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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (2/3): 53-62    DOI: 10.13523/j.cb.2011050
技术与方法     
强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能
周惠颖1,周翠霞1,2,张婷1,王雪雨1,张会图1,冀颐之3,*(),路福平1,*()
1 天津科技大学生物工程学院 工业发酵微生物教育部重点实验室 天津 300457
2 泰山学院生物与酿酒工程学院 泰安 271000
3 北京联合大学生物化学工程学院 生物质废弃物资源化利用北京市重点实验室 北京 100023
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-xia1,2,ZHANG Ting1,WANG Xue-yu1,ZHANG Hui-tu1,JI Yi-zhi3,*(),LU Fu-ping1,*()
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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摘要：

地衣芽孢杆菌2709由于易于培养、GRAS状态和完善的蛋白质分泌能力,是已经投入工业生产碱性蛋白酶的菌株。为改善该菌株的发酵生产性能,提高菌体对培养基成分的利用和碱性蛋白酶产量,对菌株的胞外分泌酶系进行完善。利用同源重组机制,在基因组复制起始位点附近引入了来源于短小芽孢杆菌的木聚糖酶基因xynA和在复制起始位点中心对称的位置引入耶氏解脂酵母来源的脂肪酶基因lipY2。整合菌株在摇瓶发酵44h时,木聚糖酶、脂肪酶酶活力分别达(58±2.07)U/mL和(207±10.62)U/mL,其分泌表达促进了地衣芽孢杆菌对发酵培养基的分解与利用,提高了培养基中还原糖、上清总氮的含量和沉淀中含氮化合物的分解;细菌生物量较地衣芽孢杆菌原始菌株提高了11.76%,同时碱性蛋白酶的发酵周期较原始菌提前了4h,碱性蛋白酶产量提高了14.41%。地衣芽孢杆菌2709分泌酶系的丰富和发酵性能的改善为在饲料行业中作为微生物制剂的地衣芽孢杆菌提供了改造的方法。
关键词： 地衣芽孢杆菌;  木聚糖酶;  脂肪酶;  碱性蛋白酶    
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
收稿日期: 2020-11-27 出版日期: 2021-04-08
ZTFLH:  Q815  
通讯作者: 冀颐之,路福平     E-mail: jiyizhi@buu.edu.cn;lfp@tust.edu.cn
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周惠颖
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引用本文:

周惠颖,周翠霞,张婷,王雪雨,张会图,冀颐之,路福平. 强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能[J]. 中国生物工程杂志, 2021, 41(2/3): 53-62.

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.

链接本文:

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

Strains or plasmids Description Source
E. coli EC135 Knockout vectors construction Chinese Academy of Science
E. coli EC135/pM.Bam Plasmid DNA methylation modifcation Chinese Academy of Science
B. licheniformis 2709(BL 2709) Wild strain CICC
Bacillus pumilus A30 PCR template of xylanase mature peptide gene CICC
pKSVT Temperature-sensitive shuttle plasmid, Kanar Hubei University
pPIC9K-mutants PCR template of lipase mature peptide gene [14]
Primer name Sequence (5'-3') Restriction sites
xyn-F CGATTGGCATTTCACCCCATCAATACGCCTTTCACATGA
xyn-R GATGAGGTGTGGGTAGGGCCATCCGACGATCAT
lip-F CGTCGATCATGCGTCAGCCATCAATACGCCTTTCACATG
lip-R GAGGTGAAGCAGAAGACGGCCATCCGACGATCAT
up-xF GAATTCCTGCAGCCCGGGGGATCCTAGGTCCCGACTAACCCTGAG BamHI
up-xR GTGAAAGGCGTATTGATGGGGTGAAATGCCAATCGA
down-xF ATGATCGTCGGATGGCCCTACCCACACCTCATCCCC
down-xR CTTTTCTACGAGCTCCACCGCGGAGCCCAAACCAAGAGGCT SacII
up-lF GAATTCCTGCAGCCCGGGGGATCCACGATGAAACGGGATCGC BamHI
up-lR GAAAGGCGTATTGATGGCTGACGCATGATCGACG
down-lF ATGATCGTCGGATGGCCGTCTTCTGCTTCACCTCAAATAAC
down-lR GAATTCCTGCAGCCCGGGGGATCCCCGGATACAATTCGGCTG SacII
T-F GATAACTCGGCGTATGTTATTCAAG
T-R GAAGTGGTGGCCTAACTACGG
V1-F CAATACCGCGCTTACCCTATC
V1-R GTAATGGAGCAGTATCCATATCTGAA
V2-F GATGCTTCCACCGGAAGTTG
V2-R AGGTATCGCCGCTGCATAC
表1  试验所使用的菌株、载体与引物
图1  构建整合载体各片段和重组载体PCR验证
图2  突变菌株PCR验证
图3  基因编辑技术原理和表达盒在基因组的插入位置
图4  BL 2709-xynA-lipY2和BL 2709的细胞生物量的变化曲线
图5  BL 2709-xynA-lipY2和BL 2709菌株发酵液中还原糖含量的变化曲线
图6  BL 2709-xynA-lipY2和BL 2709菌株发酵培养基中总氮的含量变化曲线
图7  BL 2709-xynA-lipY2菌株木聚糖酶活力变化曲线
图8  BL 2709-xynA-lipY2和BL 2709菌株脂肪酶活力变化曲线
图9  BL 2709-xynA-lipY2和BL 2709碱性蛋白酶活力变化曲线
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