Expression & Bioactivities Analysis Of Recombinant Ester Metabolic Enzyme EHT1,EEB1 And TGL3 Of S. Cerevisiae

Esterases are the important ester metabolic enzymes for brewer's yeasts to produce flavor. Now few people do the research about esterase of S. cerevisiae. So the catalytic activity of recombinant enzymes measured in vitro, understanding the catalytic function, the differences of catalytic activity and the regulation of metabolic enzymes, are very important to the development and utilization of ester.In this paper, 5 recombinant esterases were obtained. The catalytic activity and the differences of flavor which they produced were compared.Three ester metabolic enzyme genes eht1, eeb1 and tgl3 with Genbank number GU471248, GU471249 and GU471250 respectively,which were cloned from S. cerevisiae were cloned into prokaryotic expression vector pET28a. After the conditions of expression were obtained,the soluble recombinant proteins His-EHT1 and His-EEB1 were expressed in E.coli BL21(DE3)and purified by nickel chelate affinity chromatography method. The protein expression level is up to 1.034mg/mL and 0.683mg/mL respectively. But TGL3 wasn't expressioned in E.coli BL21(DE3). The three genes were cloned into eukaryotic expression vector pPIC9K. The linearized plasmids were transformed into the engineered Pichia pastoris GS115 by the method of electroporation , the recombinant engineered Pichia pastoris GS115/ pPIC9k-EHT1,GS115/ pPIC9k-EEB1å'ŒGS115/ pPIC9k- TGL3 were obtained .The fermentation condition of the three kinds of recombinatant strain in the shaking flask to produce ester metabolic enzymes from Pichia pastoris was that pH6.0, temperature 28℃,final concentration of methanol was 0.5% (added once each 12h) and the highest productivity of the three proteins was obtained in 96~120h. In this condition, the expression levels of EHT1, EEB1, TGL3 reached 100.34mg/L, 113.62 mg/L and 88.73mg/L,respectively.The secondary structure of the three ester metabolic enzymes was predicted used bioinformatics analysis software. The result indicated that the secondary structure of EHT1 was highly similar with the EEB1. Also the glycosylation sites were predicted, that 4 glycosylation sites were found in EHT1, and 2 for EEB1 and 2 for TGL3. The activity of the different recombinant proteins was measured, the result cetified that the activity of eukaryotic expression recombinant proteins was higher than prokaryotic expression recombinant proteins. The activity of EHT1 which was expressioned by GS115 reached 74.569U/mg, while the EEB1, TGL3 up to 81.614 U/mg, 34.570 U/mg, respectively.The recombinant engineered Pichia pastoris GS115/ pPIC9k-EHT1,GS115/ pPIC9k-EEB1 were fermented in the condition including methanol, besides phenylethanol, many kinds of volatile compounds such as ethyl octanoate, ethyl hexanoate and ethyl decanoate were detected compared to the fermentation without methanol after. This indicated that the recombinant proteins EHT1 and EEB1 were responsible for medium-chain fatty acid ethyl ester biosynthesis. This work provides basic foundation for forture making use of the ester metabolic enzyme.