Cloning And Expression Of Mono-and Diacylglycerol Lipase-Encoding Gene

A cDNA fragment of 860bp was amplified by RT-PCR method using total RNA extracted from Penicillium camembetlii-PG3. Analysis of nucleotide sequence of the fragment suggested that it was the same as the gene(mdlA) encoding mono- and diacylglycerol lipase(MDGL). The target gene was then inserted into expression vector pET-28a and expressed in E.coli BL21 with IPTG induction. The recombinant protein was highly expressed as inclusion body and accumulated up to 45.2% of bacterial total protein. SDS-PAGE analysis showed that relative molecular mass of recombinant protein was about 41000. No activity of the purified recombinant protein was detected towards olive oil and 13.5U/mL was reached(method of alkalimetrictitration) when using monostearoylglycerol as substrate.mdlA was then inserted into methanol-inducible expression vector pPIC9K. The linearized recombinant plasmid was transformed into chromosome of Pichia pastoris GS115 strain by electroporation. Some...