Abstract
Abstract A beta-glucosidase-like enzyme-encoding gene (bglH) of an endophytic Bacillus pumilusstrain (CL16) was clonedusing a shotgun genomic library constructed in Escherichia coli. The nucleotide sequence of the entire cloned frag-ment (2484 bp) was determined and characterized. An incomplete open reading frame (ORF) of 534 bp (ORF1) des-ignated bglP and a complete ORF of 1419 bp (ORF2) designated bglH, located in the fragment, are organized in anoperon. The protein deduced from 1419 bp (ORF2) had 472 amino acid residues without a characteristic signal pep-tide sequence, suggesting that the enzyme is localized in the cytoplasm. The amino acid sequence deduced frombglH gene had high similarity with β-glucosidases from the glycosyl hydrolase family 1. Over-expression of the B.pumilus bglH gene in E. coli showed a 54 kDa protein whose identity was confirmed by mass spectrometry(MALDI-TOF). Key words: Bacillus pumilus , bgl H, glucosidase, glycosyl hydrolase 1, PTS.Received: November 28, 2005; Accepted: August 17, 2006.
Highlights
Cellulose comprises the major carbohydrate polymer of the plant cell wall
The β-glucosidases are widespread in microorganisms where they metabolize various carbohydrate substrates, including cellobiose, produced as a consequence of cellulose hydrolysis, and aromatic β-glucosides such as arbutin and salicin that are produced by a variety of plants (Tajima et al, 2001; Spiridonov and Wilson, 2001; Park et al, 2002; Marques et al, 2003; An et al, 2005)
Nucleotide sequence, determined using the DYEnamic ET DYE Terminator Cycle Sequencing Kit (Amersham Biosciences, Germany) on MegaBACE 1000 (Amersham Pharmacia Biotech, Germany), showed high similarity with the bglH gene from B. subtilis subsp. subtilis strain 168 (E value = 1e-17). This fragment was successfully used as a probe for screening the bglH gene in a shotgun genomic library constructed from B. pumilus strain CL16 (Lima et al, 2004)
Summary
Cellulose comprises the major carbohydrate polymer of the plant cell wall. It is an unbranched polymer composed of anhydro-1,4-D-glucopyranoside units linked by β-glucosidic bonds. The Bacillus pumilus strain CL16 showed high cellulase activity and was selected for further studies. During the study described in the present paper we isolated and characterized a new locus of β-glucoside sugar utilization genes from the endophytic B. pumilus CL16 strain.
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