Expression Of Porcine Pancreas Phospholipase A₂ In Apospory Aspergillus Niger SH-2

Phospholipase A2(PLA2, EC 3.1.1.4) family of enzymes catalyze the hydrolysis of the sn-2 ester of glycerophospholipids to produce lysophospholipids and free fatty acids such as arachidonic acid, oleic acid, etc. Phospholipase A2 plays an important role in human inflammation and other physiological functions. In addition, it has been widely used in industry production(eg, oil degum degumming,) and food processing(eg, egg yolk treatment,). It was that porcine pancreas PLA2 produced by Aspergillus niger was allowed to use as a food additive by Chinese Ministry of Health in 2008. To date, the porcine pancreas PLA2 was mainly extracted from the pig pancreas which was limited in large-scale production due to its low extraction efficiency. And there is no commercial porcine pancreas PLA2 produced by microorganism in domestic market. A.niger, one of the GRAS strains, has the advantage of post-translation modification, secretion ability and high protein production in heterologous protein expression. A.niger SH-2 owns high protein expression capacity and its morphology could be easily controlled in liquid state fermentation. So it has a broad prospect to express porcine pancreas PLA2 in A.niger.The constructed PLA2 expression plasmid has been successfully transformed into the host apospory A.niger SH-2 in this study. The recombinant protein was identified to be PLA2 by SDS-PAGE and protein mass spectrum. The specific activity of recombinant enzyme reached 165.60 U/mg after affinity chromatography and desalting.Subsequently multicopy expression plasmid was constructed, making the expression of PLA2 improved by 1 fold. The transcriptional level of glucoamylase gene gla A, PLA2 and hac Ai in PLA2 transformants were detected by q RT-PCR. The unfolded protein response(UPR) involves a complex signalling pathway in which the transcription factor hac A plays a central role. Transcriptional level of hac Ai was up-regulated when the transcriptional level of PLA2 and gla A was high.Optimization of fermentation process of PLA2 transformants was conducted in 5 L tank. Mycelial morphology was affected by the concentration of residual glucose in fermentation liquor. Enzyme activity of PLA2 was improved by three times, reaching to 17.10U/m L which was higher than the enzyme extracted from animal pancreas(14.70 U/m L). The concentration of total protein in fermentation liquor was 27.80mg/m L. These data lay the foundation for fermentation process optimization.