title: Cloning and extracellular expression of a raw starch digesting α-amylase (Blamy-I) and its application in bioethanol production from a non-conventional source of starch.
abstract: The aim of this study was to clone and efficiently express a raw starch-digesting α-amylase enzyme in the culture media and also to investigate the potential application of this recombinant enzyme in the digestion of non-conventional raw starch for bioethanol production. A raw starch digesting α-amylase gene isolated from Bacillus licheniformis strain AS08E was cloned and extracellularly expressed in E. coli cells using the native signal peptide. The mature recombinant α-amylase (Blamy-I) consisting of 483 amino acid residues was found to be homogenous with a mass of 55.3 kDa (by SDS-PAGE analysis) and a predicted pI of 6.05. Structural and functional analysis of Blamy-I revealed the presence of an extra Ca(2+) -binding region between the A and C domains responsible for higher thermostability of this enzyme. The statistical optimization of E. coli culture conditions resulted in an approximately eightfold increase in extracellular expression of Blamy-I as compared to its production under non-optimized conditions. Blamy-I demonstrated optimum enzyme activity at 80 °C and pH 10.0, and efficiently hydrolyzed raw starch isolated from a non-conventional, underutilized jack fruit seeds. Further utilization of this starch for bioethanol production using Blamy-I and Saccharomyces cerevisiae also proved to be highly promising.
authors.affiliation: Department of Molecular Biology and Biotechnology, Microbial Biotechnology and Protein Research Laboratory, School of Science, Tezpur University, Tezpur 784028, Assam, India.
authors.forename: Jetendra K
authors.initials: JK
authors.lastname: Roy
authors.affiliation: Department of Molecular Biology and Biotechnology, Microbial Biotechnology and Protein Research Laboratory, School of Science, Tezpur University, Tezpur 784028, Assam, India.
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Manhar
authors.affiliation: Department of Molecular Biology and Biotechnology, Microbial Biotechnology and Protein Research Laboratory, School of Science, Tezpur University, Tezpur 784028, Assam, India.
authors.forename: Dhrubajyoti
authors.initials: D
authors.lastname: Nath
authors.affiliation: Department of Molecular Biology and Biotechnology, Microbial Biotechnology and Protein Research Laboratory, School of Science, Tezpur University, Tezpur 784028, Assam, India.
authors.forename: Manabendra
authors.initials: M
authors.lastname: Mandal
authors.affiliation: Department of Molecular Biology and Biotechnology, Microbial Biotechnology and Protein Research Laboratory, School of Science, Tezpur University, Tezpur 784028, Assam, India.|School of Biological Sciences, University of Northern Colorado, Greeley, CO, USA.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Mukherjee
mesh_terms: D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli; D000431:Ethanol / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013213:Starch / Q000378:metabolism*; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics
chemical_list: D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D000431:Ethanol; D013213:Starch; D000516:alpha-Amylases; C501919:alpha-amylase, Bacillus licheniformis
keywords: Bacillus licheniformis; Bioethanol; Jackfruit seed starch; Raw starch digesting α-amylase; Recombinant DNA