不同碳源诱导绿色木霉产双功能酶的表达差异分析

Differential expression analysis of bifunctional cellulase-chitosanasefrom Trichoderma viride XW01 induced by different carbon source

采用酶活力分析、SDS-PAGE、RT-PCR等手段对不同碳源诱导Trichodrma viride XW01产双功能酶的差异表达进行分析。结果表明,酶的合成受培养基中碳源的影响。绿色木霉在不溶性多糖类独立碳源诱导条件下对双功能酶CCBE(66kDa)的合成量要优于可溶性碳源;其中羧甲基纤维素钠(CMC-Na)为碳源时发酵液中壳聚糖酶活力最高,发酵96d后活力达8.29U/mL;壳聚糖次之,麸皮再次之;3种碳源均直接诱导绿色木霉产壳聚糖酶,以CBHE为主;微晶纤维素不能直接诱导产生壳聚糖酶,而是通过其水解产物间接诱导绿色木霉产生壳聚糖酶,发酵液中CBHE所占比例较低,组分中壳聚糖酶CCBEⅡ(52kDa)和Csn(45kDa)含量较高;且绿色木霉两种酶活力的合成和表达调节并不是协同发生的。可溶性淀粉和乳糖为碳源时,绿色木霉生长迅速,但是基本上不产双功能酶。

Trichoderma viride XW01 was cultured with different carbon sources,and the chitosanase and cellulase activities in crude enzyme solution was determined regularly.The composition of crude enzyme solutions were analyzed by SDS-PAGE.According to mRNA analysis,the gene of bifunctional cellulase-chitosanase CBHE（66 kDa） were amplified by RT-PCR using the total RNA of T.viride XW01（which was cultured on induced medium） as template.The results showed that when T.viride XW01 was cultured on dissoluble carbon source（CMC-Na,chitosan,Avicel,Bran） for several days,the highest chitosanase and cellulase activities were achieved,by CMC-Na and Avicel,reaching 8.29 U/mL,respectively.The chitosanase activity of crude enzyme solution induced directly by CMC,chitosan and Bran mainly attributed to bifucntional CBHE（66 kDa）,while chitosanases from avicel was not induced directly by avicel,but by its hydrolyzing products,and the activity was mainly derived from other chitosanases（CCBEⅡ,52 kDa） and Csn（45 kDa）.The gene of CBHE were amplified stably by RT-PCR with RNA template.It showed that the synthesis and expression regulation of cellulase and chitosanase from T.viride XW01 were not coordinately regulated.