Background
Hemicellulose is one of the copious polymer in lignocellulosic biomass (LCB). It is primarily composed of xylan linked by β-1,4 glycosidic bonds. Xylanase preferentially cleaves the β-1,4-glycosidic bonds in the xylan backbone resulting in complete hydrolysis of the biomass. Thermostable variants of glycoside hydrolases act as robust catalysts, not only in degradation but also during processing, to obtain specific carbohydrate-containing chemicals and materials (Ramasamy et al. in Madras Agric J 107(special):1. https://doi.org/10.29321/MAJ.2020.000382 , 2020).
Conclusion
The strains B. subtilis VSDB5 and B. licheniformis KBFB4 obtained from hot springs of Himachal Pradesh produced potent and alkali-tolerant thermostable xylanases, which belong to the GH11 family. The enzyme can be supplemented in industrial applications for biomass conversion at high temperatures and pH (or in processes involving alkali treatment).
Results
The xylanase production by two thermophilic bacteria isolated from thermal springs was evaluated. In addition, the gene encoding this industrially vital enzyme was isolated and characterized, and its protein structure was analyzed. The thermophilic bacteria producing xylanases were isolated from augmented sawdust and banana fiber biomass from hot springs of Himachal Pradesh and identified as Bacillus subtilis VSDB5 and Bacillus licheniformis KBFB4 using 16S rRNA gene sequencing. The persistent xylanase activity revealed that the enzyme is secreted extracellularly with the maximum activity of 0.76 IU mL-1 and 1.0 IU mL-1 at 6 h and 12 h of growth by KBFB4 and VSDB5, respectively, under submerged fermentation. Both the strains exhibited the maximum activity at pH 6 and a temperature of 50 °C. The xylanases of KBFB4 and VSDB5 were thermostable and retained 40% of their activity at 60 °C after incubation for 30 min. Xylanase of VSDB5 had wide thermotolerance and retained 20% of its activity from 60 to 80 °C, whereas xylanase of KBFB4 showed wide alkali tolerance and retained 80% of its activity until pH 10. The xylanase (xynA)-encoding gene (650 bp) cloned from both the strains using specific primers showed 98 to 99% homology to β-1,4-endoxylanase gene. Further in silico analysis predicted that the xylanase protein, with a molecular weight of 23 kDa, had a high pI (9.44-9.65), which explained the alkaline nature of the enzyme and greater aliphatic index (56.29). This finding suggested that the protein is thermostable. Multiple sequence alignment and homology modeling of the protein sequence revealed that the gene product belonged to the GH11 family, indicating its possible application in bioconversion.