Study On Enzymatic Degradation Of Straw Cellulose And Cloning And Expression Of Endoglucanase Gene Of Bacillus. Sp. Bs-1

Cellulosic materials are one of the cheapest and most abundant renewable resources in the nature. Effective use of cellulose has great and practical significance for the solving the environmental pollution, food shortages, shortage of feed resource, energy crisis and other issues which the world faces today.Cellulase is a group of enzymes in general that can hydrolyzeβ-1, 4 - glycosidic bond and hydrolyzes the cellulose into cellobiose and glucose. It is not a single enzyme but rather multi-component enzymes that cooperates one another. The catalytic mechanism of cellulase is not yet entirely clear. Microorganism that can produce cellulase has low ability to produce enzyme, and the components of the produced enzyme are incomplete or unbalanced. At present the expression levels of the most cellulase genes are low. The main problem in genetic engineering of cellulase is how to achieve effective expressions of different components and how to improve the expression level.In this paper various pretreatments to sweet sorghum straw have been carried out, including mechanical processing, heat treatment, inorganic solvents treatment, organic solvents treatment and ultrasonic treatment. Then the cellulose of the sweet sorghum straw has been hydrolyzed by cellulase. DNS method is used to determine the concentration of reduced sugar, and the effects of various pretreatments have been compared. The efficiency of the different pretreatments on the enzymatic degradation of cellulose was analyzed as the following result. Dry and high temperature treatment has the best effect. The effect of ultrasonic treatment is relatively high, effect of dilute acid and alkali treatment is relatively low.Some soil samples have been collected from a paper mill nearby Wuliangsuhai in Inner Mongolia. Bacteria have been screened using selective CMC-Na medium and their cellulase activities have been identified using Congo red medium. A bacteria strain which has high cellulase activity has been selected and purified. A molecular identification was carried out for this bacteria strain using the method of 16S rDNA. The Blast with the data in the NCBI shows that the 16S gene of this bacteria strain has 98% homology with Bacillus subtilis. The bacteria strain has been identified as a bacillus through the additional morphological observation using microscope. The strain has been named as bs-1.Using the genomic DNA of Bacillus. sp. bs-1 as templates, its endoglucanase gene is amplified by PCR, and then the DNA fragment was connected with the pMD19-T vector. Sequence analysis shows that the full length of DNA fragment is 1503 bp, which encodes a polypeptide containing 500 amino acids. The alignment shows that its endoglucanase gene has nucleotide homology of 93% to 96% with other Basillus and its endoglucanase has amino acids homology of 90% to 98% with other Basillus. The endoglucanase gene was cut from the recombinant plasmid pMD19-C by BamHⅠand HindⅢ, and then cloned in the expression vector pET-30a. The recombinant plasmid pET-30a-C was transformed into the expression host cell E. coli BL21 (DE3). The expression was induced using IPTG. The result of polyacrylamide gel electrophoresis shows that the protein band of expression is found at the position of about 55KD. The enzyme activity of the endoglucanase was determined as 270U/mL by DNS-method, which is as 1.5 times as the enzyme activity of the starting strain (175.54U/mL).