Expression System Optimization Of Phospholipase A1 Gene From Serratia Marcescens PL-06

Phospholipase A1 is a class of enzymes that can hydrolyze phospho-lipids, participate in a variety of regulation of physiological function in vivo and widely present in a variety of organisms. phospholipase A1 can hydrolyze fatty acids phospholipids generated hemolytic phospholipids and have other characteristics,so is widely used in food processing, phospholipid transformation, oil degumming, pharmaceutical and other industries, phospholipase A1 has a huge industrial prospects.Currently when Serratia marcescens phospholipase A1 gene is exogenous expressed, the expression level is low, easy to form inclusion bodies and inhibit the growth of recombinant bacteria, so finding the right expression of soluble this problem is very important.The main aspects of this paper were as following:(1)The phopholipase A1 gene from Serratia marcescens PL-06 named plaA was cloned,ligased with pET-22b(+)、pET-28a(+)and then transferred to E.coli BL21(DE3) and E.coli BL21(DE3) pLysS. optimized induction conditions for phopholipase A1 expression were obtained as follows:ampicillin concentration 30 ug/mL, IPTG concentration 0.25 mmol/mL, inducing temperature 34℃,strain density OD600 0.3 and induction time 4h. Under the optimized conditions, maximum phospholipase A1 enzyme activity was observed to be 8.6 U/mL and increased by 43.3% than before.Furthermore the growth inhibition of recombinant bacteria AP22 is more serious than AP22pLysS.Description phospholipase Al expressed in BL21 (DE3)pLysS can reduce growth inhibition, but the measured extracellular enzyme activity is not high.(2) Cloned phospholipase A1+accessory protein gene (plaB), and then connected to the E. coli secretion expression vector pET-22b (+) and the pET-28a (+), transformed into E. coli BL21 (DE3) and BL21 (DE3)-pLysS.we concluded that the growth inhibition is the most serious when plaA gene co-expressed with accessory protein plaS gene, extracellular enzyme has also been reduced,the growth also inhibited when express the accessory proteins gene separately. indicating that accessory proteins plaS can inhibit the growth of host bacteria.(3)Cloned phospholipase A1 gene (plaA) and then connected to the vector pPIC9K, transformed into P.pastoris KM71.optimized induction conditions for phopholipase Al expression were obtained as follows: methanol concentration 0.3%, temperature 28℃, strain density OD600 60, PH 5.0. Under these conditions, the recombinant extracellular phospholipase A1 activity up to 3.6U/mL, and increased by 56.5% than before.In short, the selection of E. coli BL21 (DE3) together with the vector pET-22b (+)can improve phospholipase A1 exocytosis expression amount. E. coli BL21(DE3)pLysS can effectively reduce the object gene inhibition on the growth of host bacteria, but extracellular enzyme is not high. The presence of accessory proteins PlaS increase the growth inhibit-ion of recombinant bacteria.