Abstract:[Objective] In order to improve the production of proteinase K and setup purification method. [Methods] We first optimized the codons of proteinase K gene and transfered it into Pichia pastoris (GS115) to realize secretory expression. And culture conditions including methanol concentration, temperature and pH were investigated. The purification methods, such as ammonium sulfate precipitation, affinity chromatography, were further optimized. [Results] Through the optimization of codons of proteinase K gene and cultural conditions, we obtained high-level expression of proteinase K. The results showed optimal fermentation condition was supplement of methanol 0.75%, fermentation temperature 25 °C and pH 7.0. The yield of proteinase K was 2.2 g per liter under the optimized fermentation condition. The efficient purification way is Ni-NTA affinity chromatograph after comparing with the other methods. [Conclusion] The results showed that proteinase K can be expressed at high level expression in P. pastoris and can be efficient purified with Ni-NTA affinity chromatograph.
