Study On Structure And Function Of Bifunctional Cellulase-Chitosanase From Trichoderma Viride

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.