| Phospholipase A2s(PLA2s, EC 3.1.1.4) widely exist in various kinds of biological tissue and can catalyze the hydrolysis of the 2-acyl ester bond of 1,2-diacyl-3-sn-phospholipids,yielding lysophosphatidylcholine and free fatty acids,and they show important practical application value in the fields of food,cosmetics and medicine. PLA2 can be extracted from the pancreas of some mammals,but it is not conducive to the promotion and application because of the little quantity and the complex extraction process. It is an effective way to achieve PLA2’s industrial application when PLA2 is produced by microbial fermentation,in addition,K. lactis has food safe status and has advantage in heterologous protein expression and application. In this study,the prokaryotic microbial heterologous pla2 gene from L. casei was expressed successfully in K. lactis GG799 for the first time.The signal peptide of L. casei PLA2’s amino acid sequence was predicted,then the pla2 gene was amplified by PCR excluding the region that encodes the putative signaling sequence,next,the expression vector p KLAC1-pla2 was constructed and integrated into the K. lactis genomic LAC4 locus,and the PLA2 expression cassette was under the direction of the K. lactis signal α-mating factor and under the control of the LAC4 promoter. The recombinant s PLA2 activity in the fermentation supernatant of K. lactis/p KLAC1-pla2 was detected on egg agar plates.In comparison with the control strain K. lactis/p KLAC1,SDS-PAGE analysis revealed a 17-k Da recombinant protein band in K. lactis/p KLAC1-pla2,which was consistent with the predicted molecular weight of the mature protein.The integrated pla2 gene copy number of K. lactis/p KLAC1-pla2 were determined by real-time quantitative PCR. It indicated that the copy number of the integrated pla2 gene ranged from 2 to 6 and positively correlated with s PLA2 activity. The recombinants that had only two pla2 gene copies exhibited lower enzyme activity,and this value was 0.27±0.06 U·m L-1. The highest level of recombinant s PLA2 activity was 1.87±0.12 U·m L-1 and was detected in the recombinant that had six integrated pla2 gene copies. This value was five-fold higher than the observed activity in the recombinant that contained only two gene copies. However, the recombinant that harbored 3 pla2 gene copies exhibited a higher s PLA2 activity than the recombinant that harbored 4 or 5 copies.s PLA2 catalytic reaction process must need millimole Ca2+,it showed the Ca2+ concentration significantly affected the s PLA2 enzyme activity,and the optimum Ca2+ concentration was 30 m M.The LAC4 promoter could be induced by either lactose or galactose,and the effect of the LAC4 promoter strength on s PLA2 activity under the condition of induced or non-induced were investigated. The results showed that the s PLA2 activity of 1.96±0.15 U·m L-1 was found in the YPG medium when the recombinants were cultivated for 84 h,and the s PLA2 activity was 1.87±0.12 U·m L-1 in the YPD medium when the sample was withdrawn after 72 h. The YPD medium was used for the rest of the study to investigate the effect of temperature, p H and dissolved oxygen concentration on the s PLA2 production. When the recombinant was cultured at 30 oC in a YPD medium culture volume of 70 m L in a 250-m L shake flask with an initial p H of 7.0,the s PLA2 activity reached 2.16±0.18 U·m L-1.Shake flask fermentation conditions of PLA2 production by recombinant was optimized using single factor test and orthogonal design experiments. The optimal fermentation medium was found to be as follows: glucose 30 g·L-1,yeast extract 20 g·L-1,peptone 30 g·L-1,KH2PO4 3 g·L-1.The best fermentation conditions were: temperature 30 oC,inoculation amount 2%,initial p H value 7.0,medium volume 90 ml in 250 ml flask,220 r/min of shake cultivation. Under the optimal conditions,the enzyme activity in shake flasks was enhanced from 1.87 U·m L-1 to 5.35 U·m L-1. |
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