Characterization Of Thermophilic Endoglucanase AcCel12B

Lignocellulose is the most abundant renewable resource on the earth. Using physical, chemical and biological methods to produce fermentable monosaccharides and oligosaccharides by microbial fermentation is one of the effective ways to solve the dependence of human on fossil resources. Currently thermostable enzymes have obvious advantages than commodity fungal cellulases, which are mostly mesophilic counterparts. Therefore, the research of thermostable enzymes system has been widely attention.Microbial degradation of celluloses involves the synergistic action of three major enzymes: endoglucanase, cellobiohydrolase and β-glucosidase. Cellulases are able to disrupt the cellulose by the cooperation of these activities. Endoglucanases produce cello-oligosaccharides of various lengths and thereby generating new chain ends within the amorphous region in the cellulose molecule. Exoglucanase removes cellobiose units from the reducing or non-reducing ends of the cellulose chain. Lastly, the cellobiose is hydrolyzed by β-glucosidases to glucose preventing the build-up of cellobiose which inhibits cellobiohydrolases. Cellulosomes contain numerous kinds of cellulases and related enzyme subunits which are held together by unique scaffoldin subunits. In addition, saccharopsphorylase, lyases, single oxidase also plays an important role in the degradation of lignocellulose.The thermophilic microorganism A. cellulolyticus 11 B used cellulose as a carbon source, were grown in the high temperature under acid condition. Genome analysis showed that it can produce abundant cellulases. Characterization and functional analysis of the structure-function relationship will not only important, but also provide us for potential industrial applications. Endoglucanase（Ac Cel12 B, E.C. 3.2.1.4） and cellobiohydrolase（Ac Cbh48, E.C. 3.2.1.91） are modular proteins, and β-glucosidase（Ac Bgl1, E.C. 3.2.1.21） has only one catalytic domain structure. Ac Cel12 B was comprised of a catalytic domain（CD） with homology to GH12 and a carbohydrate binding domain（CBM₂） connected by a long and proline/serine-rich linker region. Ac Cbh48 was comprised of CBM₃a, CD with homology to GH48, FN3 and CBM₂ connected by three Linkers. The mechanism of linker between each domains is not clear. Here we cloning and overexpression of endoglucanase, cellobiohydrylase and β-glucosidase from thermophile Acidothermus cellulolyticus 11 B. We systematically determined the substrate specificities, product specificities and synergistic effects of the enzymes. The truncated mutant of endoglucanase was cloned and the properties were characterized.This paper is mainly to obtain the following results:（1） Cloned the Ac Cel12 B, Ac Cbh48 and Ac Bgl1 from A. cellulolyticus 11 B. The cellulases were constructed and functionally expressed in E. coli BL21, respectively. Ac Cbh48 has not been expressed and purified successfully.（2） The optimum temperature of Ac Cel12 B is 72 oC, and the optimum p H is 4.3 for RAC. The thermal stability analysis showed that it remained 50% activity after 2h incubation at 70 oC. The optimum temperature of Ac Bgl1 is 70 oC, and the optimum p H is 7.0 for p NPGlc. Ac Bgl1 hydrolysed oligosaccharides to glucose processivity.（3） We detected synergistic effect of Ac Cel12 B and Ac Bgl1. The major productions of Ac Cel12 B in the hydrolysis of the RAC were cellobiose and cellotriose. β-glucosidase splits cellobiose and cellotriose into glucoses. The highest synergistic effect was obtain for the hydrolysis of RAC for 2 h using a combination of Ac Cel12 B and Ac Bgl1 at a molar ratio of 1:4.（4） The characterization of truncated mutant（Ac Cel12B△PS） demonstrates that Ac Cel12B△PS demonstrated excellent thermal stability and high enzyme activities as Ac Cel12 B. The deletion of PS decreased the specific activity, hydrolysis power factor and thermal stability of enzyme. While, the p H stability of Ac Cel12B△PS was enhanced under neutral conditions.In summary, the study on the function of Linker is helpful to further understand the catalytic mechanism of cellulase. The high synergistic efficiency with cellulase brings a huge potential to theoretical research.