Dataset Information

Cloning, secretory expression and characterization of recombinant ?-mannanase from Bacillus circulans NT 6.7.

ABSTRACT: The mannanase gene of B. circulans NT 6.7 was cloned and expressed in an Escherichia coli expression system. The B. circulans NT 6.7 mannanase gene consists of 1,083 nucleotides encoding a 360-amino acid residue long polypeptide, belonging to glycoside hydrolase family 26. The full-length mannanase gene including its native signal sequence was cloned into the vector pET21d and expressed in E. coli BL21 (DE3). ?-Mannanase activities in the culture supernatant and crude cell extract were 37.10 and 515 U per ml, respectively, with most of the activity in the cell extract attributed to the periplasmic fraction. In contrast, expression of mannanase was much lower when using the B. circulans NT 6.7 mannanase gene without its signal sequence. The optimum temperature of recombinant ?-mannanase activity was 50°C and the optimum pH was 6.0. The enzyme was very specific for ?-mannan substrates with a preference for galactomannan. Hydrolysis products of locust bean gum were various mannooligosaccharides including mannohexaose, mannopentaose, mannotetraose, mannotriose and mannobiose, while mannose could not be detected. In conclusion, this expression system is efficient for the secretory production of recombinant ?-mannanase from B. circulans NT 6.7, which shows good characteristics for various applications.

SUBMITTER: Piwpankaew Y

PROVIDER: S-EPMC4141934 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

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

Cloning, secretory expression and characterization of recombinant ?-mannanase from Bacillus circulans NT 6.7.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets