Optimization Of Phytase Production By Recombinant Pichia Pastoris And Studies On Its Thermostability

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). Phosphorus is the animal's necessary mineral, playing an important role in the normal metabolism. A mass of research indicated that adding the Phytase in pigs and poultry diet not only can increase the utilization of phosphorus to 60%,but also can decrease the discharge quantity of phosphorus in feces to 40%.It 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 it still exists technology problems,and one of the main problem is Phytase thermostability.The research is based on the lab saved Pichia pastoris.At the first,using the partial factors method in flask level screen 7 factors:induced time,induced pH,induced methanol amount,growth time,inoculation amount,growth pH and induced temperature.The results of the experiment with the MiniTab soft is that we have 4 significant factors: induced pH, induced methanol amount,growth time and inoculation amount.Then make them single factor optimization and the optimum fermentation conditions in flask level is:induced time 72h,induced pH 4,induced methanol amount 1.5%,growth time 20h,inoculation amount 10%,growth pH 5,induced temperature 28℃that provide the data base and reference for high density fermentation.Optimizing the induced time,induced pH,induced methanol strategy and induced temperature in 30L fermentation tank and the optimum condition in it is:growth time 20h, speed of methanol 5mL/(L·h),the speed of glycerin in induced phase 1mL/(L·h),induced pH 4,induced time 84h,induced temperature 24℃.The Phytase activity is obviously increased after the optimization,the Phytase can reach 486U/mL,improving 289% than before.Then studying the basic Phytase character.Mesuring the optimim pH with pH 3-8 buffer solution,the Phytase in our lab remain above 60% residual activity and analyzing the effect of 4 kinds of inorganic and 4 kinds of saccharides on the thermostability of Phytase,the results is that adding 0.8mmol/mL NH4+and 0.4mmol/mL sucrose can improve the Phytase thermostability,after 10min disposed respectively in 50-90℃, the Phytase remain above 80% residual activity and after deposit of 2 months it remains above 50% activity.At last putting the Phytase into actual production and application using in grass carp breeding comparing with commeicial Phytase and calcium dihydrogen phosphate,the results is that the adding ratio 0.08% can completely replace the calcium dihydrogen phosphate.In this research we bring out Phytase fermentation and product procession which increase Phytase activity,increase thermostability of Phytase and the adding amount of the Phytase which replace calcium dihydrogen phosphate.It can be sure than our lab's Phytase have the commercial value and application prospect and provide the scientific base for its large scale production.