丝状真菌瑞氏木霉内切葡聚糖酶的定向进化

EG III gene was cloned from the T. reesei cDNA bank by PCR and expressed in S. cerevisiae. The results indicated that the amount of EG III secreted by the strain with SSO2-overexpression was highest among the different recombinant S. cerevisiae strains, showed that SSO2 is a rate-limiting component of the secretory machinery in the process of EG III secretion. The EG III expression level was increased 5.3 times by deletion of the 98 bp in 5' untranslated region (UTR) of eg3 mRNA sequence. This result suggested that the regulation region could exist in the 5' untranslated region of EG III mRNA. In addition, it was observed that deletion of the 3'-UTR of EG I cDNA resulted in no active EG I produced by recombinant yeast. .The technique of double-layered plate was developed for screening the mutant library of endoglucanase III from T. reesei generated by the methods of directed evolution. Error-prone PCR was used to randomly mutate a mesophilic cellulase, EG III from T. reesei, obtaining a cold adaptive mutant, w-3. DNA sequence analysis indicated that w-3 was a truncated form of native EG III by the deletion of 25 consecutive amino acids at C terminus of protein. The activation energy of w-3 and its parent calculated from Arrhenius equation is 13.3kJomol-1 and 26.2kJomol-1, respectively. The increased specific activity of w-3 was most likely resulted from increased Kcat value and decreased activation energy.. The optimal pH of the enzyme from mutant MA11-2 changed from 4.8 to 5.4. The activity decresed in the acidic range, and increased in the alkaline range. By comparing the DNA sequence with the parent gene, it was found that 321Asn was replaced by Thr. Some alkali-tolerentβ-1,4-glycanase-producing strains were isolated, and one xylanase gene was cloned. By random and site-directed mutagenesis, mechanism of alkali-tolerance was studied.

采用低突变频率易错PCR随机诱变丝状真菌瑞氏木霉内切葡聚糖酶基因，进一步用高保真度DNA改组技术重组由随机突变产生的有利突变，筛选耐碱酶突变体，对酶进行体外定向进化。在此基础上研究酶的耐碱机理，进行结构与功能研究，同时构建过量生产内切葡聚糖酶的瑞氏木霉工程菌，为改造工业用酶提供新的实验方法和产品，具有理论和实际应用双重重要意义。