平菇漆酶基因在毕赤酵母中的分泌表达及酶学性质研究

采用RTPCR技术克隆到一个平菇(Pleurotusostreatus)漆酶基因的全长cDNA,命名为lccPo1,其序列提交GenBank,登录号为AY450404。将其ORF克隆到毕赤酵母表达载体pHBM906,转化3株毕赤酵母GS115、KM71和SMD1168,该漆酶基因在3种毕赤酵母菌株中均实现了分泌表达。3种摇瓶培养条件①25℃,1.0%(VV)甲醇;②20℃,1.0%(VV)甲醇;③20℃,0.5%(VV)甲醇,进行比较研究后发现适当提高甲醇浓度有利于漆酶在低温条件下表达,而降低培养温度到20℃则可以提高漆酶的产量2～6倍。3株重组毕赤酵母在其最适反应条件下测得三者粗酶液最高漆酶酶活分别为3.19UmLGS115(pHBM565)]、2.56UmLKM71(pHBM565)]和2.49UmLSMD1168(pHBM565)]。对重组酶进行相关的酶学性质分析表明,三者的最适反应pH值约为4.2,最适反应温度约为60℃。重组毕赤酵母GS115(pHBM565)所产酶的热稳定性稍好,在pH稳定性方面三者没有太大差异。

Expression of a laccase gene from Pleurotus ostreatus in Pichia pastoris and characterization of the recombinant enzyme

A Pleuotus ostreatus laccase gene was cloned by RT-PCR and designated as lccPol. Its sequence was submitted to GenBank with the accession number AY450404 obtained. The open reading frame was transformed into three Pichia pastoris strains GS115, KM71 and SMD1168, respectively, under control of the AOX1 promoter by using the vector pHBM906. LCCPo1 can be expressed by all three P. pastoris recombinant strains. Three different strategies for shake-flask cultures were compared: (1) (25 degrees C, 1.0% methanol), (2) (20 degrees C, 1.0% methanol), (3) (20 degrees C, 0.5% methanol). The laccase activity could be improved by increasing the methanol concentration befittingly. The results showed that the cultivation temperature had a marked effect on the production of active heterologous laccase. 2 - 6 folds higher laccase activities were obtained when the cultivation temperature was kept at 20 degrees C instead of 25 degrees C. The highest activities, 3.19U/mL GS115 (pHBM565)], 2.56U/mL KM71 (pHBM565)], and 2.49U/mL SMD1168 (pHBM565)], were gotten when the induction were performed at 20 degrees C with 1.0% (V/V) methanol supplied. The temperature and pH optimum for the recombinant laccase produced by three strains were 60 degrees C and pH4.2, respectively.