Optimizing the Culture Conditions for <i>Amphibacillus xylanus</i> Glutamate Dehydrogenase Gene Engineering Bacteria

doi:10.13523/j.cb.20191007

China Biotechnology

2019, Vol. 39

Issue (10): 58-66 DOI: 10.13523/j.cb.20191007

Optimizing the Culture Conditions for Amphibacillus xylanus Glutamate Dehydrogenase Gene Engineering Bacteria

CHEN Zi-han,ZHOU Hai-sheng,YIN Xin-jian,WU Jian-ping,YANG Li-rong(

)

College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

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Abstract

Firstly,five expressing NADH-dependent glutamate dehydrogenase E.coli engineering bacterias are constructed,get a glutamate dehydrogenase AxyGDH from Amphibacillus xylanus.The optimum temperature is 60℃ and pH is 8.0,the specific enzyme activity is (903.1±24.6)U/mg,the half-life is 167h at this condition.Then, decide the enzyme production conditions of E.coli BL21(DE3)/pET-28a(+)-AxyGDH: Inducer IPTG concentration 0.7mmol/L,induction temperature 25℃.The optimized medium composition: Glycerol 11.3g/L, yeast powder 16.3g/L, MgSO₄·7H₂O 0.62g/L,NaCl 0.5g/L, Na₂HPO₄·12H₂O 17.1g/L,KH₂PO₄ 3g/L,NH₄Cl 1.5g/L.At last,the fermentation enzyme activity of AxyGDH is (9 066±45)U/ml in the 10L tank fermentation when controlling the specific growth rate is 0.2h ^-1,which is 51.1 times than that of LB medium.It lays the foundation for glutamate dehydrogenase low cost production.

Key words： Glutamate dehydrogenase Heterologous expression Specific enzyme activity Stability Fermentation

Received: 12 March 2019 Published: 12 November 2019

ZTFLH:

Q815

Corresponding Authors: Li-rong YANG E-mail: lryang@zju.edu.cn

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	Zi-han CHEN
	Hai-sheng ZHOU
	Xin-jian YIN
	Jian-ping WU
	Li-rong YANG

Cite this article:

CHEN Zi-han,ZHOU Hai-sheng,YIN Xin-jian,WU Jian-ping,YANG Li-rong. Optimizing the Culture Conditions for Amphibacillus xylanus Glutamate Dehydrogenase Gene Engineering Bacteria. China Biotechnology, 2019, 39(10): 58-66.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191007 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I10/58

Fig.1 SDS-PAGE of glutamate dehydrogenase Lane M:Marker; Lane 1-3:CsyGDH; Lane 4-6:AxyGDH; Lane 9-7:CdiGDH; Lane 12-10:PenGDH; Lane 15-13:BsuGDH

Fig.2 Enyme activity(a) and stability(b) of cell crude extract

Fig.3 SDS-PAGE of purifying AxyGDH Lane M:Marker; Lane 1; Lane 2: AxyGDH

Fig.4 Optimal temperature(a)and pH(b)of AxyGDH

Table 1 Enzyme activity half life of AxyGDH at different temperature

Fig.5 Influence of IPTG concentration(a)and induction temperature(b)on activity

Fig.6 Effect of carbon(a) and nitrogen(b) source on enzyme activity and bacteria growth

Table 2 Factors and levels for response surface method

Fig.7 Response surface and contour map of X₁,X₂(a);X₂,X₃(b);X₁,X₃(c) to enzyme activity

Fig.8 Effect of different specific growth rate on enzyme production(a)bacteria growth process and enzyme production curve when controlling the specific growth rate is 0.20h^-1(b)


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