The Expression Of Phytase Gene In Pichia Pastoris And The Optimization Of Fermentation Conditions

Phytase(EC.3.1.3.8) is the enzyme that catalyzes phytic acid into inositol and phosphate.It belongs to the subfamily of acid phosphatases,and has valuable applications in the feed additive industry.Many efforts have been made to produce economical phytase enzymes as feed additives.To date,a number of organisms including plants,animals and microorganisms(especially of fungal and bacterial origin) were screened to produce phytases.Based on the former researches,a technique was established in this study to clone the phytase gene through CODEHOP PCR followed by TAIL-PCR method.The mRNA of Aspergillus niger and Aspergillus oryzae were transcriptd respectively to obtain their cDNA which were then used as template for the cloning of the full length phytase genes as well as partial sequences of the 5' UTR and 3'UTR from A.niger and A.oryzae.The coding region of the phytase genes with no signal peptide sequence were amplified and transformed into Pichia pastoris GS115.The expression of the phytases was methanol induced and phytase property analysis indicated that A.niger rPhyA and A.oryzae rPhyA belong to the subfamily of histidine acid phosphatases.The high expression of the recombinant P.pastoris was achieved under optimum conditions of 50℃and pH5.5 with phytase activities to be 680U/ml for A.niger rPhyA and 400U/ml for A.oryzae rPhyA.According to the practical problems in industrial processes and the former reports which used malt powder as the main source of the medium,in this study,a high productive Pichia pastoris strain with A.niger phyA gene was selected for further study.Wheat bran,wheat husk,fishmeal and other cheap agricultural wastes were analyzed as nitrogen source to screen the proper low cost materials for the industrial production of phytase.By batch fermentaion studies,wheat husk was selected and the basic fermentation conditions for phytase production were determined.The optimum conditions for batch fermentation showed to be 10g of wheat husk added per 100g malt powder with 2%of the methanol in the medium which volume was 1/10 of the flask used,the shaker speed was 250r/min and total fermentation stage was 72 hours.