Optimization And Scale-up Of Phytase Production By Recombinant Pichia Pastoris

Phytase?E.C.3.1.3.8?is one kind of enzyme which can catalyze Phytic acid?inositol hexaphosphate?and Phytic acid salt hydrolyzing into inositol and phosphoric acid?phosphate?.A mass of research indicated that adding the Phytase in poultry diet can reduce the amount of adding phosphorus in feed.It's very important to improve the efficiency of livestock production and reduce the environment pollution.At present,Phytase has been commercialized as feed additive applying in practical production,but supply of phytase output is limited,it has been limited in industrial applications,the phytase production research working on raising the yield.Pichia expression system is a fully developed and widely used eukaryotic expression system developed recently,use pichia as a host to express phytase by high cell-density cultivation,can effectively improve the phytase production.Foreign protein expression level related with factors such as the strain characteristics,exogenous gene copy number and stability.At while,the optimization of fermentation process is an important way to increase the production rate and reduce the production cost in downstream production process.The idea of this study is,according to Invitrogen"Pichia experiment Manual"and the"Pichia fermentation process Manual"respectively,first has the main components of culture medium and fermentation conditions optimized using multi-gradient experiments of single level optimization in flask,to find the optimal level.The results of the experiment is induced time 120h,induced pH 5.5,induced methanol amount 1.5%,medium volume 10%,induced temperature 28?,glycerol concentration 6%in mixed carbon source and sorbitol concentration 12%in mixed carbon source that provide the data base and reference for high density fermentation.After optimized cultivation,the phytase producing capabilities of recombinant Pichia pastoris is obviously better than before,which improved by 31.9%.Based on the result of Optimize in flasks,recombinant Pichia pastoris produe phytase in3 L and 15 L tank has been studied and two factors,induced temperature and initial induced pH,were optimized,in order to find the suitable fermentation condition,there-by to improve phytase production.The phytase activity is obviously increased after the optimization,the phytase can reach 18708 U/mL,improving 38.3%than before in 3L tank.And phytase can reach 23189 U/mL in 15 L tank,which phytase activity was increased by 24.0%and OD₆₀₀was increased by 10.5%rather than 3 L tank.In addition,the methanol feeding rate of recombinant pichia in the induction period were optimized in 15 L tank in this study.In order to determine the most suitable methanol feeding rate for the production of phytase,the optimal fermentation conditions of recombinant Pichia pastoris producing phytase,the phytase production of recombinant Pichia pastoris under different methanol feeding rate was investigated by controlling the three methanol feeding levels of 3.5 g/?L·h??5.5 g/?L·h??7.5 g/?L·h?.The results showed that under the condition of 5.5 g/?L·h?methanol feeding rate,by the end of fermentation,the phytase activity got to be 26386 U/mL,which was 106.83%of the 3.5 g/?L·h?methanol feeding rate and 122.17%of 7.5 g/?L·h?methanol feeding rate.It suggests that the methanol feeding rate is the most favorable for the production of phytase by fermentation.In this paper,the the recombinant pichia pastoris phytase expressed fermentation conditions were optimized,explored suitable fermentation process.At the same time,through the detection of related parameters in the fermentation process,it alleviated difficult problems such as the recombinant pichia pastoris growth slowly in inducing phase,low phytase expression activity level.This study provide wider space for mass industrial production,technology and theoretical guidance for phytase.