Mitra, Suranjita; Mukhopadhyay, Bidhan Chandra; Mandal, Anisur Rahaman; Arukha, Ananta Prasad; Chakrabarty, Kuheli; Das, Gourab Kanti; Chakrabartty, Pran Krishna; Biswas, Swadesh Ranjan
Cloning, overexpression, and characterization of a novel alkali-thermostable xylanase from Geobacillus sp WBI

An endo--1,4-xylanase gene xynA of a thermophilic Geobacillus sp. WBI from hot compost was isolated by PCR amplification. The gene encoding 407 residues were overexpressed in E. coli and purified by Ni-NTA chromatography. The purified enzyme (47kDa) had a broad pH optimum of 6.0 to 9.0, and was active between 50 and 90 degrees C. The enzyme retained 100% of its activity when incubated at 65 degrees C for 1h under alkaline condition (pH 10.0) and retained 75% activity at pH 11.0. The K-m and V-max of the enzyme were 0.9mgml(-1) and 0.8 mu molml(-1)min(-1), respectively. In molecular dynamics simulation at 338K (65 degrees C), the enzyme was found to be stable. At an elevated temperature (450K) specific -helix and -turns of the proteins were most denatured. The denaturation was less in WBI compared with its highest homolog G. stearothermophilus T-6 xylanase with difference of six residues. The results predict that these regions are responsible for the improved thermostability observed over related enzymes. The present work encourages further experimental demonstration to understand how these regions contribute thermostability to WBI xylanase. The study noted that WBI produces a xylanase with unique characteristics, specifically alkali-thermostability.


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