Cloning and expression of a Bacillus α-glucosidase gene in Escherichia coli, Zymomonas mobilis and Pseudomonas putida

Abstract

A gene bank constructed from the Gram-positive bacterium, Bacillus brevis 27-7 (NRRL B-4389) was screened by a fluorimetric plate assay method to identify α-glucosidase production. A B. brevis-derived recombinant plasmid, which produced the highest levels of α-glucosidase activity and appeared stable in Escherichia coli, was designated pNSW350. E. coli RR1(pNSW350) was used to optimise conditions for the α-glucosidase assay in E. coli. The enzyme assay results showed that the properties of α-glucosidase in RR1(pNSW350) and in B. brevis were similar, with the pH range being broader in RR1(pNSW350) than in B. brevis. The cloned α-glucosidase gene from B. brevis was contained on a 4.5 kb BamHI/HindIII fragment of B. brevis DNA and was found to be induced in E. coli by maltose. The α-glucosidase gene was subcloned into the vector pRK404 and the resultant broad-host-range recombinant plasmid was mobilised into Zymomonas mobilis and Pseudomonas putida. α-Glucosidase activity was detected in Zymomonas and Pseudomonas transconjugants. Pseudomonas transconjugants were able to grow on maltose as the sole carbon source, while Z. mobilis transconjugants failed to grow. Enzyme assays showed that Pseudomomas transconjugants exhibited a 1.3-fold higher level of α-glucosidase activity and Zymomonas transconjugants a 0.6-fold lower level of activity than found in B. brevis when grown in the presence of maltose. α-Glucosidase activity was induced by maltose in both Zymomonas and Pseudomonas transconjugants. Show more