Cloning of β-(1,3-1,4)-Glucanase Gene in Probiotic Bacterium Bacillus coagulans

Abstract

Endo-β-(1,3-1,4) glucanase (or lichenase, EC3.2.1.73) is an endoacting enzyme that hydrolyses mixed linkage β-(1,3-1,4)-glucan from the walls of starchy seed endosperm. Bacillus coagulans is a lactic acid producing and sporeforming probiotic bacteria. The aim of this work is to clone and express lichenase gene in B. coagulans to create better probiotics for poultry. The Lichenase gene was inserted into the Escherichia coli-Bacillus sp. shuttle vector pMK3. The construct pMK3Lic was introduced into B. coagulans DSM1 by electrotransformation. Insert analysis of pMKLic obtained from recombinant B. coagulans confirmed the lichenase gene fragment on agarose gel electrophoresis. Despite of cloning of lichenase gene in B. coagulans, no the lichenase enzyme activity was detected on lichenan overlay plates. Both the extracellular and intracellular lichenase enzyme activity wasn’t detected from cultures of B. coagulans carrying pMK3Lic when analyzed by SDS-PAGE zymogram. In the study, an electrotransformation protocol for B. coagulans was developed and transformed strain DSM1 with plasmid pMK3, an E. coli-Bacillus sp. shuttle vector. A gene was also successfully transferred into B. coagulans. The shuttle vector and electroporation method could be used in other genetic engineering studies of B. coagulans.