title: Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.
abstract: A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, China.|Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, China.|Research Center of Food Biotechnology of Xiamen City, Xiamen, China.
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
authors.forename: Wenguang
authors.initials: W
authors.lastname: Zheng
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, China.|Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, China.|Research Center of Food Biotechnology of Xiamen City, Xiamen, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Ni
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, China.
authors.forename: Han
authors.initials: H
authors.lastname: Liu
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, China.|Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, China.|Research Center of Food Biotechnology of Xiamen City, Xiamen, China.
authors.forename: Anfeng
authors.initials: A
authors.lastname: Xiao
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, China.|Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, China.|Research Center of Food Biotechnology of Xiamen City, Xiamen, China.
authors.forename: Huinong
authors.initials: H
authors.lastname: Cai
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002087:Butyrates / Q000378:metabolism; D002681:China; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D045482:Hot Springs / Q000382:microbiology; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008994:Monoacylglycerol Lipases / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D002087:Butyrates; D011994:Recombinant Proteins; C033592:4-nitrophenyl butyrate; D004950:Esterases; D008994:Monoacylglycerol Lipases
keywords: Biochemical characterization; Cloning; Thermostable esterase