A thermoalkalophilic new species of Bacillus, similar to Bacillus arseniciselenatis DSM , produced extracellular xylanase under solid. Xylanase is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, one of. Partial characterization of an extracellular xylanase isolated by chromatography from Bacillus subtilis gave a molecular weight of 32, and optimum pH and.
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To receive news and publication updates for International Journal of Microbiology, enter your email address in the box below. Kamble and Anandrao R. This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A thermoalkalophilic new species of Bacillussimilar to Bacillus arseniciselenatis DSMproduced extracellular xylanase under solid state fermentation when wheat bran is used as carbon source.
The enzyme was active on birchwood xylan and little active on p -nitrophenyl xylopyranoside but not on Avicel, CMC, cellobiose, and starch, showing its absolute substrate specificity.
For birchwood xylan, the enzyme gave a Km 5. In addition, the xylanase was also capable of producing high-quality xylo-oligosaccharides, which indicated its application potential not only in pulp biobleaching processes but also in the nutraceutical industry. Xylan is found mainly in the secondary cell wall and is considered to be forming an interphase between lignin and other polysaccharides. It is likely that xylan molecules covalently link with lignin phenolic residues and also interact with polysaccharides, such as pectin and glucan.
Xylanases are of industrial importance, which can be used in paper manufacturing to bleach paper pulp, increasing the brightness of pulp and improving the digestibility of animal feed and for clarification of fruit juices.
Applications of xylanase avoid the use of chemicals that are expensive and cause pollution [ 9 ]. Microorganisms are the rich sources of xylanases, produced by diverse genera and species of bacteria, actinomycetes, and fungi.
Several species of Bacillus and filamentous fungi secrete high amounts of extracellular xylanases [ 10 ]. Xylanase secretion often associates with low or high amount of cellulases. To use xylanase for pulp treatment, it is preferable to use cellulose-free xylanases, since the cellulase may adversely affect the quality of the paper pulp [ 11 — 15 ].
The most practical approach is the screening for naturally occurring microbial strains capable of secreting cellulose-free xylanases under optimized fermentation conditions. To use xylanase prominently in bleaching process it should be stable at high temperature and alkaline pH [ 1617 ].
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Industrial production of enzymes on large scale is associated mainly with substrate. The use of agriculture residues as low-cost substrates for the production of industrial enzymes is a significant way to reduce production cost.
The technique of fermentation using solid state substrate has the great advantage over submerged fermentation due to absence or near absence of aqueous phase that provides natural habitat for growth of microorganisms, economy of the space, simplicity of the media, no complex machinery, equipments and control systems, greater compactness of the fermentation vessel owing to a lower water volume, greater product yields, reduced energy demand, lower capital and recurring expenditures in industry, easier scale-up of processes, lesser volume of solvent needed for product recovery, superior yields, absence of foam build-up, and easier control of contamination due to the low moisture level in the system [ 1018 ].
In consideration with these facts the present study aims to characterize extracellular alkalothermophilic xylanase produced by Bacillus arseniciselenatis DSM when grown in solid state fermentation.
To our knowledge, this is the first report describing the production of thermoalkalophilic cellulase-free xylanase by Bacillus arseniciselenatis DSM In addition, this xylanase was found to be able to degrade xylan into xylo-oligosaccharides.
Soil samples were collected from coastal areas of Mandovi, Goa, India. Enrichment was done using birchwood xylan Sigma Chemicals, Germany as a sole source of carbon. Twenty five bacterial cultures were screened for xylanolytic ability by adding dye-labelled substrate, for example, xylan-brilliant red 3BA in xylan agar medium [ 19 ]. Prominent selected isolate was identified on the basis of morphological, cultural, biochemical properties [ 20 ] and 16S rRNA sequencing.
Multiple alignment of sequences and calculations of levels of sequence similarity were performed by using ClustalW2 program. A phylogenetic tree obtained was analyzed for closely related organism.
The evolutionary history was inferred using the neighbor-joining method [ 14 ]. The bacterial isolate was maintained in liquid medium as well as solid medium in basal salt solution BSS containing 0. The selected strain was further tested for their abilities to produce extracellular xylanase under solid state fermentation.
Wheat bran was used as the substrate. Centrifugation will remove xylanase from substrate.
Isolation and Characterization of a Xylanase from Bacillus subtilis
Supernatant was filtered through Whatman No. Prior to centrifugation, the samples were withdrawn for determining viable number of cells by standard viable plate count technique. Xylanase activity was measured according to Bailey et al. The reaction was terminated at zero time in the control tubes. Cellulase activity was measured according to Ghose with necessary modifications [ 23 ].
International Journal of Microbiology
The reaction was terminated at zero time in control tubes. An autozero was set in spectrophotometer using buffer solution. Total soluble protein was measured according to Lowry et al.
Protein concentration was determined using bovine serum albumin BSA as a standard. Protein precipitation by salting out technique using ammonium sulphate NH 4 SO 4 2 was carried out with constant gentle stirring [ 26 ]. The protein was eluted with the 0. Coomassie brilliant blue R was used to stain the gel. The protein molecular weight markers used were of medium range containing Initial reaction rates using birchwood and oat spelt xylan as substrate were determined at substrate concentrations of 0.
The kinetic constants, Km and Vmax, were estimated using the linear regression method of Lineweaver and Burk [ 28 ]. Hydrolysis products were detected by thin layer chromatography TLC [ 29 ]. D-xylose X 1xylobiose X 2xylotriose X 3and xylotetraose X 4 were applied as standard.
To evaluate thermal stability, 0. The effect of pH on enzyme activity was determined by incubating xylanase at various pH ranging from 6. To evaluate the stability of the enzyme at each pH, the purified enzyme was incubated into the respective buffer over a pH range of 6. About 25 bacterial strains, which formed clear halos around their colonies on xylan agar plates, were picked up for further studies, isolated from soil collected at selected study site.
It was identified on the basis of various morphological and biochemical characteristics as shown in Table 1. The sequence was deposited in Gene Bank Accession No. The phylogenetic relation of this isolate is as shown in Figure 2. It is closely associated with Bacillus sp. It was also isolated from soil sample collected at coastal areas of Mandovi, Goa. When the strain was grown on wheat bran for 3 days of incubation at pH 8. Several workers reported the suitability of wheat bran for xylanase production in SSF [ 3132 ].
Hence there was increase in possibility of cellulase contamination when grown on wheat bran. From twenty selected strains, five were able to produce cellulase along with xylanase in SSF. This was due to the presence of cellulose in substrate wheat bran used in SSF. Proteins precipitated within this range had maximum xylanase activity and was used for purification.
Xylanase was further purified enaim DEAE cellulose ion exchange column. Xylanase was purified 3. The present results were supported by previous work. Xylanase produced by Bacillus sp. The action of the purified xylanase towards various substrates was studied. The enzyme was active on birchwood xylan, little active on p -nitrophenyl xylopyranoside but not on Avicel, CMC, snzim, and starch Table 3.
Similarly, xylanase with absolute substrate specificity was purified from Trichoderma viride by Ujiie et al. The Km and values of xylanase were 5. Xylanases isolated from Aeromonas cavie ME-1 and Bacillus sp. The present results indicated that xylanase cleaved the substrate to liberate mainly xylooligosaccharides, but not able to act on resulting oligosaccharides to xylanaee xylose, suggesting that it is a endoxylanase.
Analysis of hydrolytic products of xylan by the xylanase of Thermoascus aurantiacus showed fnzim xylan was degraded to various xylo-oligosaccharides without a significant accumulation of xylose [ 45 ].
The end products were xylobiose, xylotriose, xylotetraose, and higher oligosaccharides when xylan was hydrolyzed with endoxylanase of alkalophilic Bacillus sp. No xylose was found in the hydrolysis products when analysed by HPLC [ 47 ]. Stability of the enzyme was the most important factor in studying characteristics.
At higher temperature values xylanase stability was gradually declined Figure 8. Xlanase, Honda et al.
C [ 47 ]. Among these xylanases, N shows maximum activity at pH ranging from 6. Stability at the extreme pH values may be due to charged amino acid residues. The enzymes stable in xylanzse conditions were characterized by a decreased number of acidic residues and an increased number of arginines [ 50 ].
Bacillus arseniciselenatis DSM xjlanase a thermoalkalophilic cellulose-free xylanase in higher amount when grown on solid state conditions using cheaply available agroresidual substrate wheat bran.
Hence it can be used for large-scale production of xylanase using such agroresidual substrates. The purified xylanase also was capable of producing high-quality xylo-oligosaccharides, indicating its application potential not only in pulp biobleaching processes but also in the nutraceutical industry.
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Table of Contents Alerts. Abstract A thermoalkalophilic new species of Bacillussimilar to Bacillus arseniciselenatis DSMproduced extracellular xylanase under solid state fermentation when wheat bran is used as carbon source. Materials and Methods 2. Phenotypic Characteristics Prominent selected isolate was identified on the basis of morphological, cultural, biochemical properties [ 20 ] and 16S rRNA sequencing. Growth Conditions of Culture The bacterial isolate was maintained in liquid medium as well as solid medium in basal salt solution BSS containing 0.
Xylanase Production in Solid State Fermentation SSF The selected strain was further enzmi for xylanasf abilities to produce extracellular xylanase under solid state fermentation. Xylanase Assay Xylanase activity was measured according to Bailey et al.