Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Abstract

Backgroundβ-Mannanase catalyzes the cleavage of β-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable β-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 β-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high β-mannanase activity at alkaline condition. In this study, this β-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied.ResultsA thermo-alkaline β-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 β-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.ConclusionsThe thermo-alkaline β-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported β-mannanases and was a promising thermo-alkaline β-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 β-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 β-mannanases in B. subtilis.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0973-0) contains supplementary material, which is available to authorized users.

Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization

“…Kinetic analysis showed that when using locust bean gum as a substrate, the V max and K m of 30a/BSM were 1238 ± 55.54 μmol/min/mg and 3.74 ± 0.40 mg/mL (Table ), respectively, indicating that BSM was a typical endomannanase with moderate catalytic ability . Though thermostability was impressively improved, the K m was not altered by molecular cyclization .…”

“…To assess the thermostability, all linear, cyclized, and relinearized enzymes were preincubated in citrate/phosphate buffer (pH 6.0) at 50−70 °C for 1 h. Aliquots were taken at different time intervals (2,5,10,20,30, and 60 min) and subjected to activity assays under the optimal conditions (pH 6.0, 50 °C). The initial activities before preincubation were taken as 100%.…”

“…Complete deconstruction of mannan requires the activities of a number of enzymes, such as endo -β-1,4-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), α- l -arabinosidase (EC 3.2.1.55), α-galactosidase (EC 3.2.1.22), and acetylmannan esterase (EC 3.1.1.6) . Among them, endo -β-1,4-mannanase is the key enzyme that randomly catalyzes cleavage of β-1,4-glycosidic bonds of the mannan backbone to release mannooligosaccharides and facilitate initial degradation . Mannanases are classified into the glycoside hydrolase (GH) families 5, 26, and 113 in the CAZy database () and mainly follow a preserving catalytic mechanism …”

“…1 Among them, endoβ-1,4-mannanase is the key enzyme that randomly catalyzes cleavage of β-1,4-glycosidic bonds of the mannan backbone to release mannooligosaccharides and facilitate initial degradation. 2 Mannanases are classified into the glycoside hydrolase (GH) families 5, 26, and 113 in the CAZy database (http:// www.cazy.org/) and mainly follow a preserving catalytic mechanism. 3 Feed enzymes, including mannanase and xylanase, are known to degrade nonstarch polysaccharides (NSPs) in plant-derived feed diets.…”

Selection ofBacillus subtilisUS191 as a mannanase‐producing probiotic candidate

“… and Zhou et al. have characterized Bacillus‐ derived β‐mannanases with optimal activity at pH 6 and 9.5, respectively.…”