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Overexpression of an Extracellular Hybrid β-1, 3-1, 4-glucanase by Recombinant E. Coli

Author: LiuZheng
Tutor: LuYingHua
School: Xiamen University
Course: Biochemical Engineering
Keywords: E. coli Heterozygous gene β-1 ,3-1, 4 - glucanase Fed-batch fermentation Medium optimization Affinity chromatography
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 145
Quote: 3
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Abstract


β-glucanase is an important industrial enzymes, which can effectively eliminate the negative impact of the cereal β-glucan in the brewing and feed industry. Domestic β-1 ,3-1, 4 - glucanase in its infancy, the performance failed to meet the practical application of the natural enzyme production strains and enzymes. However, the application of molecular biology, genetic engineering methods to provide an opportunity. Β-1 ,3-1, 4 - glucanase mainly to improve the yield and performance of the enzyme in building a genetically engineered strain. The research and application of industrial biotechnology has conducted a series of exploration, aimed at finding a sustainable development for industrialization, large-scale production of β-1 ,3-1, 4 - glucanase method. The research topics include: 1. Fermenter stream fed-batch culture of recombinant Escherichia coli manufacturer of JMl09-pLF3 β-1 ,3-1, 4 - glucanase the original optimized medium based on optimization of the fermentation process in 7 L fermentation The tank on the fed-batch fermentation for enzyme production. The results showed that, between 12 ~ 32 h constant rate of flow to the fermenter plus double concentrated medium nitrogen source, a great role in promoting cell growth and enzyme production. The maximal activities increased to 1680 U / mL, 231.40% higher than the batch fermentation; maximum cell density of 7.67 g / L batch fermentation 3.44 times. 2. Efficient secretory expression of β-1 ,3-1, 4 - glucanase recombinant Escherichia coli build better thermal stability heterozygous beta-1 ,3-1, 4 - glucanase expressed genes ( bgl) inserted into the plasmid pET-22b (), and inserted into the the kil-Km secretion cassette, to get a new β-glucanase expression plasmid pET-22-BGL-(KIL-Km). The recombinant plasmids were transformed into E. coli E coli JM109 and BL21 (DE3). Proved the functionality expressed by the recombinant strain containing the plasmid pET-22-BGL-(KIL-Km) of the efficient secretion of the target protein by SDS-PAGE. Response surface method optimization and reorganization of E.coli BL21 (DE3)-pET-22-bgl-(kil-Km) enzyme production medium in the TB medium on the basis of single factor experiments lactose as the best carbon source, casein peptone was the best nitrogen source. Box-Behnken design method and RSM response surface analysis investigated the main factors lactose concentration, the casein peptone concentration and carbon and nitrogen than three factors interaction on the basis of single factor optimization. Optimization of enzyme production medium formula to determine recombinant E.coli BL21 (DE3)-pET-22-bgl-(kil-Km) (g / L): casein peptone 33.39, lactose 8.03, glycerol 9.15 NaCl 10.0, KH 2 PO 4 2.31, K 2 HPO 4 12.54. Optimal medium at 37 ° C, 150 rpm for shake flask cultures 32.5 h, the activity reached 1242.94 U / mL, 3.3 times higher than the initial medium. Separation and purification and characterization of the recombinant enzyme characterization using Ni 2 6 × His tag-specific binding properties of affinity chromatography purified recombinant enzyme, the target protein purified 7.69-fold , purified recombinant enzyme activity of 1706.2 U / mL, than live 11261.6U/mg, the activity recovery of 86.44%. Analysis by SDS-PAGE, it was found with 6 His-tagged β-glucanase a molecular weight of about 26 kDa. Recombinant beta-1 ,3-1, 4 - glucanase enzymatic properties: optimum reaction temperature and heat temperatures are 50 ° C and optimum pH between 6.0 to 7.0, stable under alkaline conditions high.

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