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Gene Cloning ，Expression and Characterization of β-fructofuranosidase Fromarthrobacter

Author: PengDuan
Tutor: WangYongHua
School: South China University of Technology
Course: Of Food Science
Keywords: the beta - fructofuranoside enzyme Cloning Expression Enzymatic Properties
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 55
Quote: 0
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Abstract


Fructo-oligosaccharides (FOS) because of their high proliferation of bifidobacteria, detox clean bowel, prevent tooth decay and improve lipid metabolism (fat and cholesterol) and other physiological functions, and gradually by consumers of all ages. Industry mainly beta-fructofuranosidase enzyme in sugar production oligofructose. beta-Fructofuranosidase enzyme sucrose as substrate and catalyzes the formation of oligofructose, its mechanism of action is sucrose, glucose-fructose group beta-(1-4) glycosidic bond cleavage to generate fructose and glucose, fructose, in The action of the enzyme further polymerization into functional oligosaccharides. beta-fructofuranosidase enzyme widely present in the biosphere, the nature of the different microbial sources of beta-Fructofuranosidase. Aspergillus niger, Arthrobacter, Aspergillus oryzae and other microorganisms capable of producing beta-fructofuranosidase enzyme, the experiment Arthrobacter 10137 for the study, its genome as a template, beta-fructofuranosidase gene was amplified and insert it into the cloning vector pMD19-T, the sequencing results showed that the gene fragment length 1488bp, encoding 496 amino acids. The gene was cloned into the expression vector the PFL-B13cl on SDS-PAGE analysis of the size of the target gene protein of approximately 67 kDa determined by colony PCR and restriction analysis, was successfully constructed recombinant Escherichia coli the PFL-B13cl-BFF. The experimental design of the single factor experiment the PFL-B13cl-the bff culture conditions, results show that: recombinant Escherichia coli vaccination pFL-B13cl-bff amount to 5%, 37 ° C cultured for 1.5 to 2 hours after the induction of 22 ° C to continue to cultivate 12h production The enzyme the best. IPTG and lactose can induce the PFL-B13cl-bff produce recombinant protein, lactose-induced concentration of 12g / L, Enzyme activity reached the maximum 5.67U/mL to 0.5mol/LIPTG induced the activity 6.14U/mL away little In this experiment, lactose as a safe and effective use of the inducing agent. In this study, recombinant Escherichia coli pFL-B13cl-bff 5L bioreactor. The results showed that the recombinant E. coli in 5L fermenter activity of 9.69 U / mL, the pH control activity increased 10.39U/mL continuous flow of glucose increased enzyme production 11.27U/mL, but the accumulation of acetate metabolism The product will result in the decline phase of the advance. The crude enzyme solution was extracted from the recombinant E. coli the PFL-B13cl-bff purified by metal chelate chromatography, SDS-PAGE analysis of 300mmol / L eluate purpose of protein bands. Its research, The beta-Fructofuranosidase enzyme having the following characteristics: optimum reaction temperature of 30 ° C ~ 40 ° C, its thermal stability temperature range of 40 ° C or below; good stability in the pH value within the range of 6.0 ~ 7.5; in the selected range of metal ions, Cu2, Ag enzyme was strongly inhibited, Mg2, Ca2 has a protective effect on the enzyme activity.

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