Cloning, characterization and sequence determination of a cellobiase gene from Cellulomonas Biazotea in Escherichia coli

Abstract

Cellulomonas biazotea has been previously shown to produce secretory cellobiases. A genomic library of C. biazotea was constructed in an expression vector pM and transformed into E. coli JM101 strain. Subsequent screening for activity on MUG substrate led to the isolation of 13 clones expressing β-glucosidase activities. Each of them had a unique restriction pattern suggesting that they were different determinants of β-glucosidases in C. biazotea. A recombinant construct, designated 9178, was found to be able to anneal with a primer designed from the N-terminal sequence of Cba2 for which Cba2 was a novel cellobiase previously purified from the culture supernatant of C. biazotea. The pNPGase activity of 9178 was found to be localized in the cell, only 4% of the activity was secreted in periplasm. A deletant of 9178 carrying a defective Tac cassette, designated MluIΔ, when compared with 9178, was found to express same level of enzyme activity. The finding implied that this cellobiase gene might be expressed by its native promoter. The employment of homologous signal peptide for protein secretion in E. coli was usually unsatisfactory which might account for the low level of secreted β-glucosidase activity. DNA sequencing revealed that the N-terminal sequence of 9178 was different from that of the Cba2. Therefore, it was a new determinant of β-glucosidase in C. biazotea, designated cba3, which contained an open reading frame of 2889bp encoding a putative peptide of 963 amino acids with a predicted molecular weight of 101436.84Da. Comparison of the predicted amino acid, showed significant homology, ranging from 40.6% to 52.4%, with the amino acid sequences of other β-glucosidases. Scanning an alignment of enzyme sequences for positions of strict sequence conservation allows the identification of potential active sites. The His⁴⁵⁶ and Asn⁵⁰⁰-Glu⁵⁰¹-Pro⁵⁰² motifs that are highly conserved in family 1 β-glucosidases, were also observed in the amino acid sequence of Cba3.

Date of Award	2003
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology