肺炎克雷伯氏菌漆酶基因的克隆及其在毕赤酵母中的高效表达

Cloning of the Laccase Gene from Klebsiella pneumoniae and its Efficient Expression in Pichia pastoris

以肺炎克雷伯氏菌(Klebsiellapneumoniae)基因组为模板,通过PCR扩增得到其漆酶基因lac;基于毕赤酵母密码子偏爱性优化后,得到新型漆酶基因lacm;将其与大肠杆菌–毕赤酵母(E. coli-P. pastoris)穿梭表达载体pPIC9K连接,构建重组质粒pPIC9K-lacm;将该质粒转化毕赤酵母(Pichiapastoris)GS115中,实现该漆酶的胞外分泌表达,发酵液中重组漆酶(rLACM)活力达0.37 U/m L.经对rLACM酶学性质分析表明:rLACM的最适温度为70℃,最适pH为8.0,在30～70℃、pH 5.0～9.0活力稳定.

The gene of lac was obtained from the genomic DNA of Klebsiella pneumoniae through PCR amplification.A novel laccase gene lacm was obtained based on the yeast codon bias optimization.It was inserted into an E.coli-P.pastoris shuttle vector pPIC9K to construct recombinant plasmid pPIC9K-lacm.The recombinant plasmid was then transformed into Pichia pastoris GS115.The recombinant laccase was successfully expressed in P.pastoris and secreted into the culture medium.In optimized fermentation conditions,the activity of the recombinant laccase(rLACM)in fermentation broth was up to 0.37 U/mL.The optimal enzyme activity of rLACM was observed at pH 8.0 and 70℃temperature.Stability studies showed that rLACM was stable when temperature was 30-70℃and pH 5.0-9.0.