Gene amplification and sequencing of hemicellulase using culture dependent technique

Abstract

The present study aims to discover hemicellulase genes from thermophilic bacteria of Anoxybacillus gonensis G2T and Halothermobacillus malaysiensis RA using culture dependent technique. The hemicellulase genes with the amplicon size of 2118 bp, 1143 bp, 1554 bp and 2190 bp from A. gonensis G2T and H. malaysiensis RA were successfully obtained. Bioinformatics analyse of primary, secondary structure and tertiary structure of these proteins were performed by using bioinformatics tools. Beta-xylosidase from A. gonensis G2T and alpha-glucuronidase from H. malaysiensis RA are intracellular proteins. While, endo-1,4-beta-xylanase and alpha-N-arabinofuranosidase from H. malaysiensis RA have signal peptide and secreted as extracellular protein. I-TASSER and Swiss Model server were used to model the tertiary structure of hemicellulase. Good structures were generated after energy minimization through YASARA server. Using Neighbor-Joining method, the phylogenetic analysis reveals that beta-xylosidase from A. gonensis G2T (bxAg) is closely related with beta-xylosidase from Geobacillus sp. WSUCF1; endo-1,4-beta-xylanase from H. malaysiensis RA (ebxHm) is distantly related with endo-1,4-beta-xylanase from Xanthomonas translucens pv translucens, family GH10 Maribacter dokdonensis DSW-8 and Saccharicrinis fermentans DSM 9555JCM 21142; alpha-N-arabinofuranosidase from H. malaysiensis RA (anaHm) is closely related with alpha-N-arabinofuranosidase from Gemmatimonas sp. SG8 28; and alpha-glucuronidase from H. malaysiensis RA (agHm) is distantly related with alpha-glucuronidase from Sphingomonas sp.WG, Caulobacter vibrioides, Xanthomonas oryzae pv. oryzae and Stenotrophomonas maltophilia.