The Effect on Heterologous Expression of Alkaline Protease AprE by Two Different Promoter and Combinatorial

doi:10.13523/j.cb.20191003

China Biotechnology

2019, Vol. 39

Issue (10): 17-23 DOI: 10.13523/j.cb.20191003

The Effect on Heterologous Expression of Alkaline Protease AprE by Two Different Promoter and Combinatorial

SHI Chao-shuo,LI Deng-ke,CAO Xue,YUAN Hang,ZHANG Yu-wen,YU Jiang-yue,LU Fu-ping LI Yu

Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457, China

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Abstract

Background: Alkaline protease is an important industrial protease with many applications, and its fermentative activity can’t meet the needs of industrial production.Objective: The aim is to improve the alkaline protease AprE production by screening promoters.Methods: Bacillus subtilis WB600, constructed in previous research, was served as the host strain for AprE production and four promoters (P1,P2,P-1-2,P-2-1) were screened to improve AprE production.Results: The results showed that four recombinant strains containing different promoters could successfully express alkaline protease AprE. After 48h of fermentation, the activity of AprE of P2 was 4 041U/ml, which was 1.23 fold of promoter P1.The highest enzyme activity of the double promoter recombinant B. subtilis WB600/P-2-1-aprE reached 6 125U/ml, which was 1.35 fold of the double promoter P-1-2.Conclusion: Collectively,an effective strategy for enhanced production of alkaline protease was provided.

Key words： Promoter Alkaline protease Heterologous expression Enzyme activity

Received: 09 March 2019 Published: 12 November 2019

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	Chao-shuo SHI
	Deng-ke LI
	Xue CAO
	Hang YUAN
	Yu-wen ZHANG
	Jiang-yue YU
	Fu-ping LI Yu LU

Cite this article:

SHI Chao-shuo,LI Deng-ke,CAO Xue,YUAN Hang,ZHANG Yu-wen,YU Jiang-yue,LU Fu-ping LI Yu. The Effect on Heterologous Expression of Alkaline Protease AprE by Two Different Promoter and Combinatorial. China Biotechnology, 2019, 39(10): 17-23.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191003 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I10/17

Table 1 List of real-time fluorescence quantitative PCR primers

Fig.1 Schematic diagram of different promoters in recombinant plasmid

Fig.2 PCR amplification of aprE, the enzyme digestion of different promoters and pWB980 M:DNA marker;1:The fragment of P1 by enzyme; 2:The fragment of P2 by enzyme; 3:The fragment of P-1-2 by enzyme ;4:The fragment of P-2-1 by enzyme;5:PCR verification of aprE; 6:The fragment of pWB980 by enzyme

Fig.3 Expression of aprE gene in B. subtilis WB600 A: PA-1-2; B: P2; C: WB600; D: P1; E: PA-2-1

Fig.4 Ratio of case in hydrolysates and colonies diameter of four recombinant strains(^** P<0.01)

Fig.5 SDS-PAGE of analysis of WB600 M: Marker ;1 - 4:PA1/ PA-1-2/ PA2/ PA-2-1

Fig.6 Comparison of enzyme activity from four recombinant strains(^** P<0.01)

Fig.7 The RNA of four recombinant strains 1:PA1; 2:PA-1-2; 3:PA2; 4:PA-2-1

Fig.8 The real time PCR result of four recombinant strains(^* P<0.05)


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