Colning And Prokaryotic Expression Of Phospholipase A1Gene

Phospholipase Al plays proven important roles in many physiological functions in vivo, it is designated as virulence in some pathogens and directly related to pathogenisis. Phosphplipase A1hydrolyze phospholipids releasing lyso-phospholipid, this characteristic give phospholipase A1perspective in food and pharmaceutical industry. Because the advantages high efficiency, low energy consumption and low pollution when phospholipase Al applied to vegetable oil degumming, phospholipase A1get widespread attention and application. The gene located downstream of phospholipase A1in Serritia spp. encode a accessory protein was reported can improve the efficient expression of phospholipase Al in E.coli. Research about the relationship between the accessory protein and phospholipase A1was important not only for the industrial application of bacterial phospholipase A1but also for the elucidation of bacterial pathogenic mechanism. The main aspects of this study were as followed. (1) The genome of Serratia marcescens PL-06which screened by our laboratory was used as the templet of PCR for amplify the gene plaA, plaS and plaB, which encode the phospholipse A1(PlaA), the assessory protein (PlaS) and the phospholipse A1+assessory protein. The sequence identity reached higher than95%. The plaB gene, which comprised by overlapped plaA and plaS, was classic overlapping gene. The structural information of PlaA and PlaS was predicted by bioinformatics softwares. The sequence of this two gene was conserved. According to the predicted results, PlaA was classic α/β hydrolases, PlaS belonged to the ANK family which was accouted broadly participated in the protein-protein reactions and it implied this structure might relevanted to the role in promoting the phospholipase A1activity.(2) The E. coli expression strains of plaA, plaS and plaB was named AP28, SP28and BP28, which was constructed by ligased them to pET-28a (+) and transferred to BL21separately. The expreesion results were as followed:i) The phospholipase Al expreesion level in AP28was much higher than in BP28. But the enzyme activity of AP28was1.2U/mL and the specific activity was78.94U/mg, while the enzyme activity of BP28was16U/mL and the specific activity achieved311.65U/mg. So the accossery protein was necessary for the high activity expression of phospholipase A1in E.coli. ii) the gene plaS expressed independently restrained the growth of the host E.coli strain, iii) The inhibition effect to the host strain was more obviously when the plaS gene co-expression with plaA in BP28and there was a lot of bacterial protein in the supernatant of fermentation broth of BP28, which might because of the work of active phospholipase.(3) The phospholipase Al expressed in AP28mainly in the form of inclusion body. After optimal the recovery rate of the target protein reached188.75μg/mL, however the enzyme activity was12U/mL, and the specific activity was63.58U/mg.(4) The constructed Bacillus subtilis WB600expression strains of the gene plaA and plaB were named as AW and B W. The gene expressed in WB600was similar as in E. coli. The phospholipase A1activity of BW was much higher than AW, the highest enzyme activity value was1.2U/mL and7.2U/mL respectively, this result indicated that the accessory protein also improved the efficient expression of phospholipase Al in Bacillus subtilis. When co-expressed accessory protein with phospholipase A1in BW, the cell growth was apparently suppressed.In conclusion, the accessory protein gene located downstream of phospholipase A1gene in Serratia marcescens PL-06was necessary for the high activity prokaryotic expression of phospholipase Al, but the expression of the accessory protein restained the growth of host stain. This provided foundation for the study about the heterologous active expression of bacterial phospholipase Al and the toxicity mechanism of bacteria which use phospholipase A1as virulence.