Production of recombinant phytase enzyme with alcohol dehydrogenase (ADH3) promoter in Pichia pastoris

Phyase which is used in paper industry, food industry, preparation of myo-inositol phosphates and especially in feed industry, is an important enzyme. Phytases are generally used in nutrition feed. Phytase separates phosphate from phytate, decreases the need of feed supplementation by phosphorus, increases the nutritional value of the feed and decreases the level of discarded phosphorus. This situation, causes to increase bioavailability and decrease environmental contamination. Phytases which are used in food and feed industry, are usually derived from bacteria and fungi. Microbial phytases are known thermostable in wide pH ranges. Recently, because of insufficient natural sources of enzymes, interest of recombinant production has increased. Pichia pastoris is a yeast which is used in commonly recombinant protein production. P. pastoris can use carbon source such as methanol, glucose, sorbitol, glycerol, mannitol, as well as ethanol. In this study, Escherichia coli phytase gene was produced under the control of alcohol dehydrogenase (ADH3) promoter in P. pastoris, which is a methylotrophic yeast. Recombinant phytase was produced to use by synthetic SNT5 and native ADH3 promoter (900 bp) and phytase yields which are produced by two promoters, were compared. The copy numbers of the obtained production strains were determined by Real Time Polymerase Chain Reaction (RT-PCR) analysis, were selected single copy clones and this clones were produced by feeding ethanol during 96 hours in shake flasks.Enzyme activities of synthetic ADH3 promoter and native ADH3 promoter are 12203 U/ml and 7613 U/ml, respectively. It is determined that the synthetic promoter shows activity approximately 60% higher than the natural promoter at the end of 96th hour. According to SDS-PAGE analysis, the molecular weight of the phytase was found approximately 55 kDa. The optimum conditions of the enzyme were determined pH of 4.0 and temperature of 60°C. The enzyme was stable above 90% at pH 2.5-6.0 during 2 hours. The phytase retained 87% and 49% activities at 60 and 70°C'de for 30 min, respectively. The phytase was activated by Ca2+ and Mg2+, but inhibited by Cu2+, Zn2+ and Fe2+. As a result of this study, P. pastoris is suitable expression system for recombinant high amount phytase production under the control of alcohol dehydrogenase (ADH3) promoter. The method used in the study has been exemplary for different promoter studies.