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Crystallization and preliminary X-ray study of alkaline beta-mannanase from the alkaliphilic Bacillus sp. N16-5.

ABSTRACT: The catalytic domain of an alkaline beta-mannanase from the alkaliphilic Bacillus sp. N16-5 has been expressed and purified. The recombinant enzyme was crystallized using the hanging-drop vapour-diffusion method at 298 K. X-ray diffraction data were collected to 1.6 A resolution. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.03, b = 63.31, c = 83.34 A. Initial phasing was carried out by molecular replacement using the three-dimensional structure of a mannanase from the alkaliphilic Bacillus sp. JAMB602 as a search model.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC2564887 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray study of alkaline beta-mannanase from the alkaliphilic Bacillus sp. N16-5.

Zhao Yueju Y Zhang Yunhua Y Gao Feng F Xue Yanfen Y Zeng Yan Y Ma Yanhe Y

Acta crystallographica. Section F, Structural biology and crystallization communications 20080930 Pt 10

The catalytic domain of an alkaline beta-mannanase from the alkaliphilic Bacillus sp. N16-5 has been expressed and purified. The recombinant enzyme was crystallized using the hanging-drop vapour-diffusion method at 298 K. X-ray diffraction data were collected to 1.6 A resolution. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.03, b = 63.31, c = 83.34 A. Initial phasing was carried out by molecular replacement using the three-dimensional struc ...[more]

PMID: 18931445

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Project description:Significant progress has been made in isolating novel alkaline ?-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these ?-mannanases. We report the catalytic domain structure of an industrially important ?-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (?/?)(8)-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 ?-mannanases. It was revealed that BSP165 MAN and other subfamily 8 ?-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to ?-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline ?-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (?/?)(8)-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK(a) calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 ?-mannanases and will help to further understanding of alkaline adaptation mechanism.

Project description:BACKGROUND:?-Mannanase catalyzes the cleavage of ?-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable ?-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline ?-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high ?-mannanase activity at alkaline condition. In this study, this ?-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied. RESULTS:A thermo-alkaline ?-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0-11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing ?-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg-1 was observed on konjac glucomannan with the Km and kcat value of 0.62 g l-1 and 1238.9 s-1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml-1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2?×?SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml-1 after 72 h cultivation with 6% inoculum size by shake flask fermentation. CONCLUSIONS:The thermo-alkaline ?-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported ?-mannanases and was a promising thermo-alkaline ?-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml-1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline ?-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other ?-mannanases in B. subtilis.

Dataset Information

Crystallization and preliminary X-ray study of alkaline beta-mannanase from the alkaliphilic Bacillus sp. N16-5.

Publications

Crystallization and preliminary X-ray study of alkaline beta-mannanase from the alkaliphilic Bacillus sp. N16-5.

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