Sequencing, Cloning, Expressing And Metabolic Flux Analysis In E.coli Of β-mannanase

β-mannanase is an important hemicellulase, which may be widely used in food, medicine, feed and oil industry. In this thesis,β-mannanase molecular biology was the core of the research. The study was carried out according to gene engineering and metabolic engineering methods. The main conclusions presented in this work were shown as follows:Producing strain identification: The strain preserved by our laboratory was identified according to the morphological, physiological, biochemical characteristics and sequencing of 16S rDNA. The result showed that the strain was Bacillus subtilis, named Bacillus subtilis TJ-200603.Sequence information: The conservativeβ-mannanase sequence of Bacillus subtilis TJ-200603 was obtained by degenerate PCR. It had 98% nucleotide homology with Bacillus subtilis G1(DQ309335). Then the whole sequence was obtained by homologous PCR. Analysis and prediction using on-line tools showed that ORF included 1104bp; 367 amino acids were coded; the first 31 amino acids translated to signal peptide; the rest translated to mature protein peptide which had 38.005kDa molecular weight and a 5.6 isoelectric point. Amino acid sequence showed:β-mannanase of Bacillus subtilis TJ-200603 belonged to Glycosyl hydrolase family26. At present,β-mannanase sequence of Bacillus subtilis TJ-200603 is submitting to GenBank database.Gene cloning and expressing in E.coli: After construction of recombinant cloning plasmid-pMD-man,β-mannanase was cloned in E.coli DH5α; after construction of recombinant expressing plasmid-pET-22b-man,β-mannanase was expressed in E.coli BL21(DE3). SDS-PAGE result showed that the protein expressed in E.coli was about 38kDa, agreed with the previous prediction. The expressed protein was located in periplasm and cell. The content was 36.03μg/mL and 97.02μg/mL respectively, and the enzyme activity was 4.8 U/mL and 6.7U/mL respectively.Metabolic flux analysis: The metabolic network ofβ-mannanase synthesis from engineering E.coli was established, then the metabolic pathway was analysed and five important metabolic nodes were discussed in detail by metabolic flux analysis.Thermostability: Based onβ-mannanase sequence, the 3D structure was obtained using Homology Modeling. We got the result that the hydrogen bond was formed between enzyme active region and hydroxyl. The optimal protective agents were found based on the above result. When sorbitol, sucrose, and chitosan were added with the constant concentration (2g/L), the relative activities ofβ-mannanase were increased to 222.8%, 201.3%, and 198.7%, respectively. The combination of these three additives, which was sorbitol of 2g/L, chitosan of 1g/L and sucrose of 3 g/L, made the relative activity ofβ-mannanase increase to 258.1%. The optimal reaction temperature, shifted from 50oC to 60oC, the decreased kinac and the prolonged half life of reaction all proved thatβ-mannanase was stabilized with additives.