Enhanced production and characterization of a beta-glucosidase from Bacillus halodurans expressed in Escherichia coli.
A putative beta-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-beta-D-1-thiogalactopyranoside, the enzyme expressed at approximately 40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A(600) 12 in auto-inducing M9NG medium, beta-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the beta-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45 degrees C and 8.0, respectively, under the assay conditions. K(m) and k(cat) against p-nitrophenyl-beta-D-glucopyranoside were 4 mM and 0.75 sec(-1), respectively. To our knowledge, this is the first report of high-level expression and characterization of a beta-glucosidase from B. halodurans.
Naz
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School of Biological Sciences, University of the Punjab, Lahore, 54590, Pakistan.
Ikram
N
N
Rajoka
M I
MI
Sadaf
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S
Akhtar
M W
MW
D001407:Bacillus / Q000145:classification / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase