Cloning and production of feed upgrading enzyme encoding genes in probiotic lactic acid bacteria

Phytases can increase the nutritional value of food and feed by liberating inorganic phosphate from phytate, the major storage form of phosphorus in plants. In this study, the phytase gene (phyC) from Bacillus subtilis VTT E-68013 was amplified by PCR technique and inserted into pMK3 shuttle vector of Escherichia coli-Bacillus sp. to construct pMK3F. The recombinant vector, pMK3F, was firstly transferred into E. coli by heat-shock method. MK3F plasmid isolated from E. coli was used to introduce into Bacillus coagulans cells by electrotransformation. HindIII and BamHI digestion of the recombinant plasmids from E. coli and B. coagulans yielded 1300 bp fragment of DNA carrying the gene encoding phyC and 7200 bp fragment of pMK3 vector on agarose gel electrophoresis. Phytase gene of recombinant vector pMK3F was also amplified by PCR and visualized by agarose gel electrophoresis to support the results. Although phytase gene was cloned in B. coagulans, phytase enzyme activity was not detected on Wheat Bran Extract/Sodium-phytate/LB/Agar plates and SDS-PAGE gel contains Wheat Bran Extract/Na-phytate as substrats. ß-(1,3-1,4)-glucanases (lichenases) hydrolyse linear ß-glucans containing ß-1,3 and ß-1,4 linkages such as ß-glucans and lichenan. Lichenase gene originated from Streptococcus bovis was ligated with pMK3 shuttle vector of E. coli-Bacillus sp. to construct pMK3Lik. The recombinant vector, pMK3Lik, was firstly introduced into E. coli and then B. coagulans. Digestion of recombinant plasmids from E. coli and B. coagulans by SmaI was showed the 1800 bp lichenase gene band on agarose gel elctrophoresis. Although E. coli cells carrying pMK3Lik was showed lichenase activity, the enzyme activity was not detected from recombinant B. coagulans cells on both LB/Lichenan/Agar plates and SDS-Lichenan-PAGE. Key words: Phytase, lichenase, Bacillus coagulans, probiotic, cloning