| Phospholipase A2(PLA2) is an enzyme that catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids. It widely exists in mammalian pancreas, snake venom and bee venom. PLA2 s can be used for degumming of vegetable oil in the oil industry which plays a critical role in the refining of edible oil. Nowadays, Lecitase® Novo(PLA1) and Lecitase® Ultra(PLA1) are the most used in oil degumming. But the cost of the enzymes is very high. In this study, we successfully expressed the PLA2 from S. violaceoruber in the methylotrophic yeast Pichia pastoris, and the enzymatic properties were determined. According to the enzymatic properties, we optimized the oil degumming conditions which laid a foundation for the further studies and industrial application. The main results are as follows:(1) The S.violaceoruber PLA2 gene(Gen Bank NO. AY359866.1) was optimized in accordance with the codon preference. The recombinant plasmid p PIC9K-PLA2 was linearized by Sal I and transformed into P. pastoris competent cells. Eight positive recombinant strains were screened on MD plate and the copy number were 16, 13, 12, 9, 7, 6, 2, and 1 respectively. The expression and activity of recombinant PLA2(r PLA2) increased with the increase of gene copy number, but were not linear with gene copy number.(2) The r PLA2 contained three types of proteins with varied molecular weights: 14.3 k Da, 18.2 k Da and 21.0 k Da. We carried out PAS staining method and Endo H method to identify the N-glycosylation of r PLA2. The result showed that the 21.0 k Da protein and the 18.2 k Da protein occurred N-glycosylation, while the 14.3 k Da protein was of no N-glycosylation.(3) Three potential N-glycocylation sites(N-34, N-85 and N-113) of r PLA2 were mutated. The results showed that N-85 did not ocurr N-glycosylation, and N-34, N-113 were of N-glycosylation. The 14.3 k Da protein did not occur N-glycosylation. The 18.2 k Da protein occurred N-glycosylation at N-34 site or N-113 site. The 21.0 k Da protein occurred N-glycosylation at N-34 site and N-113 site. Moreover, we investigated the effects of N-glycosylationon on r PLA2 expression and enzyme activity in supernatant. The results showed that N-glycosylation has little effect on the expression and secretion of r PLA2. The N-34 and N-113 sites mutation could improve the enzyme activity in different extent. The enzyme activity of r PLA2 w which did not occur N-glycosylation was the lowest.(4) The enzymatic properties of this enzyme were determined. The optimum temperature of r PLA2 was 50 °C. When the temperature was above 62 °C or below 42 °C, the enzyme activities were less than 70%. At low temperatures, r PLA2 was relatively stable. The residue enzyme activities could keep above 60% at the temperature below 35 °C. The enzyme exhibited maximum activity at p H 6.0. It was stable at a p H range of 4.5–6.2 which meet the requirements of oil degumming(p H 4–6). The residue enzyme activities could keep above 80% at the p H range of 5.5–6.5 which demonstrated that p H has a noticeable effect on r PLA2. The p I of the r PLA2 was 5.87. Ca2+ showed a significant positive effect on the activity of r PLA2.(5) We also carried out oil degumming experiments using r PLA2 and obtained the optimized conditions: 40 °C, p H 6.0, water content 4%, enzyme dosage 0.6 U?g-1。The phosphorus concent of degummed oil was down to 9.6 mg?kg-1 within 6 h under the optimized conditions which is very promising for the industrial application. |
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