 1397–1403 (2003)
Abdel-Fattah, Y., Soliman, N., El-Toukhy, N., El-Gendi, H., Ahmed, R.: Production, purification, and characterization of thermostable α-amylase produced by Bacillus licheniformis isolate AI20. J. Chem. 2013, 11 (2012)
Asoodeh, A., Alemi, A., Heydari, A., Akbari, J.: Purification and biochemical characterization of an acidophilic amylase from a newly isolated Bacillus sp. DR90. Extremophiles 17, 339–348 (2013)
Asoodeh, A., Emtenani, S., Emtenani, S., Jalal, R., Housaindokht, M.R.: Molecular cloning and biochemical characterization of a thermoacidophilic, organic-solvent tolerant α-amylase from a Bacillus strain in Escherichia coli. J. Mol. Catal. B 99, 114–120 (2014)
Bernhardsdotter, E., Garriott, O., Pusey, M.: Enzymic properties of an alkaline chelator-resistant α-amylase from an alkaliphilic Bacillus sp. isolate L1711. Process Biochem. 40, 2401–2408 (2005)
Hagihara, H., Igarashi, K., Hayashi, Y., Endo, K., Ikawa-Kitayama, K., Ozaki, K., Kawai, S., Ito, S.: Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38. Appl. Environ. Microbiol. 67(4), 1744–1750 (2001)
Kalpana, B.J., Pandian, S.K.: Halotolerant, acid-alkali stable, chelator resistant and raw starch digesting alpha-amylase from a marine bacterium Bacillus subtilis S8–18. J. Basic Microbiol. 54(8), 802–811 (2014)
Mamo, G., Gessesse, A.: Purification and characterization of two raw-starch-digesting thermostable α-amylases from a thermophilic Bacillus. Enzyme Microb. Technol. 25(3), 433–438 (1999)
Sajedi, R.H., Naderi-Manesh, H., Khajeh, K., Ahmadvand, R., Ranjbar, B., Asoodeh, A., Moradian, F.: A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104. Enzyme Microb. Technol. 36(5), 666–671 (2005)
Nagarajan, D.R., Rajagopalan, G., Krishnan, C.: Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103. Appl. Microbiol. Biotechnol. 73(3), 591–597 (2006)
Emtenani, S., Asoodeh, A., Emtenani, S.: Gene cloning and characterization of a thermostable organic-tolerant α-amylase from Bacillus subtilis DR8806. Int. J. Biol. Macromol. 72, 290–298 (2015)
Dheeran, P., Kumar, S., Jaiswal, Y.K., Adhikari, D.K.: Characterization of hyperthermostable alpha-amylase from Geobacillus sp. IIPTN. Appl. Microbiol. Biotechnol. 86(6), 1857–1866 (2010)
Liu, B., Wang, Y., Zhang, X.: Characterization of a recombinant maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616. Enzyme Microb. Technol. 39(4), 805–810 (2006)
Mitchell, W.J.: Carbohydrate uptake and utilization by Clostridium beijerinckii NCIMB 8052. Anaerobe 2(6), 379–384 (1996)
Mitchell, W.J.: The phosphotransferase system in solventogenic Clostridia. J. Mol. Microbiol. Biotechnol. 25(2–3), 129–142 (2015)
Moon, H.G., Jang, Y.S., Cho, C., Lee, J., Binkley, R., Lee, S.Y.: One hundred years of clostridial butanol fermentation. FEMS Microbiol. Lett. 363(3), 1–15 (2016)
METACYC: A member of the BioCyc database collection. https://metacyc.org (2020). Accessed 26 Feb 2020
FAOSTAT: Food and Agriculture Organization of the United Nations https://fao.org (2019). Accessed 10 Aug 2019.
Potatopro: Food Innovation online crop https://potatopro.com (2019). Accessed 5 Mar 2019
Nimcevic, D., Schuster, M., Gapes, J.R.: Solvent production by Clostridium beijerinckii NRRL B592 growing on different potato media. Appl. Microbiol. Biotechnol. 50(4), 426–428 (1998)
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This work was supported by the Science and Engineering Research Board, Department of Science and Technology, Government of India (ECR/2016/000722); and Faculty Startup Grant (FSG/2016/108) from SAU, New Delhi, India.
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Mahato, R.K., Fatema, I.T. & Rajagopalan, G. Thermostable, Solvent, Surfactant, Reducing Agent and Chelator Resistant α-Amylase from Bacillus Strain IBT108: A Suitable Candidate Enables One-Step Fermentation of Waste Potato for High Butanol and Hydrogen Production. Waste Biomass Valor 12, 223–238 (2021). https://doi.org/10.1007/s12649-020-01017-1
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DOI: https://doi.org/10.1007/s12649-020-01017-1