【作者】 刘萍；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学， 2009， 博士

【摘要】 纤维素酶是公认的一种具有非专一性水解壳聚糖的代表性酶类。本实验室已从绿色木霉来源的商品纤维素酶制剂中分离纯化出一种具有水解纤维素和壳聚糖活性双功能酶。为了进一步确证纤维素酶中该双功能酶的存在,本研究采用分子生物学手段,对产纤维素酶的绿色木霉菌株的双功能酶基因进行克隆和体外重组表达,并探讨该双功能酶的基因结构与双功能性质的关系。本研究对于进一步了解双功能酶结构与功能的关系具有重要的学术意义,同时对于提高该双功能酶的活力也具有重要的应用价值。研究了绿色木霉菌株(Trichoderma viride)在羧甲基纤维素钠(CMC)诱导下产高活性酶的发酵条件;采用柱层析系统对发酵液中的双功能酶组分进行分离纯化,从酶学性质和酶结构方面与本实验室前期从商品纤维素酶中分离出的双功能酶进行了对照比较;结果表明:在CMC诱导培养4天时发酵液中壳聚糖酶活力和纤维素酶活力同时达到最大值;所纯化的双功能酶与从商品酶中纯化的双功能酶为同一蛋白。该双功能酶的Maldi-Tof-Tof Mass序列测定结果表明:该双功能酶的两个主要肽段的序列与GenBank里糖苷水解酶7族(GH-7)的绿色木霉产CBHⅠ(gi|295937)的氨基酸序列具有很高的同源性,推测该CCBE可能属于GH-7族。以T.viride RNA为模板,采用RT-PCR、SMART-RACE方法克隆了双功能酶CCBE的全长cDNA序列,并进行毕赤酵母表达,结果显示:CCBE的全长cDNA序列与五个在GenBank中登录的木霉属GH-7族的CBHⅠ基因(Trichoderma.sp)具有88-100%的同源性,但与已知的壳聚糖酶和几丁质酶基因却无任何序列相似性,而重组表达产物具有强的壳聚糖酶活力,证实了绿色木霉所产CCBE属于GH-7族,两活力产自同一基因,其mRNA可能存在选择性剪接现象。采用RT-PCR法测定了不同诱导时间绿色木霉CCBE的mRNA表达水平,同时研究了不同内含子保留的剪接子基因在重组毕赤酵母中的转录表达情况,结果表明:绿色木霉CCBE的mRNA存在依赖于诱导时间的选择性剪接现象,四种剪接基因型DC(保留有两内含子)、CCBEIN1(内含子1保留)、CCBEIN2(内含子2保留)和CC(不含内含子)的出现以及所占比例随诱导时间而变化。各剪接子在毕赤酵母体内的转录产物相同,均不含内含子;四个重组表达产物的分子量大小相同,均具有强的壳聚糖酶活力,但纤维素酶活力却截然不同:只有CCBEIN1和CC具有CMCase活力,且前者活力大小约为后者的一半,由此推测内含子的保留尤其是内含子2的保留可能通过改变各表达产物的蛋白构象从而影响其活力表达。对CCBE不同内含子保留的剪接基因的毕赤酵母重组表达产物水解壳聚糖和CMC的酶学性质、水解产物进行研究,并采用删除突变对双功能酶不含内含子的基因型进行C-末端删除分析,探讨了基因结构与双功能性质的关系;结果表明:不同内含子保留的双功能酶基因的体外重组酶作用两底物的性质相近但并不完全一致,作用壳聚糖的最适温度和最适pH分别集中于55-60℃和5.0-5.2;而作用CMC则为55-60℃和4.2,水解方式均为外切;与从绿色木霉中纯化的CCBE基本保持一致,其中重组酶CC的性质与纯化CCBE的一致性最高。内含子1的保留通过改变重组酶部分CMC催化结构域的正确折叠而抑制其CMC降解活力,但对其底物结合位点无影响;而对于壳聚糖酶活力,其使酶与壳聚糖结合能力下降,但对其催化域的构象影响不大;内含子2的保留改变了纤维素结合结构域的正确构象,从而完全失去CMCase活力,但不影响重组酶壳聚糖酶活力的催化结构域和底物结合结构域,而是使其催化域附近的底物进入通道变小,从而使得大分子难以进入。由此证实了: CCBE作用壳聚糖的催化结构域和底物结合位点均位于CCBE的N-端及序列中上游位置,与作用CMC的截然不同,后者则位于C-端及序列中下游部位。本论文研究验证了绿色木霉在纤维素诱导条件下会产生具有壳聚糖和纤维素降解活性的双功能酶,采用分子生物学手段确证了纤维素酶中双功能酶的存在;同时发现了绿色木霉所产的双功能酶基因具有内含子保留的选择性剪接现象,探讨了双功能酶不同内含子保留的基因结构与双功能性质的关系。更多还原

【Abstract】 Cellulase is recognized as a typical non-specific chitosan-degrading enzyme.In our previous study, a bifunctional enzyme with chitosanolytic and cellulolytic activity (CCBE) has been purified from the commercial cellulase produced by Trichoderma viride. in order to confirm the existence of CCBE in cellulase, in this study, the gene of CCBE from cellulase hyper-producing T.viride were cloned and expressed in vitro using the molecular biological methods, and the relationship between the gene structure and bifunctional properties of CCBEwas also discussed, which is of important academic value in elucidating the relationship between structure and function of CCBE and of practical significance in improving the activity of this enzyme.A cellulase-producing T.viride strain was chosen to determine the production condition of high active enzyme induced by CMC, showing that both of the chitosanase and cellulase activities approached maximum after induction for 4 days. The bifunctional enzyme in the culture was then purified and identified as identical to that from commercial cellulase, convincing that CCBE could be produced by T.viride with induction of CMC.Maldi-Tof-Tof Mass analysis showed that two major peptide peaks of apparent mass 1764.000 and 1932.7490 of CCBE digested by trypsin was found high homology with cellobiohydrolaseⅠ(CBHⅠ) gi|295937from T.viride . Accordingly, CCBE was presumed as an enzyme belonging to GH-7.Taking the total RNA of T.viride as template, the full-length cDNA sequence of CCBE was cloned by RT-PCR and SMART-RACE, and then expressed in Pichia patoris. Sequence analysis indicated that the full- length cDNA sequence of CCBE had 88-100% identity with the four CBHⅠgenes (Trichoderma. sp), while no similarity with chitosanase or chitinase genes reported in GenBank, which confirmed that the CCBE from T.viride belonged to GH-7,and its mRNA probably underwent alternative splicing .The differential expression level of CCBE mRNA was tested during different inducing periods of T.viride, indicating that mRNA of CCBE underwent a unique alternative splicing which is dependent on the inducing time. The appearance and proportion of four splicers varied with the inducing time increasing. The four splicers were assembled artificially by GDS technique and then expressed in Pichia patoris. The transcripts of four different transformants showed sequence-identical and had no introns retaining, which means that the amino acid sequence and molecular weights of the four recombinant enzymes were identical. Activity analysis showed that all of the splicers had comparable strong chitosanase activity, while their cellulolytic activity varied. CCBEIN1 had moderate CMCase activity compared to its chitosanase activity, and the dual activities of CC were comparative, however, CCBEIN2 and DC almost lost their CMCase activity, indicating the retained introns, especially intron 2 changed the conformation of their recombinant enzymes to make them lost their CMCase activity.The characteristics and hydrolysis products of four recombinant enzymes toward chitosan and CMC were similar but not completely uniform. The optimum temperature and pH of four enzymes toward chitosan were in a range of 55-60℃and pH 5.0-5.2, respectively, while toward CMC, both CCBEIN1 and CC exhibited optimal condtions among 55-60℃and at pH4.2; among these, the recombinant CC showed highest consistent with the purified CCBE.The retained intron 1 in genes partially changed the folding of the catalytic domain of the recombinant enzymes on CMC,but had no effects on their CMC binding domain;while chitosan binding capacity lowered when intron 1 retained and its catalytic domain for chitosan hydrolysis varied not significantly.However, genes with intron 2 made its expressing products show narrower loop channels, while their CBD conformation also changed to lose the binding capacity of CMC, resulting in the lost of CMCase activity of CCBEIN2 and DC.From the above, it is confirmed that the catalytic and substrate-binding domains of CCBE on chitosan hydrolysis located in the upper-middle reaches of N-terminal, which is different with those of CMC, locating in the lower reaches.In this study, it is convinced that bifunctional enzyme with chitosan- and CMC- degrading activity could be produced by T.viride with the induction of CMC.The results confirmed the existence of CCBE from cellulase and elucidated the relationship between the gene structure and bifunctional properties of CCBE.更多还原