Cloning, Expression And Enzymatic Properties Of Cellulase Gene From Bacillus Subtilis

The study of cellulase about cloning and expression mainly focused on single gene in recent researches, but in the degradation of crystalline cellulose a variety of cellulase synergy was needed. Glucanase enzyme system played an important role in the process of degrading the complex fiber polymer into small molecule carbohydrates. It would be an effective way to improve the degradation rate of cellulose by transforming glucanase genes with different functions into Escherichia coli by gene engineering technique. Improving the expression mechanism of cellulase by using molecular techniques would accelerated the application process of cellulase in industrial and agricultural production, which had a great significance in solving the problem of energy crisis, environmental pollution and reducing the pressure of food fight between human and animals. This study was mainly about screening of Cellulase genes of Bacillus subtilis, expressing of single Cellulase gene and the fusion Cellulase gene and also estimating of the enzyme activity. The research is as follows:1. In order to isolate the cellulase gene of Bacillus subtilis, the soil samples were screened by Congo red staining method firstly. Fourteen strains with the better capability producing cellulase have been isolated; Enzyme activity assay results showed that the strains B.subtilis X1, B.subtilis X2 had a strong degradation capacity of carboxymethyl cellulose substrate, CMCA were 6.93 U/mL and 6.61 U/mL respectively; Through the morphological, physiological, biochemical identification and 16 S r DNA molecular identification of the two strains, the results showed that, B.subtilis X1, B.subtilis X2 were both Bacillus subtilis.2. According to the submitted gene sequence of endoglucanase(ID:KF240848.1) in GeneBank, a pairs of primers was designed. Using the B.subtilis X1 genome as the template, the endoglucanase gene named Cel042 was amplified by PCR method, then cloned into T vector and sequenced. The analysis of sequence showed that, the target gene was 1506 bp, the ORF coding region was 1500 bp, encoding a protein of 499 amino acids, The sequencing result of the endoglucanase gene from Bacillus subtilis was compared with those published on GeneBank, the homology was up to 98.5%. The endoglucanase gene from Bacillus subtilis was obtained in this study. Cel042 was cloned into expression vector pET32a(+), and transformed into Escherichia coli BL21(DE3). After that, it was induced to express protein. SDS-PAGE results showed that a fusion protein about 74 k Da had been expressed, The CMCA was 29.98 U/mL. The analysis result showed that the maximum enzyme activity was 4.3 times of the enzyme activity of the original strain B.subtilis X1, which indicated that the endoglucanase gene Cel042 from Bacillus subtilis was effectively expressed in Escherichia coli.3. According to the submitted gene sequence of endoglucanase(KM:009051.1) in GeneBank, a pairs of primers was designed. Using the B.subtilis X2 genome as template, the β-1, 3-1, 4-Glucanohydrolase gene which named as Cel22 was amplified by PCR method, then T cloning and sequencing had been done. Sequence analysis showed that the target gene was 729 bp, encoding a protein of 242 amino acids. Homology analysis showed that the homology between the amplification of beta-1, 3-1, 4 glucanase gene and the JN039020.1 announced in GeneBank was up to 99%. The results showed that a beta-1, 3-1, 4 glucanase gene from Bacillus subtilis was obtained in this test, which named as Cel22. Then the Cel22 gene was subcloned into pET32a(+), and transformed into BL21(DE3). SDS-PAGE results showed that a fusion protein about 44 kDa was expressed. The CMCA of beta-1, 3-1, 4 glucanase up to 20.87 U/mL, which is 3.2 times to the strain B.subtilis X2.4. Two pairs of primers with the peptide sequence GSGGGS for Cel042 and Cel22 were designed. Both the double cellulase fusion gene Cel042-Cel22 and the pET32a(+) were digested to construct the recombinant expression vector pET32aCel042-Cel22, then transformed it into Escherichia coli BL21(DE3) for expressing. SDS-PAGE results showed that a fusion protein about 101 k Da was expressed. Enzyme activity assay showed that the activity of fusion cellulase was 57.62 U/mL, enzymatic properties showed that the optimum temperature was 50℃, the optimal pH was 6; the purified enzyme could maintain more than 75% activity in pH 4~8. The enzyme activity effect could be maintained for 70% when the temperature between 40℃and 70℃. Mn2+ could improve the activation of the enzyme. Hg2+ and ethylene diamine tetraacetic acid(EDTA) could significantly inhibited of the enzyme. The fusion expression of different cellulases laid the foundation for the researches on the new way of cellulose degradation.