Expression Of An Endo-β-1,4-glucanase EndS-1 From Sinorhizobium Meliloti M5NICS In Escherichia Coli And Pichia Pastoris, And Study On It By Point Mutation

Cellulose, a polymer of ?-1, 4-linked glucose units, is the major polysaccharide constituent of plant cell walls and one of the most aboundant organic compounds in the world. It has been considered enormous potential as chemicals and renewable source of energy. Cellulases are the enzymes which hydrolyse theβ-1,4 linkages in cellulose. Many organisms have been reported as good sources for the production of cellulases, including fungi, bacteria and animals. Today, cellulases, major derived from filamentous fungi such as Trichoderma, Aspergillus species, are commonly used in many industrial applications, especially in textile, food, brewing, new materials as well as in the pulp and paper industries. However, to discover new cellulases from other organisms with novel characteristics, to study its properties expressed in industrial expression systems, to modify the gene product and study whether it can be applied is still needed.A few of yeas ago, EndS gene encoding a endo-β-1,4-glucanase (EC 3.2.1.4),a nonglycosylated protein,which mainly has CM-cellulase activity,was isolated from Sinorhizobium meliloti M5NICS, and had been successfully expressed in Escherichia coli. The expressional level of EndS is not so high as reported by P. Michaud. However, we had obtainned a mutant EndS-1 that has three amino acid residues different from EndS during expression the EndS in E. coli.by using constitutive expression system. As the result we found that the EndS-1 enzymes produced from E. coli showed the highest hydrolytic activity of 23.44 IU /mg against CMC at pH 6.4-7.0 under 60oC,and has very high thermostability, more than 50% of the enzyme activity of EndS-1 was retained when the incubation time was even prolonged to 6 hours at 60 oC,pH 7.0. The expression level of EndS-1 was not high in 250ml flask during fermention-optimzation.To investigate whether the enzyme EndS-1 can be higher expressed in eukaryotic industrial expression systems, we chose the methylotropic yeast Pichia pastoris as cellular host and vector pPIC9K for the enzyme secretory expression. In this work, we report cloning and expressing of the EndS-1 endoglucanase gene in the yeast P. pastoris GS115 , purification of the enzyme expressed by Pichia pastoris, characterization, and comparison of the properties of the endoglucanase expressed in Pichia pastoris and E. coli. As a result of experiment we found that the endo-β-1,...