Enhancing the Expression of the Substrate by the Extracellular Secreted Enzymes and Improving the Alkaline Protease Production in <i>Bacillus licheniformis</i>

doi:10.13523/j.cb.2011050

China Biotechnology

2021, Vol. 41

Issue (2/3): 53-62 DOI: 10.13523/j.cb.2011050

Enhancing the Expression of the Substrate by the Extracellular Secreted Enzymes and Improving the Alkaline Protease Production in Bacillus licheniformis

ZHOU Hui-ying¹,ZHOU Cui-xia^1,²,ZHANG Ting¹,WANG Xue-yu¹,ZHANG Hui-tu¹,JI Yi-zhi^3,^*(

),LU Fu-ping^1,^*(

)

1 Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education,College of Biotechnology,Tianjin University of Science &Technology, Tianjin 300457, China
2 College of Biology and Brewing Engineering, Taishan University, Taian 271000,China
3 Beijing Key Laboratory of Biomass Waste Resource Utilization, College of Biochemical Engineering,Beijing Union University, Beijing 100023,China

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Abstract

Bacillus licheniformis 2709 is a strain of alkaline protease that has been put into industrial production due to its easy cultivation, GRAS status and perfect protein secretion ability. In order to improve the fermentation production performance of the strain, increase the utilization of culture medium components and the production of alkaline protease by the bacteria, the extracellular secretion enzyme system has been improved. With the help of homologous recombination mechanism, the xylanase gene (xynA) from Bacillus pumilus was introduced near the origin of replication and the lipase gene (lipY2) from Yarrowia lipolytica was introduced into the center of the origin of replication. The results showed that the activity of xylanase and lipase reached(58±2.07)U/mL and (207±10.62)U/mL, respectively when the strain was fermented in shake flask for 44h. Their efficient secretion and expression ability promoted the utilization rate of the fermentation substrate by Bacillus licheniformis, thus increasing the content of reducing sugar and total nitrogen in the medium and decomposing nitrogen compounds in the precipitate. Compared with the original strain, the bacterial biomass of the mutant strain was increased by 11.76%, the fermentation cycle of alkaline protease was shortened by 4h, and the yield of alkaline protease was increased by 14.41%. The enrichment of the secreted enzymes and the improvement of fermentation performance of Bacillus licheniformis 2709 provide a method for modification of Bacillus licheniformis as a microbial agent in the feed industry.

Key words： Bacillus licheniformis Xylanase Lipase Alkaline protease

Received: 27 November 2020 Published: 08 April 2021

ZTFLH:

Q815

Corresponding Authors: Yi-zhi JI,Fu-ping LU E-mail: jiyizhi@buu.edu.cn;lfp@tust.edu.cn

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	Hui-ying ZHOU
	Cui-xia ZHOU
	Ting ZHANG
	Xue-yu WANG
	Hui-tu ZHANG
	Yi-zhi JI
	Fu-ping LU

Cite this article:

ZHOU Hui-ying,ZHOU Cui-xia,ZHANG Ting,WANG Xue-yu,ZHANG Hui-tu,JI Yi-zhi,LU Fu-ping. Enhancing the Expression of the Substrate by the Extracellular Secreted Enzymes and Improving the Alkaline Protease Production in Bacillus licheniformis. China Biotechnology, 2021, 41(2/3): 53-62.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2011050 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I2/3/53

Table 1 Strains,vectors and primers used in this study

Fig.1 Identification of the construction of integrative vector fragments and the recombinant vector by colony PCR (a) Identification of the construction of integrative vector fragments 1: The upstream homologous sequence of position I; 2: The xynA expression cassette sequence; 3: The downstream homologous sequence of position I; 4: The upstream homologous sequence of position II; 5: The lipY2 expression cassette sequence; 6: A homologous sequence downstream of position II (b) Identification of the construction of recombinant vector 1: PCR verification of integration position I upstream homologous sequence, downstream homologous sequence and the xynA expression cassette; 2: PCR verification of integration position II upstream homologous sequence, downstream homologous sequence and the lipY2 expression cassette M: 1kb DNA Ladder

Fig.2 Identification of the mutant strain by colony PCR M: 1kb DNA Ladder (a) Identification of the single-exchange strains for integration position I 1-4: The correct single-exchange strain for integration position I; 5:The negative control BL 2709 strain (b) Identification of the double-exchange strains for integration position I 1-4: The correct double-exchange strain for integration position I; 5: The negative control BL 2709 strain (c) Identification of the single-exchange strains for integration position II 1-4: The correct single-exchange strains at the integration position II; 5: The negative control BL 2709 strain (d) Identification of the double-exchange strains for integration position II 1-4: The correct double-exchange strains at the integration position I; 5: The negative control BL 2709 strain

Fig.3 Gene editing method based on temperature-sensitive plasmid and gene expression cassette in B. licheniformis (a) Schematic diagram of gene editing method based on temperature-sensitive plasmid (b) Genome locations to be inserted the xynA and lipY2 gene expression cassette in B. licheniformis

Fig.4 The cell biomass of the BL 2709-xynA-lipY2 and BL 2709

Fig.5 The content of reducing sugar in strain fermentation of the BL 2709-xynA-lipY2 and BL 2709

Fig. 6 The content of total nitrogen of fermentation in the fermentation medium of BL 2709-xynA-lipY2 and BL 2709 (a) The content of total nitrogen of fermentation supernatant in the fermentation medium of BL 2709-xynA-lipY2 and BL 2709 (b) The content of total nitrogen of fermentation precipitate in the fermentation medium of BL 2709-xynA-lipY2 and BL 2709

Fig.7 Xylanase activities of the BL 2709-xynA-lipY2 and BL 2709

Fig.8 Lipase activities of the BL 2709-xynA-lipY2 and BL 2709

Fig.9 Alkaline protease enzyme activity of the BL 2709-xynA-lipY2 and BL 2709


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