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Optimization and purification of mannanase produced by an alkaliphilic-thermotolerant Bacillus cereus N1 isolated from Bani Salama Lake in Wadi El-Natron.

ABSTRACT: An alkaliphilic-thermotolerant Bacillus cereus N1 isolated from Bani Salama Lake, Wadi El-Natron, Egypt, was proved to produce mannanase enzyme. Optimization of the fermentation medium components using Plackett-Burman design was applied. Glucose and inoculum size were found to be the most significant factors enhancing the production of the enzyme. On applying optimized medium in the fermentation process, an enzyme productivity of 42.2 UmL-1 was achieved with 6.4 fold increase compared to the basal one. Mannanase was also extracted and purified using chromatography such as ion-exchange chromatographic and gel filtration methods. It was indicated that, the mannanase activity extracted and purified from the isolate B. cereus N1 was reduced to 321.6 U (about 36% of the whole mannanase in the culture filtrate) in comparison with the initial mannanase activity (900 U) and the total protein content reduced to 52 mg (the initial total protein content was 220 mg). However, the specific activity for the mannanase from B. cereus N1 at the end of the purification steps was found to be about 628 Umg-1 compared to 4.2 Umg-1 at the initial culture filtrate. It was also indicated that the mannanase enzyme was purified almost 149-fold.

SUBMITTER: El-Sharouny EE

PROVIDER: S-EPMC4433781 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Optimization and purification of mannanase produced by an alkaliphilic-thermotolerant Bacillus cereus N1 isolated from Bani Salama Lake in Wadi El-Natron.

El-Sharouny Ebaa Ebrahim EE El-Toukhy Nabil M K NM El-Sersy Nermeen Ahmed NA El-Gayar Abeer Abd El-Aal AA

Biotechnology, biotechnological equipment 20150113 2

An alkaliphilic-thermotolerant Bacillus cereus N1 isolated from Bani Salama Lake, Wadi El-Natron, Egypt, was proved to produce mannanase enzyme. Optimization of the fermentation medium components using Plackett-Burman design was applied. Glucose and inoculum size were found to be the most significant factors enhancing the production of the enzyme. On applying optimized medium in the fermentation process, an enzyme productivity of 42.2 UmL-1 was achieved with 6.4 fold increase c ...[more]

PMID: 26019646

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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:Wadi El-Hitan, the UNESCO World Heritage Site, of the Fayum Depression in the northeast part of the Western Desert of Egypt, has produced a remarkable collection of Eocene vertebrates, in particular the fossil whales from which it derives its name. Here we describe a new genus and species of marine catfishes (Siluriformes; Ariidae), Qarmoutus hitanensis, from the base of the upper Eocene Birket Qarun Formation, based on a partial neurocranium including the complete left side, partial right dentary, left suspensorium, two opercles, left pectoral girdle and spine, nuchal plates, first and second dorsal spines, Weberian apparatus and a disassociated series of abdominal vertebrae. All of the elements belong to the same individual and some of them were found articulated. Qarmoutus gen. nov. is the oldest and the most complete of the Paleogene marine catfishes unearthed from the Birket Qarun Formation. The new genus exhibits distinctive features not seen in other African Paleogene taxa, such as different sculpturing on the opercle and pectoral girdle with respect to that on the neurocranium and nuchal plates, denticulate ornamentation on the skull bones arranged in longitudinal rows and forming a radiating pattern on the sphenotic, pterotic, extrascapular and the parieto-supraoccipital, indentations or pitted ornamentation on the nuchal plates as well as the parieto-supraoccipital process, strut-like radiating pattern of ornamentation on the opercle from the proximal articulation to margins, longitudinal, curved, reticulate ridges and tubercular ornamentations on the cleithrum, sinuous articulation between the parieto-supraoccipital process and the anterior nuchal plate, long, narrow, and arrowhead shaped nuchal shield, very small otic capsules restricted to the prootic. Multiple parsimony and Bayesian morphological phylogenetic analyses of Ariidae, run with and without "molecular scaffolds", yield contradictory results for the placement of Qarmoutus; the genus is either a phylogenetically basal ariid, or it is deeply nested within the ariid clade containing New World species of Sciades.

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

Optimization and purification of mannanase produced by an alkaliphilic-thermotolerant Bacillus cereus N1 isolated from Bani Salama Lake in Wadi El-Natron.

Publications

Optimization and purification of mannanase produced by an alkaliphilic-thermotolerant <i>Bacillus cereus</i> N1 isolated from Bani Salama Lake in Wadi El-Natron.

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