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Keywords:

  • genomic library;
  • β-glucosidase;
  • bgl operon;
  • β-xylosidase;
  • termite

Abstract

The gut of the termite Reticulitermes santonensis contains an interesting diversity of prokaryotic and eukaryotic microorganisms not found elsewhere. These microorganisms produce many enzyme-digesting lignocellulosic compounds, probably in cooperation with endogenous enzymes. Regarding cellulose and hemicellulose digestion in the termite gut, much remains to be learned about the relative contributions of termite enzymes and enzymes produced by different microorganisms. Here we grew bacterial colonies from termite gut suspensions, identifying 11 of them after PCR amplification of their 16S rRNA genes. After constructing in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained, we performed functional screening for α-amylase, xylanase, β-glucosidase, and endoglucanase activities. This screen revealed a clone producing β-glucosidase activity. Sequence analysis showed that the cloned genomic DNA fragment contained three complete ORFs (bglG, bglF, and bglB) organized in a putative bgl operon. The new β-glucosidase (BglB), identified with its regulators BglG and BglF, belongs to glycoside hydrolase family 1. The new β-glucosidase was expressed in E. coli and purified by affinity chromatography. The purified enzyme shows maximal activity at pH 6.0 and 40 °C. It also displays β-xylosidase activity.