Modification And Expression Of Endoglucanase Gene From Rhizopus Stolonifer Var.Reflexus

Endoglucanase is one component of the cellulase, mainly acts on the amorphous regions of cellulose chains. It catalyze the cellulose to yield short chains of oligosaccharides of unequal length by facilitating the random cleavage of the internal β-1,4-D-glycosidic linkages, and plays a crucial role in the process of cellulose degradation. Endoglucanase can be widely applied to industrial production of producing fuel ethanol and pulp treatment, and has a good application prospect. At present, how to improve the yield and the activeity of endoglucanase has become a hot research topic.The Rhizopus stolonifer var. reflexus TP-02mentioned in this paper is a filamentous fungal strain separated from ecological forests in Mount Huangshan. In our previous studies, the strain was found to produce high levels of cellulase activity. Here we reported the isolation of a novel endoglucanase gene eg2from Rhizopus stolonifer TP-02and its modification, the structural and functional relationships were also studied, and then engineering bacteria was successfully constructed in order to improve the enzyme activity. Main research results are as follows:1) Successfully completed the synthesis of Rhizopus stolonifer cDNA first line, and a novel endoglucanase gene eg2was cloned from it, the GenBank accession number of eg2is JX315341. The gene was connected to pET28a vector and transformed into Escherichia coli BL21, and the positive clones were screened by CMC-Na selective medium. Fermentation experiments of the recombinant showed the activity of endoglucanase peaked at0.723IU/mL after21h fermentation under the induction of IPTG2) The structure and function of eg2was predicted and analyzed by bioinformatic softwares. Sequence analysis of the gene revealed an open reading frame of954bp, which encoded317amino acids. The gene product endoglucanase II (abbreviated as EG II) contains a typical cellulose binding domain (CBM1) and two catalytic domain (GH45feature sequences), belonging to Glycoside Hydrolase Family45of the cellulose superfamily. A putative signal peptide of23amino acids was predicted in the N-terminal region of EG II, which was in theory for soluble good globulin.3) The sequences of CBM1and GH45were compared and analyzed, and their structural models were simulated respectively. The modification sites were determinated as follows:N39S, V136D, T251G, D255Q P256S and E260D. Focus on the catalytic mechanism of EG II, a preliminary discussion on the relationship of structure and function of the catalytic region GH45was completed. The binding mode of substrate and GH45region before and after mutation was studied by Discovery Studio programe through simulated mutation and molecular docking. Results showed that catalytic center has a large groove on the surface to allow substrate into and reaction with polar groups. Nevertheless, residues participated in the reaction are too much and are unevenly distributed, which may lead to an inhomogenous force arid thus may not accurately pulled the glycosidic bonds. After mutagenesis, a special tunnel was formed with the opening of mutation loops that make sure the substrate could quickly enter into the catalytic center accurately. The polar groups inside the tunnel acted on substrate and degraded it to release oligosaccharides, which may avoid substrate accumulation.4) Mutation groups were designed and completed by overlapping PCR, and12strains of E.coli recombinant were screened. Molecular modification of EG II was accomplished, and fermentation experiments showed that the activity of all the mutants is higher than the original strain. Moreover the EGⅡ-E mutant contained all6mutations reaching the highest activity (1.321IU/mL) after21h fermentation, which increased by82.7%than the parent strain. The fermentation characteristics of recombinants showed that mutations of2catalysis residues, V136D and E260D, have enhanced the endoglucanase activity; the mutation of N39S accelerated the rate of carbohydrate binding and shortened the peak time significantly; while other3mutations in the loop region only affect the enzyme activity slightly.5) The E.coli recombinants constructed above were induced by IPTG for21h, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Recombinant not induced served as negative control. The results of SDS-PAGE showed the molecular weight of EG II is45kDa. Furthermore, the bands of EGⅡ-E were the thickest, which have the highest expression.6) Besides, eg2was cloned and linked into pPIC9K, and then transfected the plasmid pPIC9K-eg2into the Pichia pastoris GS115. The positive clone was sceened by MD plate and CMC-Na medium with congon red. The recombinant Yeasts were induced by methanol, and the ability of endoglucanase could achieve the peak of1.754IU/mL after84h fermentation, which is2.43times of the E.coli recombinants.