Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (11): 257-266.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1508
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LIU Shan1,2(), YE Wei2, ZHU Mu-zi2, LI Sai-ni2, DENG Zhang-shuang1(), ZHANG Wei-min2()
Received:
2020-12-13
Online:
2021-11-26
Published:
2021-12-03
Contact:
DENG Zhang-shuang,ZHANG Wei-min
E-mail:shanliu1995@126.com;dzs163@163.com;wmzhang@gdim.cn
LIU Shan, YE Wei, ZHU Mu-zi, LI Sai-ni, DENG Zhang-shuang, ZHANG Wei-min. Cloning,Expression and Characterization of a Novel Acyltransferase GPAT[J]. Biotechnology Bulletin, 2021, 37(11): 257-266.
Fig.1 PCR verification of gene g7779 A:Cloning of gene g7779. B:PCR verification of bacterial solution. M:DNA marker 2K Plus. 1:Gene g7779. P:Positive control. 2-8:Positive monoclonal of pET28a-g7779
Fig.2 Purification and Western-Blot analysis of GPAT A:Optimization of the expression condition of GPAT. M:Blue Plus II protein marker. 1, 3, 5, 7, 9 and 11:Supernatant of condition ①, ②, ③, ④, ⑤ and ⑥. 2, 4, 6, 8, 10 and 12:Sediment of condition ①, ②, ③, ④, ⑤ and ⑥. B:Purification of GPAT. M:Blue Plus Ⅱ protein marker. 1:Uninduced total protein. 2:Total protein of supernatant after induction. 3-5:Binding buffer. 6:68 mmol/L imidazole eluent. 7:116 mmol/L imidazole eluent. C:Western-Blot analysis of GPAT. M:EasySee Western marker. 1:Uninduced total protein. 2:Total protein of supernatant after induction. 3:Binding buffer. 4:68 mmol/L imidazole eluent. 5:116 mmol/L imidazole eluent
Fig.5 Characterization of enzymatic properties of GPAT A:Optimal reaction temperature of GPAT. B:Optimal reaction pH of GPAT. C:Thermostability of GPAT. D:Enzyme kinetics of GPAT
Fig.7 Secondary structure analysis of GPAT The order of vertical lines from long to short are:α - helix(blue),extend strand(red),β - turn(green)and random coil(purple)
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