| Cellulose is the most widespread and abundant carbon matrix precursor on the earth whose utilization ratio is relatively low. With the development of gene engineering technology, the gene encoding cellulase has been cloned into bacteria, yeast, fungus and plants in the hope of obtaining recombinant cellulase with high activity.Mulberry bacterial disease is the major and serious disease that does harm to the twigs and leaves of mulberry, and is widespread in sericultural areas. The bacterium causing mulberry disease was confirmed as Pseudomonas syringae by the isolation and characterization of the bacterium which show it belongs to genus pseudomonas and that mainly causes the rot and rapture of the twigs of mulberry. The invasion of Pseudomonas syringae into plants mainly depends on its cell wall degrading enzymatic system consisting of one or several cellulases, and the system plays an important role in the degradation of cell wall and thus enhancement of pathogenicity.In the study, the gene encoding cellulase of the bacterium causing mulberry disease was cloned and expressed in E. coli in the hope of obtaining engineering bacterium producing recombinant cellulase with high activity. According to the sequence of the gene encoding cellulase of Pseudomonas syringae submitted in NCBI, specific primers were designed, with BantH I and EcoR I restriction sites introduced into, and used to amplify the gene encoding cellulase. The PCR products were digested with BamH I and EcoR I, ligated with pET28a vector digested with the same enzymes, and then sequenced. The result showed that the open reading frame of the gene is 1173 bp in size, encoding 390 amino acids. The cloned gene was found to encodeβ-1,4-D-Glucanohydrolase by comparing its amino acid sequence with those in NCBI.Based on the cellulase gene structure, the full-length(P1/P2) and core peptide(P3/P4) were constructed recombinant vectors with pET28a, respectively. Then the constructed recombinant vectors were transformed into BL21, and IPTG was used to induce the expression of cellulase. Western Blotting and MALDI-MS were used to detect the expression product following SDS-PAGE, and two specific bands were detected, with their molecular weight being 42 kD and 35 kD, respectively, which are consistent with the theoretical values of full-length and core peptides of the gene of interest. Bacteria induced by IPTG were harvested, washed by buffer NaAc-HAc, broken by ultrasound and centrifuged, and then supernatant was collected. DNS method was used to determine the activity of the expression product under different reaction conditions. While the activity of the full-length peptide(P1/P2) increased with temperatures ranging from 30 to 40℃, peaked at 40℃, and then decreased gradually, the activity of the core peptide (P3/P4) was found to be the highest at 30℃, The activity of the core peptide (P3/P4) was also found to be the highest when pH value was 7．The amount of reduced sugar produced by the degradation of cellulose by cellulase increased with time, and when the reaction time was between 12 h and 15 h, while the activity of the full-length peptide enhanced significantly, that of the core peptide had a decreased tendency. Moreover, its filter paper activity was 2．3×10~3 U/L. Results showed that the cellulase produced by the recombinant bacterium was neutral cellulase with relatively high activity, and thus the bacterium can be further constructed to be an engineering bacterium.
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