title: AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. strain EGB.
abstract: A novel α-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The Km and Vmax of recombinant AmyM for soluble starch were 6.61 mg ml(-1) and 44,301.5 μmol min(-1) mg(-1), respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Zhoukun
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Jiale
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Biying
authors.initials: B
authors.lastname: Zhang
authors.affiliation: College of Bioscience and Bioengineering, Jiangxi Agriculture University, Nanchang, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Ye
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: College of Food Science, South China University of Technology, Guangzhou, People's Republic of China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China czl@njau.edu.cn.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
mesh_terms: D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009231:Myxococcales / Q000201:enzymology* / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D009844:Oligosaccharides; D011994:Recombinant Proteins; C016549:maltohexaose; D000516:alpha-Amylases