Effects On Phytases Expressed By Two Type Engineering Yeasts

Phytase is a class of phosphatases that can catalyze the hydrolysis of phytate into myo-inositol and phosphate, when added to food and feed, they can decrease chelation of microelements by phytate, relieve anti-nutrition of phytate, increase absorption and utilization of phosphorus, improve nutritional value of feed and food, and Simultaneously alleviate environment pollution caused by phosphorus. Therefore, the study and application of phytase in food and feed has drawn extensive attention in the world.Phytase has been studied extensivly in other countries, however, native study on phytase is relatively limited for its late set out, most researchers have constructed engineered yeast strains to express phytase, and these strains are mostly nonmethanol utilizing phenotype. We has constructed a strain of methanol utilizing phenotype, and found that enzymatic characteristics of the recombinant phytase we express with this strain is different from that of wild type or that obtained by other reseachers, especialy, the functional pH range is broader. This study concentrated on the effects of transformation method and integration type on enzymatic characteristic of phytase, and is a basic work for expression of phytase gene in Pichia pastoris. Based on the laboratory's former research, we carried out the following studies:1. Isolation and linearization of Plasmid DNAPlasmid pPIC9K-phyA was extracted with Plasmid Mini Kit, digested with DraI, verified with electophoresis, extracted with phenol/chloroform, precipitated with ethanol, solved in 10μL TE, and measured up to 500ng/μL with ultraviolet spectrophotometer.2. Preparation and transformation of protoplastThe protoplast were prepared in hyperosmotic buffer with Zymolyase, incubated with plasmid DNA at the presence of PEG and CaCl₂, grown on hyperosmotic plates for 24 hours, and lots of colonies appeared, this result indicate that it is achievable to obtain lots of transformants with protoplast transformation.3.Phenotype and PCR screen of transformantsAfter grown to proper colony at 30℃, colonies were selected on MM and MD plates, the strains growth normally on MD while slowly on MM plates were primarily identified as MutS. The total DNA were extracted and amplified with primers 5' A0X1 and 3 'AOX1, and 21 strains with targeted band in PCR product were identified as Mut^S transformants.4. Comparation of enzymatic characteristics(1) The phytase expression of Mut⁺ recombiant increased and achived peak after 168 hours induction, and the corresponding enzyme activity reached peak value of 143958.3U/mL, while that of Mut^S recombinant achived peak value after 192 hours, and the enzyme activity reached peak value of 141058.3U/mL. SDS-PAGE shows that the molecular weights of both products were among 70kDa and 97kDa.(2) Measurement of optimum pH shows that both phytases have two optimum pHs, respectively 2.5～3.0 and 5.0～5.5, the peak value at pH2.5 is equal to that at 3.0, while the peak value at pH5.0 is equal to that at 5.5, both activities of the two phytases were high within the pH range of pH4.5～6.5 and decreased dramatically at pH7.0, and the functional pH ranges of the two phytases were both broaded significantly. Activity measurement at pH5.5 and different temperature shows that the optimum temperatures of the two phytases were both aroud 55℃, and both displayed high activity within the temperature range of 50℃～55℃, indicate that both of the functional temperature ranges were broaded. Measurement of thermotolerance shows that the thermotolerance of both phytase were higher than that of wild type.Togetherly, the phynotype of engineered yeast has no significant effect on enzymatic characteristic of the phytase it expressed.