Alkaliphilic Nocardiopsis sp. strain F96 produced three beta-1,3-glucanase isozymes of different molecular masses (BglF1, BglF2 and BglF3). The N-terminal amino acid sequences of BglFs indicated that these isozymes were the products of a single gene. The beta-1,3-glucanase gene (bglF) was cloned from the chromosomal DNA of strain F96. The bglF gene encoded a polypeptide of 270 amino acids including a signal sequence. The deduced amino acid sequence of mature BglF exhibited the highest homology to those of glycoside hydrolase (GH) family 16 beta-1,3-glucanases, suggesting that the enzyme belonged to the GH family 16. The mature region of bglF gene was functionally expressed in Escherichia coli. The optimum pH and temperature of purified recombinant BglF were pH 9.0 and 70 degrees C, respectively. This enzyme efficiently hydrolyzed insoluble beta-1,3-glucans and showed the highest activity toward a beta-1,3-1,4-glucan rather than beta-1,3-glucans. These results suggested that BglF would be a novel beta-1,3-glucanse. Mutational analysis revealed that Glu123 and Glu128 should be the catalytic residues of BglF.
Endo-1,3--glucanase, an enzyme that hydrolyzes the 1,3--glycosyl linkages of -glucan, belongs to the family 16 glycosyl hydrolases, which are widely distributed among bacteria, fungi and higher plants. Crystals of a family 16 endo-1,3--glucanase from the alkaliphilic Nocardiopsis sp. strain F96 were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2 1 , with unit-cell parameters a = 34.59, b = 71.84, c = 39.67 Å , = 90.21 , and contained one molecule per asymmetric unit. The Matthews coefficient (V M ) and solvent content were 1.8 Å 3 Da À1 and 31.8%, respectively. Diffraction data were collected to a resolution of 1.3 Å and gave a data set with an overall R merge of 6.4% and a completeness of 99.3%.
beta-1,3-Glucanase F (BglF) from alkaliphilic Nocardiopsis sp. F96 is a single domain enzyme composed of only a catalytic domain. Chimeric BglFs with some carbohydrate-binding domains were constructed and characterized. By connecting the C-terminal additional domain of beta-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain of chitinase J from alkaliphilic Bacillus sp. J813, binding ability and hydrolyzing activity toward insoluble beta-1,3-glucans were both improved.
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