The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 °C and 37 °C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.

编码啤酒酵母Kex2蛋白酶衍生物Kex2-667的基因（编码Kex2蛋白酶的N端第20至667个氨基酸残基，包含前肽，催化结构域，P结构域和Ser / Thr富集区）及其第225个氨基酸残基突变体K225L在毕赤酵母中过表达。通过透析和阴离子交换色谱法（Q-FF）纯化蛋白酶。他们的性质进一步研究。对于催化效率，Kex2-667-K225L的Kcat / Km值比Kex2-667高3倍。在pH5.6孵育8小时后，两者在25°C和37°C时都非常稳定，而Kex2-667仍占总活性的近90％，而Kex2-667-K225L仅占80％。从pH4.0到pH9.0，Kex2-667-K225L的稳定性低于Kex2-667。由于突变位点K225位于钙离子结合位点之一，因此导致了更紧密的钙离子结合区，这可能是Kex2-667-K225L的催化效率提高而稳定性稍差的原因减少了。