| The neutral protease (NPR) is a protease which suitable pH is 6.0~7.5. It can catalyze the cleavage of peptide bonds in proteins. Since the neutral proteases have many features and advantages in industry such as high speed and wide range of conditions of catalyzed reaction without pollution, they have been extensively used in leather treatment, detergent, food and pharmaceutical industry.To improve production of the neutral protease and to understand the function of the "pro" sequence , both the active domain gene and the full-length gene (included the " pro " sequence ) of the neutral protease from Bacillus subtilis have been cloned and expressed in the Pichia host SMD1168. Moreover, the fermentation conditions of the engineering yeast strain and the properties of the recombinant neutral protease were also studied in our research. This study would offered the basis to the protein engineering of the neutral protease and its overexpression and application in the future.This dissertation consisted of four parts:1. Cloning both the active domain gene and the full-length gene of neutral protease from Bacillus subtilis;2. Expression both the active domain gene and the full-length gene of neutral protease in Pichia pastoris;3. Optimization of the fermentation conditions of the recombinant Pichia pastoris;4. Studing on properties of the crude recombinant neutral protease.The detailed results and conclusions were as follows:1. The specific primers were designed according to the known nucleotide sequence of the nprE. The full-length gene fragment encoding the neutral protease was cloned successfully from B. subtilis genome DNA with the specific primers. " Sequence analysis showed that the homology between the cloned full-length gene of neutral protease and the reported nprE gene was very high (98%). Another specific primer was designed according to the nucleotide sequence of the cloned full-lengthgene. And the active domain gene of the neutral was cloned successfully too,2. The expression vector pPIC9K-NPL and pPIC9K-NPS were constructed respectively by ligating the cloned gene with 3'-terminal of a-factor signal sequence on the pPIC9K in EcoR I site. Both of the two recombinant expression vectors were introduced into Pichia pastoris SMD1168 by electroporation. The positive transformants were characterized by protease activity determination, SDS-PAGE, and PCR amplification against yeast total DNA. It demonstrated that the full-length gene of the neutral protease was expression and modification correctly in SMD1168. The transformant named NL19 secreted recombinant neutral protease with the highest enzyme activity of 24100U/mL. But the expression product of the recombinants which contained only the active domain gene of neutral protease were biologically inactive because of lacking the "pro" sequence in neutral protease gene. The experiment of fermentation and PCR method show that NL19 had a good genetic stability.3. Improved expression level of recombinant neutral protease by P. pastoris was achieved by optimization both the growth phase and induction phase culture conditions. The results indicated that the optimum fermentation condition of the growth phase is as follows: 2% glycerol, 2.5% (NHj) 2SO4 0.00004%Biotin, 10%lmol/L phosphate buffer, pH=5.5, 240rpm, 3048hr, 10% inoculation. The orthogonal test results of induction phase showed that the optimum desired fermentation condition is as follows: 1.5% methanol, 0.5% glycerol, 2% (NtLj) 2804, 0.00004%Biotin, 10%lmol/L phosphate buffer, pH=7.0, 240rpm, 30癈, 48hr, 1:1 inoculation, supplement the same concentration of methanol and glycerol every 24 hours. Five times repeat fermentation experiments of strain NL19 under the optimum conditions show that the average neutral protease activity is 29878U/mL which correspond with results of the orthogonal test.4. The molecular mass of the neutral protease was 44.3kDa. The optimum substrate is casein. Optimum pH of the neutral protease was between 7.0-7.5, and it was stable in th...
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