Study Of Molecular Directional Evolution Of Endoglucanase Of Trichoderma. Spp.

Cellulose is the most important carbon resourse in the world, accounting for half of the plant dry mass ,which is produced by photosynthesis. Due to the lack of an effective means to deal with cellulose, its resources become a tremendous waste. Through the complex cellulase digestion, cellulose can be effectively degradated and become some useful produce, such as glucose and ethanol. Endoglucanase is the important components of the cellulase complex, which can degradate cellulose into easy handling and small molecule of the non-reduction end of cellulose, and occupys an important position in cellulose-energy projects. To find effective conversion of cellulose ways, to effectively use a lot of waste of cellulose resources, and to effectively degradate cellulose into energy, it is the purpose of this experiment. The cellulose endonuclease research has great practical and application background for effective useing of cellulose, solving the cellulose pollution and waste, and suppling bioenergy resourse.DNA shuffing gradually become mature and improved, since its adventure in 1994. There were many successful examples to use DNA shuffling to improve cellulase in foreign countries. Matsumura I attainedβ-glucuronide mutant with high patience to PFA and formalin treatment, after three cycles of mutation, DNA shuffling and screening, in 1999; Furukawa K established a mutation treasury integrated to bacteria surface display system by DNA shuffling, which was used to screen clone with CMC activity, in 2000; Kumamaru T gained new gene with catalic and degradational ability toβ-glucuronide increased by 500 times, after three DNA shuffling cycles of E.coliβ-galactosidase, in 2001. As the endoglucanase studied deptly and chimeric diversity library of bacteria, bioinformatics, computer-aided drug design technologies applied, it was believed that theoretical research of cellulose-based would play a more significant role in the national energy and environmental pollution's treatment.Plasmid-pASKint100 is a procaryon display system, which utilize the integration of outer membrane protein intimin and foreign protein to display on the surface of bacteria, and E. coli K12 is its host. Plasmid was a gift of Professor Andreas Christmann in Germany. A.Christmann did a lot of tests and have successfully integrated foreign protein into plasmid, which was detected by sensitive fluorescent immune detection technology. The use of procaryon display system to display peptides and enzymes has a large number of application examples (see Summary), and it is very mature, high yield, low cost and strong renewability. and it avoids the enzyme release process of rupture from membrane, reduce the loss of enzyme, and has more efficient catalysis, when comparing with enzyme of intracellular expressing. Plasmid' relevant nature would be seen in Fig1.Currently most of microbe used in the production of cellulose are fungi, and more research fungi are Trichoderma, Aspergillus, and Rhizopus. The filamentous fungus-Trichoderma (Trichoderma.sp) is recognized as the highest One of cellulose production. Trichoderma reesei and Trichoderma koningii were used as producing bacterium of endoglucanase in this study.In this trial, First, homologous sequences comparement, anagenesis study, the secondary and tertiary molecular structure analysis, and other bioinformation technology is used to analyze the restructuring EG sequences. It explores the endoglucanase's active site and structure domain from another angle, and has a deeper understanding of endonuclease to find endonuclease availability. According to bioinformatic analysis, in vitro molecular evolution means-DNA shuffling is used to transform the endonuclease gene, to find the ideal of cellulose mutation through a large amount of beneficial mutations chimeras and efficient means of screening, hope to gain improvement in the activity and stability of enzyme. And with the help of bacteria display system, enzyme could display on the surface of bacteria.Main methods used in this study are: the utilization of Clustalx and other bioinformatics software to analyze EG sequence's biological information, the classic DNA shuffling, the staggered extension shuffling, RT-PCR, and so on. Major findings are: When two Trichoderma's endoglucanases catalyze and degradate cellulose, the critical amino acids are two Glu; Some aromatic amino acids play an important role in combination with cellulose; A number of amino acids' amides play an important role in the electron transfer of the endoglucanases' degradating cellulose; Two Trichoderma's endoglucanases both have a special three-dimensional catalytic domain and binding domain, and they both have a signal peptide;the 28S and 18S straps of Trichoderma's RNA prepared are clear, and RNAs' OD260/280 are about 2.0, which can be used for downstream operations; the T.r and T.k PCR purification produce attained by anti-retroviral are sequenced as EG gene; the pASKint100 plasmid transforms into E.coli and 2ug products gains after double digestion and phosphorylation. Two DNA shuffling are applied to reorganized EG, there is a clear band at the EG size of fragment (about 800 bp) in electropherogram; after restructuring fragment transforming into E.coli, plasmid is extracted, then Nae1 identification is done, and last, the heterogenesis fragment and plasmid pASKint100's connection is confirmed.Finally, conclusions are made as follow by refering and analyzing. Bioinformatic analysis was an important guide and assist to experiment; EG family has a similar three-dimensional structure and catalytic mechanism;After primers are designed with bioinformations, a amount of products are obtained by retrotranscription and they are sequenced as endoglucanase sequence, which confirms the importance of combining between the theory of bioinformatic analsis and experimental work; After two endoglucanase sequences are treated by DNA shuffling, they are restructured, which confirms that DNA shuffling technology can change the sequence of a fragment; After EG fragments conjuncting with the prokaryon display system-pASKint100, they do not show significant biological activity in E. coli, which may result from the limitation of the prokaryon display system in the correct fold of displayed EG.