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Study on Recombinant Carboxypeptidase B

Author: WuShan
Tutor: LiSuXia；ZhangYueQing
School: East China University of Science and Technology
Course: Biological Engineering
Keywords: procarboxypeptidase B inclusion bodies refolding purification
CLC: Q55
Type: Master's thesis
Year: 2012
Downloads: 72
Quote: 0
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Abstract


In this paper, we mainly investigated the expression of recombinant porcine procarboxypeptidase B (pCPB) in Escherichia coli, and the purification, enzymatic property and stability of recombinant porcine carboxypeptidase B (CPB). The high cell density fermentation of recombinant strain was also studied. In addition, we expressed recombinant human pCPB in Pichia pastoris GS115.The recombinant porcine pCPB was expressed as inclusion bodies, which needed to be dissolved, refolded and activated with trypsin to obtain active CPB. The high purity recombinant porcine CPB was got after anion exchange chromatography DEAE-FF purification. Then enzymatic property and stability of recombinant porcine CPB were studied further. The Km and Vmax were 0.27 mmol/L and 222.22μmol/min, respectively. The optimum pH was 7.65, the optimum catalytic temperature was 25℃. It was stable when pH was between 5.0 and 9.0 or temperature was lower than 30℃. Cu2+ and EDTA inhibited the activity of CPB while Co2+ could promote it nearly one time, other metal ions investigated had little influence on CPB.The fermentation of recombinant strain was carried out in a 10-L fermenter with 5 L-working volume, based on shake flask cultures. The culture media contained glucose as carbon source, tryptone and yeast extract offering nitrogen and growth factor. In addition, inorganic nitrogen source, inorganic salt and phosphate adjusting pH were included. In the fermentation process, glucose and yeast extract were added to supplement nutrient, and ammonia to adjust pH. Finally we got 312 g wet cell and 36 g inclusion bodies after cell disruption.We constructed expression strain of recombinant human pCPB. The host strain was Pichia pastoris GS115. The expression vector was secretory type pPIC9k. After inducement with 0.5% methanol, recombinant human pCPB was expressed as soluble protein and secreted to the culture medium. The concentration of recombinant protein reached 0.13mg/ml, with the activity of 0.4U/ml after activation.

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CLC: > Biological Sciences > Biochemistry > Enzymes
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