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Cloning and Expression of pel9A Encoding a Thermostable Pectate Lyase

Author: ZhenDongXiao
Tutor: LiHuaZhong
School: Jiangnan University
Course: Microbiology
Keywords: Heat pectin lyase Clostridium stercorarium Restructuring Plasmid Culture Expression Purification Nature of the enzyme
CLC: Q78
Type: Master's thesis
Year: 2006
Downloads: 76
Quote: 0
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Abstract


The pectin lyase can be used for the extraction of raw materials, coffee and tea fermentation of the essential oils and carotenoids Medical containing of pectin wastewater treatment and crude oil extraction process. Also found that in recent years the pectin lyase can also be used for the purification of plant viruses, pulp bleaching and textile bio-refining. By PCR amplification the structural gene pel9A producing heat pectin lyase from heat-resistant Clostridium in Clostridium stercorarium, and cloned into the expression vector pET28a to construct the expression of recombinant plasmid pPel9A. This recombinant plasmid was transformed into the recipient strain E. coli BL21 for expression, and cultured at 37 ° C for 2h in 0.8 mmol / L under the presence of IPTG at 37 ° C for 5h induction of protein expression by SDS-PAGE analysis of the molecular weight of 130 kDa, consistent with the expected molecular weight. Cells by sonication, the measured pectinase activity 15U/mg crude protein. Plasmid stability studies have shown that E. coli BL21 (pPel9A) in the case of no selection pressure 37 ° C for continuous adapter 5, the plasmid is lost only 15%, indicating that the plasmid is basically stable. Single factor and orthogonal experiments, optimized for culturing the recombinant bacteria E. coli BL21 (pPel9A) enzyme production medium composed of glucose, 10 g / L ammonium sulfate 2 g / L, protein peptone 2 g / L citric acid 1.0 g / L, potassium dihydrogen phosphate, 12.5 g / L MgSO4 · 7H2O 1 g / L, sodium chloride and 2 g / L, TES 5 mL / L, pH 7.0. After optimization, the shake flask cultures bacterial amount of 2.696 g / L (DCW). 5 L Desktop stirred fermenter, additional carbon sources on the growth of recombinant bacteria. Control the dissolved oxygen above 30%, the initial Portuguese sugar 10g / L, in the logarithmic growth phase complement plus 10g / L glucose, culture to 8h, bacterial amount of 6.388 g / L (DCW), pectate lyase activity for 35u/ml culture medium. The crude enzyme solution was purified by HiTrap Chelating HP nickel affinity the column and Supedex200 gel column. Heat partially purified pectin lyase enzymatic properties showed that the optimum pH of 7.0, the determination of the enzymatic activity of the enzyme solution at different pH buffer at 4 ° C for 12 hours showed that in the pH 5 to within the range of 9 to stabilize enzyme optimum temperature of 65 ° C, and the temperature stability range of 40 ~ 75 ° C. Different metal ions on the enzyme activity of Fe2 completely inhibited the activity of the enzyme, Mg, K, Co2, Ni2 and Na different degrees of inhibition of enzyme activity, Ca2 within the range of 0.01 to 0.2 mmol / L significant role in activation of the enzyme, the highest concentration of activated as 0.05 mmol / L.

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