Functional Characterization Of Cellulase Enzymes In Cytophaga Hutchinsonii

With the development of the word economy,global resource crisis is being more and more serious.It is supposed that available petroleum will run out in the coming decades,and resource crisis has already been a general and urgent problem.Cellulose, the most abundant component of plant biomass,is not fully utilized.Therefore,efficient bioconversion of cellulose has attracted universal attention in recent years.Although cellulosic biomass has distinct advantages in costs and scale-up production,its hydrolysis is very difficult,especially for crystalline cellulose.The low efficiency of cellulase has been one of limiting factors responsible for high costs of cellulose hydrolysis.Cytophaga hutchinsonii use a novel strategy for cellulose utilization,so the research of its cellulolytic mechanism is meaningful to utilization of renewable resources.In this thesis,we studied catalytic characteristics of cellulase enzyme expressed in E.coli and binding characteristics of carbohydrate binding module (CBM).The major results of the thesis are as follows.1.Bioinformatic analysis of cellulolytic enzymes encoding genes in C. hutchinsonii genomeAll sequences related to cellulolytic enzymes were analyzed by various bioinformatic softwares.Results showed that C.hutchinsonii glycohydrolases that may be involved in cellulose digestion belong to glycohydrolase family 3,5 and 9.Meanwhile,there are only three kinds of carbohydrate binding modules(CBMs), CBM4,CBM6 and CBM9 in C.hutchinsonii genome.Surprisingly,none of proteins related to endo-1,4-glucanases contain CBMs domains and most of CBMs are found on glycohydrolases which may be involved in hemicellulose digestion.According to current research,only CBM9 could bind to crystalline cellulose,so we analyzed all CBM9 sequences detailedly.Our work revealed that there are 10 CBM9 in the genome of C.hutehinsonii,which could be divided into three classes named CBM9-A,CBM9-B and CBM9-C.2.Heterologous expression and primary characterization of cellulolytic enzymes of C.hutchinsoniiBecause cellulolytic enzymes' expression is difficult,we tried to use three expression systems,including E.coli,Pichia and Bacillus subtillis systems.Endo-1,4 - glucanases CHU₁280 was successfully expressed in E.coli system.The production of CHU 1280 was low and could not be purified,so we tested its catalytic characteristics by mixture.We found that CHU₁280 could hydrolyze CMC efficiently and degrade tetrasaccharides to disaccharides,pentasaccharides to disaccharides and trisaccharides, but it could not degrade disaccharides and trisaccharides.3.Characterization of binding properties of CBM9 in C.hutchinsoniiCBMs,as an important component in cellulose binding proteins,play a great role in cellulose degradation.Only two reports studied CBM9 binding characteristics.One was by biochemical methods,and the other was to analyze its crystal structure.In this thesis,we expressed CBM9 in E.coli.After purification,we studied its binding characteristics and found that it could bind to insoluble xylan and Avicel,and combining capacity to xylan was stronger.CBM9 could also bind to soluble xylan.Though CBM9 has three Ca²⁺ binding sites,Ca²⁺ concentration did not affect CBM9 to bind to sugars.Therefore,we speculated Ca²⁺ did not enjoy in combination, but played a role to stable protein structure.The three dimensional structure of CBM9 was constructed by homology modeling. From the final modeled structure,it is obvious that CBM9 in C.hutchinsonii could overlay with template well in sheet skeletons,but the loops in the active site were different with an additional Tyr112.Further site-directed mutagenesis revealed that simultaneous mutations of Tyr75 and Tyr112 resulted in a decrease in the binding capacity.We presumed that aromatic tings of two aromatic amino residues in CBM9 active site did not perfectly parallel the sugar ring which weakens the hydrophobic interaction between CBM and the sugar chain while Tyr75 and Tyr112 coordinate to ensure the efficient binding.