| Cellulose is the most abundant renewable resource on the earth, which can be utilized in a low rate now. And burning it directly will cause serious environmental pollution. There are a large numbers of microorganisms capable of degrading cellulose in the natural environment, so it can be used to degrade the cellulose and produce a series of useful by-products to reduce the pollution to the environment. The hydrolysis of cellulose needed cellulase. Cellulose was endonuclease and exonuclease hydrolysis of cellobiose and oligosaccharides, and the above product by cellobiose hydrolysis generate the glucose. Cellobiases was widely used which not only can be applied to bio fuels, solving the problem of shortage of fuel, but also can be used in food, chemical, biological manufacturing, and other industrial production. So there are momentous theoretical and practical significance for the research about the cellobiase. The main results of this paper are as follows:1. The strain getting through UV mutagenesis, the cellobiase gene (GenBank Accession No:KP307454) was cloned by RT-PCR method from high yield cellulase Aspergillus niger strain C112. The strain get through UV mutagenesis. The nucleoside sequence of cellobiase was 2934 bp containing noncoding sequence, encoding 860 amino acids with the PI of 4.70. The theoretical molecular weight of the protein was 93.33 kD. The homology of target gene of Aspergillus niger(GenBank Accession No:JX982101.1) reached 99%. The total number of acidic amino acid residues (Asp+Glu) was 99. The total number of basic amino acid residues(Arg+Lys) was 64. Thus, the protein is an acidic protein. The content of glycine (Gly) was the highest in 20 kinds of amino acids, which was 10.7%. And the second was alanine (Ala), which was 9.07%.2. The pPIC9K was used as the expression vector of Aspergillus niger cellobiase gene, and screened the positive clones. The transformation of pPIC9K plasmid with the target gene was transformed into Pichia pastoris SMD1168 by means of chemical transformation. The protein bands of 93.3 kD were detected by SDS-PAGE protein electrophoresis, consistent with the expected target size.3. Analysis of the production of cellobiase from recombinant Pichia pastoris, by YG as culture medium. Fermentation supernatant was taken every 12 h, testing the activity of cellobiase. When the fermentation time was 96 h, cellobiase activity was the highest, cellobiase enzyme activity reaching to 30.37 U/mL. Cellobiase optimum temperature is 50 ℃, and optimum pH was 5.0. After treatment with 1 mM Mg2+, the relative activity of the cellobiase was the highest, the relative enzyme activity about 110.2±6.5%. Treatment with 1mM Mg2+、K+、Ca2+ and Pb2+ could effectively increase the activity of enzyme,1 mM Na+ and Fe2+ had little effect on enzyme activity, and 1mM Fe3+、Li+、Zn2+、Cu2+、Mn2+ and Ag+ had inhibitory effect on the cellobiase. Chemical reagent,2 mg/mL SDS and 10 mM EDTA had little effect on the activity of the cellobiase, and 1 mM β-mercaptoethanol can effectively improve the enzyme activity of the cellobiase. The results showed that some metal ions could effectively improve the activity of the cellobiase.4. The methanol-induced expression of cellobiase in a recombinant Pichia pastoris strain with the cellobiase gene of Aspergillus niger that had been constructed in our previous work was investigated during shake flask cultivation. It needs to determinate the kind and concentration of carbon source, the kind and concentration of nitrogenous source. Through the monofactorial experiments, the induction time, inoculation amount, pH, and methanol amount on the expressed activity of cellobiase, protein expression and the biomass of mycelia, then the optimized parameters of main factors were determined by response surface methodology. The results showed that the optimal bran was 2.5%(W/V), and corn steep powder was 3.0%(W/V). The optimal fermentation conditions were 96 h cultivation at an inoculation amount of 15%(V/V), initial pH 5.0, and methanol 1.0%(V/V), the theoretical value of cellobiase activity were 32.80 U/mL. When repeated experiments were done, the cellobiase activity were (32.24±0.51) U/mL and the exogenous protein expression were (131.57±1.52) mg/L which had closed to the predicted value.5. The 5L fermentation tank was used to magnifing the process of producing cellobiase by recombinant Pichia pastoris. Combining the data of the strains in 5L fermentation tank through batch fermentation with mathematics, the model of bacteria generated produced cellobiase by batch fermentation and the synthesis of cellobiase and the matrix consumption dynamics model were established as follows:(1) Microbial growth kinetics model(2) Kinetics of substrate Consumption model(3) Cellobiase kinetics modelThe dynamic process of batch fermentating cellobiase by recombinant Pichia pastoris can be described by the model above, which can support theories of scale-experimental in the future. |
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