Diversity And Catalytic Mechanism Of CBHs From Pencillium Decumbens

Due to their application potential in second generation bioethanol production, starting from lignocellulosic raw materials, cellulase is currently in the centre of attention. Cellulase has very high biotechnological importance in the lignocellulose bioconversion to liquid fuels and other useful products. Especially, cellobiohydrolases are key components in the multienzyme cellulase complexes, which are responsible for deep conversion of cellulose to soluble sugars. In the culture media of typical cellulolytic fungi, CBHs are the most abundant among the secreted proteins, often accounting～60%w/w. The bottlenecks in the bio-conversion are the low efficiency and high cost of cellulases to hydrolyze the lignocellulose, which could not meet the needs of industrial biomass utilization. The CBHIs with higher specific activities and higher stability are needed. These studies could provide a good theoretical basis for CBH modification.To investigate the cellobiohydrolases and their reaction mechanism, this paper systematically studies the CBHs diversity, focusing on function diversity, characterisitics diversity, mechanism diverisity and modules diversity.1. A non-hydrolytic CBHI-A, as a synergism factor, purified from Penicillium decumbensA synergism factor in cellulose degradation was purified from P. decumbens. The protein was identified as exoglucanase (CBH)1and designated as CBHI-A. The protein exhibited no enzymatic activity against CMC,pNPC, and salicin. The hydrolysis of lignocellulose and filter paper by CBHI-A alone produced no cellobiose and reducing sugar, but CBHI-A has strong synergism with different commercial cellulase preperations. In the supplementation experiment, although the dosage of CBHI-A was small, the glucose yield released from the pretreated corn stover (water washed) increased10%to40%. Surprisingly, the addition of CBHI-A to the NS50013sample (incomplete cellulase system) from Novozymes increased glucose yield by approximately60%, with pretreated corn stover as the substrate. HBI and CrI of the cotton fibers treated with CBHI-A decreased by19%and34%.The diameters of the cotton fibers treated with CBHI-A for seven days was96nm, approximately7%bigger than that of the cotton fibers before treatment. The results showed that CBHI-A could disrupt the hydrogen bonds of cellulose and make the high-crystalline cellulose more amorphous.2. The effects of N-glycosylation, together with its site and chain length, on the characteristics diversity of CBHIFour cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had same amino acid sequence but display different characteristics in molecular mass, specific activity and enzymatic characterisitics. CBHI diversity was investgated at gene and protein levels. The method of N-glycan structure analysis has been set up by peptide mass spectrum data. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data of different glycoforms of CBHI. The result implies that longer N-glycan chains at the Asn-137of CBHI increased cellobiohydrolase activity. Secondly, the N-glycan sites were site-directed mutanted and homologously expressed in P. decumbens. Only the N-glycosylation at Asn-137can improve cellobiohydrolase activity by40%, whereas rCBHI with N-glycosylation only at Asn-470exhibited no enzymatic activity. Thirdly, the deglycosylated CBHI was unstable and the half-lives of deglycosylated CBHI decreased to approximately40min. Whether or not the protein is glycosylated, together with N-glycosylation site and N-glycan structure, affects CBH activity. N-glycosylation not only influences CBH activity but may also brings a new feature to a nonhydrolytic CBHI glycoform (CBHI-A).3. Analysis of CBHI catalytic phenomenon diversity in the hydrolysis of the crystalline cellulose, armphous cellulose and cellulose derivativeTo investigate the mechanism of CBHI, the tryptophan residue at four glycosyl binding sites was successfully replaced by alanine in order to evaluate real mechanism in catalytic reaction. Firstly, the hydrolysis activity of all mutated CBHI significantly reduced against Avicle and PASC. It impled that the four glycosyl binding sites were important for correct position of glycan chain in the catalytic tunnel of CBHI. Trp398makes stracking interactions in the-2site that are believed to be important for the distortion of the substrate in the-1site during catalysis. Cleavage of β-1,4linkage occurs at the-2and-1sites. The mutant CBHI-W398A could not help substrate distort to-1site (W407). Therefore, the mutant CBHI-W398A could not have cleavage of β-1,4linkages in cellulose glucan. The mutant CBHI-W398A has no hydrolysis activity towards Avicel and PASC. Secondly, the result of hydrolysis of cellulose by CBHI was different with that ofpNPC by CBHI. Because the tytophan in the tunnel, using the binding force between tytophan and pNP, tract pNPC to enter into tunnel of CBHI. Even if CBHI had the mutant at W398, pNPC with the binding force between pNPC and W407was still able to reach the catalytic sites in the tunnel. In conclusion, the results provide strong evidence for the conclusion that there is no direct relationship between cellulase activities on soluble substrate and insoluble substrate. The results indicated that the activity on pNPC could not represent that on cellulose substrates. Our experiment also provide inspiration that cellulase mutant must be selected on the natural lignocellulose, while the screening of cellulase may deviate from correct direction on lignocellulose analogues and derivatives. This implied that the substrate analogue should not be used for classification and detection of enzymatic activity, which explained why some CBHIs had high activity against pNPC and low hydrolysis towards lignocellulose.4. The characteristics analysis and application of a novel Cellobiohydrolase Ⅱ (PdCel6A) purified from Penicillium decumbensAn acidic Cel6A, cellobiohydrolase (CBH) Ⅱ, was purified from P. decumbens and was designated as PdCel6A. The deduced internal amino acid sequence of the novel CBH showed a high identity with CBH Ⅱ from Aspergillus fumigatus. Surprisingly, PdCel6A exhibited characteristics comparable to that of CBH Ⅰ, as well as CBH Ⅱ. Similar to CBH Ⅰ, the novel CBH had a specific activity of1.9IU/mg against p-nitrophenyl-β-D-cellobioside. This is thus far the first report concerning CBH I-like CBHII with high activity against pNPC. The enzyme retained about80%of its maximum activity after6h of incubation at pH2.0. Considering these properties and using delignified corncob residue as a substrate, ethanol concentration increased by20%in the simultaneous saccharification and fermentation process when supplemented with low doses of PdCe16A (0.2mg/g substrate). To our knowledge, this is the first report involving a CBH I-like CBH Ⅱ. This provides new insight into the role of CBH Ⅱ in cellulose degradation.5. The potential role in catalysis of linker of PdCel6A uncovered by changing its length and flexibilityThe cellulose-binding module and catalytic domain of PdCel6A showed a high degree of sequence similarity with other fungal Cel6As. However, PdCel6A has11more amino acids in the linker region than other Cel6As. To evaluate the relationship between the longer linker and its catalytic activity, linker lenghth and linker flexibility were respectively investgated. Fistly,11amino acids were deleted from the linker region of PdCel6A. The shortened linker of PdCel6A resulted in the disappearance of the enzymatic activity against pNPC, while the rate of crystalline cellulose degradation was dramatically increased. Secondly, six amino acids in the linker of PdCel6A have been site-mutanted to six prolines. With disappearance of PdCel6A flexibility, PdCel6A lose activity against pNPC and Avicel. The studies implied the flexibility of linker has the potential role in catalytic of PdCel6A in addation to the connection between CBM and CD. Our data provide direct evidence that a longer linker effects on the cellulolytic activity of PdCel6A dramatically.