以增加催化结构域的基因重构方法提高里氏木霉外切型葡聚糖酶Ⅱ的活性

分别通过增加CBHⅡ和EGⅣ催化结构域的方式对里氏木霉QM9414外切型葡聚糖酶Ⅱ进行基因重构。第一种重构方式是在CBHⅡ基因的上游增加EGⅣ的催化结构域基因，第二种重构方式是在CBHⅡ基因的下游增加其自身的催化结构域基因。通过PCR和基因重构手段获得重组质粒pPICZαA -cdE –cbh2和pPICZαA- cbh2-cdC，并在毕赤酵母GS115中表达，获得重组菌株P.pastoris PEC11和P.pastoris PCC16，在经改良的摇瓶培养条件下能表达具有较高CMCNa酶活性的融合蛋白，培养液的CMC活性分别达到3.87U/ml和7.66U/ml，其中P.pastoris PCC16表达的重构酶活性是毕赤酵母表达的单一CBHⅡ酶活性的2倍。表明采用增加结构域的方式可以有效提高纤维素酶的活性。

Enhancements of the Cellulolytic Activity of Trichoderma reesei Cellobiohydrolase Ⅱ through Adding Catalytic Domain

The gene structure of the T.reesei cellobiohydrolase Ⅱ(cbh2) was reconstructed by fusing with additional genes which encode catalytic domains from EGⅣ(cdE ) and CBHⅡ(cdC ), respectively. The target genes were obtained through PCR and the gene cdE was ligated on the 5’ end of cbh2 while cdC was ligated on the 3’ end through gene fusion, generating reconstructed genes cdE –cbh2 and cbh2-cdC, respectively. The reconstructed genes were expressed in Pichia GS115 and results the recombinant strains P.pastoris PEC11 and P.pastoris PCC16 with the most efficient activities. When cultivated in the optimized incubation condition, the CMC activities of the cultivation supernatant of P.pastoris PEC11 and P.pastoris PCC16 attained 3.87U/ml and 7.66U/ml, respectively. The modified CBHⅡwith an additional catalytic domain from itself improved the CMC activity about 2-fold.