Purification, Gene Cloning, Expression, And Function Analysis Of Endo-1,4-beta Cellulases From Rhizoctonia Solani AG-1-IA

Cellulose can be degradated by many kinds of micrograms, such as fungi, bacteria, and so on. In the process of degradation, there is an essential enzyme namedβ-1, 4-endo-cellulase, which belongs to cellulase family. The internal amorphous area of cellulose polysaccharide chain is cut randomly byβ-1, 4-endo-cellulase, resulted in oligosaccharides of different length and new ends.Maize sheath blight has become an important disease in China's main corn production areas. R. solani is the main pathogen. A variety of cell wall degrading enzymes are produced and released in vivo. Polygalacturonase (PG), polymethylgalacturonase (PMG) andβ-1, 4-endo-cellulase are the three main cell wall-degrading enzymes. Cellulose is one of the main components of corn andβ-1,4-endo-cellulase is one of its degrading enzymes.R. solani AG-1-IA was isolated from cornfield. So far,β-1, 4-endo-cellulases of R. solani have not yet been studied. Growing in improved Marcus medium containing CMC,β-1,4-endo-cellulase can be produced by R. solani. The enzyme was purified to homogeneity from the culture supernatant of the strain by fractional ammonium sulphate precipitation, Phenyl-Sepharose chromatography and DEAE-Sepharose chromatography. The molecular mass of a single band of the enzyme was estimated to be 61.1kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum reaction pH value of purified beta-1,4-endo glucanase was 4.0, and the optimum reaction temperature was 45℃. According to incubation on maize, the purified beta-1,4-endo glucanase can cause great damage to the plant tissue.Degenerate primers was designed based on the conserved domains of other reportedβ-1, 4-endo-cellulases, and partial cDNA fragment encoding theβ-1, 4-endo-cellulase was obtained through RT-PCR. RACE-PCR and TAIL-PCR was used to generate full-length cDNA clones. Then partial DNA encoding theβ-1, 4-endo-cellulase was cloned which includes three introns. They have been registered in GenBank with accession number FJ550135 and FJ538292 respectively. The gene ofβ-1,4-endo-cellulase from R. solani and expression vector pPIC9K were digested ligated in vitro to construct eukaryocyte expression plasmid pPIC9K/eg. The recombinant vector was transformed to Pichia pastoris GS115 competent cell after linearization by restriction enzyme Sal I. The recombinant P. pastoris PGS-D was obtained and the expressedβ-1, 4-endo-cellulase was purified. The expression level was up to 0.51mg/mL. The molecular mass of a single band of the enzyme was estimated to be 30.0kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum reaction pH value of the expressed beta-1,4-endo glucanase was 4.0, and the optimum reaction temperature was 55℃. According to incubation on maize, expressed beta-1,4-endo glucanase can kill maize plant cells.