In this study, screening and isolation of xylanocellulolytic enzymes producing positive microbes from termitarium and termite gut microbiome were done using cost-effective agricultural wastes. The enrichment of xylano-cellulolytic microbes was done in three steps using wheat bran and waste paper. The qualitative screening of xylanase and cellulase producing micro-organisms was done on nutrient agar plates containing wheat bran and waste paper, respectively. Xylanase and cellulase positive colonies were analysed by observing the zone of substrate (wheat bran and waste paper) hydrolysis around the colonies. A total of 30 bacterial isolates were obtained from termite gut and termitarium, respectively. Xylan and cellulose degrading potential of the positive isolates was also quantitatively estimated using agro-wastes-based medium. All the bacterial isolates displayed cellulase and xylanase activities in the range of 0.45-6.80 and 51-380 IU/ml, respectively. This is the first report mentioning the isolation of xylano-cellulolytic microbes from termite gut and termitarium using very simple cost-effective methodology.
This study shows the presence of five isozymic forms of alkaline xylanase from Bacillus pumilus using fast flow rate microfiltration, ultrafiltration, Q-sepharose, and phenyl sepharose chromatographic techniques. Polyacrylamide gel electrophoresis, highperformance liquid chromatography, and zymographic studies also revealed the purity of five isoforms of alkaline xylanases. Isoforms-X-I, X-III, and X-V exhibited optimum activity at pH 8.5, whereas X-II, X-IV showed maximum activity at pH 9. All isoforms were optimally active at temperature 55 C. Isoforms were found to be stable at pH 7-11, showed 92-100% residual activity after 3 hr, treatment time for most industrial applications. The isoforms retained nearly 80-86% residual activity after incubating at 45 C for 3 hr. Molecular weights of xylanase I-V, were 13.1, 15.3, 18.4, 20.1, and 21.0 kDa, respectively. Mg 2+ ions were found to be potent activator for all isozymic forms. The Km and Vmax values of X-I, X-II, X-III, X-IV, and X-V were 6.71, 6.66, 7.14, 5.88, 6.25 mg/ml and 2,000, 1,695, 1,666.66, 1,428.57, and 1,408.45 IU/mg protein, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the monomeric nature of all isoforms. The lowmolecular masses, significantly enhanced activity in the presence of industrially suitable-low cost activator, better stability of all isoforms at pH 7-11 and at higher temperature, also presence of multiple forms of alkaline xylanase, makes this enzyme suitable for textile-paper industries. This is also the first report mentioning the purification of five isozymic forms of alkaline xylanase using fast flow rate techniques.
Simultaneous production of alkaline xylanase and all seven types of pectinases by a bacterial isolate, under solid-state fermentation was checked in this study. Under optimized conditions, high concurrent production of xylanase (22,800 ± 578 IU/g substrate) and pectinase (4,832 ± 189 IU/g substrate) was achieved. The different types of pectinases produced were exo-polymethylgalacturonase (782 IU/g), endo-polymethylgalacturonase (6.42 U/g), exo-polygalacturonase (2,250 IU/g), endo-polygalacturonase (11.57 U/g), polymethylgalacturonate lyase (53.99 IU/g), polygalacturonate lyase (59.78 IU/g), and pectin esterase (5.78 IU/g). Wheat bran resulted in the highest titer of both enzymes. The maximum xylanase-pectinase yield was detected after 7 days of incubation with 2 mM MgSO 4 and 1.5 g/L K 2 HPO 4 at wheat bran to moisture ratio 1:1.5 (w/v), media to flask volume ratio 1:25, pH 7.0, temperature 37 • C, and inoculum size 15%. Xylanase was most stable at pH 8.0, retained more than 75% activity up to 24 H, whereas pectinase was most stable at pH 9.0, having full activity even after 24 H. At 45 • C, the xylanase showed 82% residual activity after 6 H of incubation. The pectinase was 97% and 61% stable up to 3 H at 50 and 55 • C, respectively. This is the first report showing the production of xylanase-pectinases by bacterium along with high titer of seven types of pectinases, suitable for industries.
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