ABSTRACT
Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.
Subject(s)
Bacillus/enzymology , Protein Structure, Secondary , beta-Glucosidase/chemistry , beta-Glucosidase/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Enzyme Stability , Escherichia coli , Hot Temperature , Hydroxylamine , Hydroxylamines , Kinetics , Molecular Sequence Data , Mutagenesis , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Point Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrophotometry, Ultraviolet , ThermodynamicsABSTRACT
The enzyme encoded by the bgIA gene of Bacillus polymyxa, a type I beta-glucosidase belonging to family I of glycosyl hydrolases, has been purified to homogeneity from an Escherichia coli culture which overexpressed the gene, and crystallized. The crystals, which diffract to 3.0 A resolution, belong to the orthorhombic space group C222(1). The cell dimensions are a = 155.4 A, b = 209.4 A, c = 209.7 A.
Subject(s)
Bacillus/enzymology , beta-Glucosidase/chemistry , Bacillus/genetics , Chromatography, Gel , Chromatography, Ion Exchange , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , beta-Glucosidase/genetics , beta-Glucosidase/isolation & purificationABSTRACT
By applying different classical and fast protein liquid chromatographic techniques, three xylanases (beta-1,4-d-xylan xylanhydrolase) were purified to homogeneity from the extracellular enzymatic complex of Bacillus polymyxa. The three enzymes (X(34)C, X(34)E, and X(22)) were small proteins of 34, 34, and 22 kDa and basic pIs 9.3, >9.3, and 9.0, respectively. X(34)C and X(34)E are closely related and seem to be isoforms of the same enzyme. However, they differ in some characteristics. The three enzymes had different pH and temperature optima. One of them, X(34)E, showed a high thermal stability. The V(max) values determined for X(34)C, X(34)E, and X(22) enzymes on oat spelts xylan were 14.9, 85.5, and 64.0 U mg, respectively, and 16.1, 62.0, and 150.6 U mg on birchwood xylan. When oat spelts xylan was the substrate used, K(m) values of 3.4, 2.4, and 1.9 mg ml were obtained for X(34)C, X(34)E, and X(22) enzymes, respectively, and 0.65, 6.3, and 0.32 mg ml were the respective K(m) values determined with birchwood xylan as the substrate. The enzymes were nondebranching endo-beta-xylanases. Xylose was one of the products of xylan hydrolysis by xylanases X(34)C and X(34)E, but this monosaccharide was not released by X(22) enzyme. However, neither of the enzymes was able to degrade xylobiose.
