Abstract: Rhizomucor miehei lipase(RML)gene was designed using yeast's prefered codon to improve its expression level in Pichia pastoris Vector and the properties of RML was studied. According to the bias in codon choice of the high expression gene in P.pastoris, the mature portion of RML gene with high specific activity was optimized and artificially synthesized by overlap extension PCR without changing the amino acid sequence. The RML gene was subcloned into expression vector pPIC9K to yield the recombinant expression vector pPIC9K-RML.The recombinant plasmid was transformed into P.pastoris GS115 by electroporation. Subsequently, the positive clone was selected by G418 and PCR and the high expression recombinant P.pastoris GS115-RML was obtained. After induced by 0.5% methanol for 168 h at 28℃, the activity of lipase achieved 122 U/mL and was 10 times higher than that of the unoptimized wild type R.miehei. The expression of RML gene in P.pastoris could be successfully improved by using codon optimization. The optimum reaction conditions of recombinant RML were temperature 45℃ and pH value 7. At these conditions, when RML reacted in 40% methanol for 100 min, the remaining activity was 96.9%.This showed that RML displayed a good stability in methanol.

Key words: Rhizomucor miehei lipase, Pichia pastoris, codon optimized, enzymatic properties