title: Cloning, heterologous expression, and enzymatic characterization of a novel glucoamylase GlucaM from Corallococcus sp. strain EGB.
abstract: The gene encoding a novel glucoamylase (GlucaM) from the Corallococcus sp. strain EGB was cloned and heterologous expressed in Escherichia coli BL21(DE3), and the enzymatic characterization of recombinant GlucaM (rGlucaM) was determined in the study. The glucaM had an open reading frame of 1938 bp encoding GlucaM of 645 amino acids with no signal peptide. GlucaM belongs to glycosyl hydrolase family 15 and shares the highest identity 96% with the GH15 glucoamylase of Corallococcus coralloides DSM 2259. The rGlucaM with His-tag was purified by the Ni2+-NTA resin, with a specific activity from 3.4 U/mg up to 180 U/mg, and the molecular weight of rGlucaM was approximately 73 kDa on SDS-PAGE. The Km and Vmax of rGlucaM for soluble starch were 1.2 mg/mL and 46 U/mg, respectively. rGlucaM was optimally active at pH 7.0 and 50 °C and had highly tolerance to high concentrations of salts, detergents, and various organic solvents. rGlucaM hydrolyzed soluble starch to glucose, and hydrolytic activities were also detected with amylopectin, amylase, glycogen, starch (potato), α-cyclodextrin, starch (corn and potato). The analysis of hydrolysis products shown that rGlucaM with α-(1-4),(1-6)-D-glucan glucohydrolase toward substrates. These characteristics indicated that the GlucaM was a new member of glucoamylase family and a potential candidate for industrial application.
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Zhoukun
authors.initials: Z
authors.lastname: Li
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Ji
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Dong
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Ye
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Jiale
authors.initials: J
authors.lastname: Wu
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China; College of Bioscience and Bioengineering, Jiangxi Agriculture University, 330045 Nanchang, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Qiongzhen
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Fu
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Shuhuan
authors.initials: S
authors.lastname: Li
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China. Electronic address: czl@njau.edu.cn.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006868:Hydrolysis; D009231:Myxococcales / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013213:Starch / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase
keywords: Cloning; Corallococcus; Glucoamylase; Glucosidic bands