CAJ | 학술논문

通过RT-PCR从绿色木霉AS3.3711中克隆得到EG Ⅲ基因,序列分析显示该基因编码序列全长为1 257 bp,编码418个aa,且自身带有21个aa的信号肽序列,与里氏木霉eg3的同源性为99.6%.将该基因连入酿酒酵母多拷贝整合型表达载体pScIKP中获得重组质粒,线性化后电转化酿酒酵母工业菌株AS2.489,通过SDS-PAGE蛋白电泳、刚果红染色和酶活测定对转化子进行了分析.结果表明:转化子有明显的重组EG Ⅲ表达带,能够产生CMC水解圈,说明eg3基因已在酿酒酵母中获得了正确的分泌表达,表达EG Ⅲ的重组菌产酶高峰出现时间为60 h,最高酶活接近120 U/mL,重组酶EG Ⅲ的最适温度为60 ℃,最适pH为6.0,转化子的遗传稳定性达到99.17%,实现了绿色木霉eg3基因在工业酿酒酵母中的稳定高效表达.
통과RT-PCR종록색목매AS3.3711중극륭득도EG Ⅲ기인,서렬분석현시해기인편마서렬전장위1 257 bp,편마418개aa,차자신대유21개aa적신호태서렬,여리씨목매eg3적동원성위99.6%.장해기인련입양주효모다고패정합형표체재체pScIKP중획득중조질립,선성화후전전화양주효모공업균주AS2.489,통과SDS-PAGE단백전영、강과홍염색화매활측정대전화자진행료분석.결과표명:전화자유명현적중조EG Ⅲ표체대,능구산생CMC수해권,설명eg3기인이재양주효모중획득료정학적분비표체,표체EG Ⅲ적중조균산매고봉출현시간위60 h,최고매활접근120 U/mL,중조매EG Ⅲ적최괄온도위60 ℃,최괄pH위6.0,전화자적유전은정성체도99.17%,실현료록색목매eg3기인재공업양주효모중적은정고효표체.
The endo-p-glucanase Ⅲ gene (eg3) was obtained from the total RNA of Trichoderma viride AS3.3711 by RT-PCR. Sequence analysis indicated that this egi gene was composed of 1 257 nucleo-tides, coding for 418 amino acid residues, and had its own signal peptide. The sequence homologies between this gene with Trichoderma Reesei egi were 99.6%. Then it was cloned into the high-copy integra-tive expression vector pScIKP, generating a recombinant plasmid named pScIKP-eg3, which was then transformed by electroporation into Saccharomyces cerevisiae AS2.489 after linearization. Transformants were detected by SDS-PAGE analysis, Congo Red method and enzyme activity assay. The results showed that transformants could secrete recombinant EG Ⅲ and produced hydrolysis halos on the Congo-Red-CMC plate, indicating that this eg3 gene was correctly expressed in S. cerevisiae. The highest enzyme activity of recombinant EG Ⅲ reached 120 U/mL after 60 h cultivation, and the optimum temperature and pH were 60 ℃ and 6.0 respectively. The genetic stability of transformants achieved 99.17% after 50 generations in nonselective medium. In conclusion, the eg3 gene of T. viride could be expressed in industrial yeast strain stably and efficiently.