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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  2024, Vol. 44 Issue (4): 23-32    DOI: 10.13523/j.cb.2308018
研究报告     
耐热碱性蛋白酶AH-101的异源表达、酶学性质与洗涤应用研究*
郝漫,邵岚莹,公为峰,惠威,史超硕,路福平,张会图**()
天津科技大学生物工程学院 天津 300457
Heterologous Expression, Characterization and Washing Application of Thermostable Alkaline Protease AH-101
HAO Man,SHAO Lanying,GONG Weifeng,HUI Wei,SHI Chaoshuo,LU Fuping,ZHANG Huitu**()
College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
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摘要：

目的:通过NCBI(National Center for Biotechnology Information)数据库筛选可以应用于洗涤工业的蛋白酶,使用地衣芽孢杆菌进行高效表达。方法:利用同源重组的方式将蛋白酶基因整合到地衣芽孢杆菌基因组中,研究重组蛋白酶的酶学特性和洗涤效果。结果:该重组酶的最适作用温度为60℃,最适pH为11.0;AH-101具有良好的耐热性,可以在20~55℃范围内保持稳定,在60℃保温1 h,剩余活性仍高于30%;此外,该酶与表面活性剂和液体洗涤剂具有良好的兼容性,在终浓度为0.1%和0.5%的表面活性剂和液体洗涤剂中孵育2 h,重组酶的酶活力损失小于30%。结论:成功构建了一株含有外源蛋白酶的地衣芽孢杆菌重组菌株,对重组蛋白酶的特性和洗涤效果进行研究,该重组蛋白酶可以作为环保型酶制剂应用到洗涤工业中。
关键词： 耐热碱性蛋白酶;  地衣芽孢杆菌;  整合表达;  洗涤工业    
Abstract:

Objective: To screen the protease which can be used in washing industry through NCBI database and express it efficiently using Bacillus licheniformis. Methods: The protease gene was integrated into the genome of Bacillus licheniformis by homologous recombination, the fermentation was studied, and the enzymatic characteristics and washing effect of the recombinant protease were investigated. Results: The optimum temperature and pH of the recombinant enzyme were 60℃ and 11.0, respectively. AH-101 has good heat resistance, which can be kept stable in the range of 20 ~ 55℃, and the residual activity is still higher than 30% at 60℃ for 1 h. In addition, the enzyme has good compatibility with surfactants and liquid detergents, and the loss of activity of the recombinant enzyme is less than 30% when incubated for 2 h in surfactants and liquid detergents with final concentrations of 0.1% and 0.5%. Conclusion: A recombinant Bacillus licheniformis strain containing exogenous protease was successfully constructed, and the properties and washing effect of the recombinant protease were investigated. The recombinant protease can be used as an environmentally-friendly enzyme preparation in the washing industry.
Key words: Thermostable alkaline protease    Bacillus licheniformis    Integrated expression    Washing application
收稿日期: 2023-08-10 出版日期: 2024-04-30
ZTFLH:  Q814  
基金资助: * 国家重点研发计划(2021YFC2100400)
通讯作者: ** 电子信箱:hzhang@tust.edu.cn   
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郝漫
邵岚莹
公为峰
惠威
史超硕
路福平
张会图

引用本文:

郝漫, 邵岚莹, 公为峰, 惠威, 史超硕, 路福平, 张会图. 耐热碱性蛋白酶AH-101的异源表达、酶学性质与洗涤应用研究*[J]. 中国生物工程杂志, 2024, 44(4): 23-32.

HAO Man, SHAO Lanying, GONG Weifeng, HUI Wei, SHI Chaoshuo, LU Fuping, ZHANG Huitu. Heterologous Expression, Characterization and Washing Application of Thermostable Alkaline Protease AH-101. China Biotechnology, 2024, 44(4): 23-32.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2308018    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I4/23

菌株/质粒 名称 特性/目的 来源
菌株 Escherichia coli JM109 质粒扩增 TaKaRa
Escherichia coli EC135 pM.Bam 质粒DNA的甲基化修饰 中国科学院微生物研究所
BLΔEBV 分泌表达AH-101的宿主菌株 天津科技大学
Bacillus licheniformis Papr-AH-101 具有整合到apr位点的AH-101基因的重组菌株 本研究
Bacillus licheniformis Papr-AH101-2 具有两个拷贝的Papr-AH-101表达盒的重组菌株 本研究
质粒 pKSVT pTU温敏型自杀质粒 湖北大学
pTU-apr-AH-101 AH-101整合apr位点载体 本研究
pTU-epr-AH-101 AH-101整合epr位点载体 本研究
表1  研究中使用的菌株和质粒
用途 引物 序列(5'-3')
PCR扩增带有apr
位点同源臂的AH-101		 AH-101-apr-F CTGCTCAACCGGCGAAAAATACTAGTAGCGAGGAGAAAAAGGAATATT
AH-101-apr-R TTTCGCAGGATAGCCAATGCGGCCGCTCAGTGGTGGTGGTGGTGGTGTTGT
GTTGCACGTCCAGCAT
PCR扩增带有epr位点
同源臂的表达盒		 AH-101-epr-F TAAAAAAGGAGGAGAGCCGGAACAGTTATTAATAACCAAAAAATTTTAAATTGG
AH-101-epr-R GATTCCGGGCTTTTTTCCGTAGCGGCCGCTCAGTGGTGGTGGTGGTGGTGTTGTGTTGCAC
GTCCAGCA
PCR克隆apr位点同源臂
Sma I/Sac I		 apr-UF CCCTTAACGAATTCCTGCAGCCCGGGATCTTGCAGGTTATCGTCAACGA
apr-UR AAGGGATTCCTCCCTTCTGTCACGCCTCTAAGGCGTTCATT
apr-DF ATGAACGCCTTAGAGGCGTGACAGAAGGGAGGAATCCCTTCTT
apr-DR TCCACCGCGGTGGCGGCCGCTCTAGAGTACGCTTCATCCTCAACCTGC
PCR克隆epr 位点同源臂
Sma I/Xba I		 epr-UF ACGAATTCCTGCAGCCCGGGATAGAAGCAACCAACCCTATTTGCG
epr-UR TTTGGTTATTAATAACTGTTCCGGCTCTCCTCCTTTTTTAGC
epr-DF CACCACCACTGAGCGGCCGCTACGGAAAAAAGCCCGGAATC
epr-DR TTGATCTTTTCTACGAGCTCCCTTTCAGGAGGGCAACGAG
重组质粒验证 pTU-F GTTTGATGATTGGTTCCCGCT
pTU-R CTGAAGCCAGTTACCTTCGGAA
apr位点双交换验证 apr-VF ACAAGAGAATGACTCCCGATGC
apr-VR TGGAAGAAGCCTTCATATTGCAG
epr位点双交换验证 epr-VF TCTTTCTTCTCTTCAGCCACTTCC
epr-VR GTCCGCTGTTTCTCCTGCTTT
表2  研究中使用的引物
图1  基因编辑过程 A:质粒的构建 B:基因组双交换
图2  重组菌株的构建过程 A:目的片段的获得,泳道1为AH-101片段,泳道2为表达盒片段 B:重组质粒的构建,泳道1为epr位点整合用载体,泳道2为apr位点整合用载体 C:基因组双交换过程,泳道1为apr位点双交换对照菌株,泳道2为apr位点阳性克隆,泳道3为epr位点对照菌株,泳道4为epr位点阳性克隆
图3  重组菌株基因组排列
图4  两种重组菌株蛋白酶活力的时间曲线
图5  重组蛋白酶的最适作用温度和温度稳定性 A: 温度对重组蛋白酶活力的影响 B: 重组蛋白酶的温度稳定性
图6  重组蛋白酶的最适作用pH和pH稳定性 A: pH对重组蛋白酶活力的影响 B: 重组蛋白酶的pH稳定性
金属离子 1 mmol/L 5 mmol/L
对照 100 100
Mg2+ 105.38 ± 1.16 115.42 ± 0.98
Zn2+ 64.86 ± 0.49 46.74 ± 1.29
Ca2+ 103.30 ± 1.01 103.23 ± 0.84
Na+ 94.89 ± 2.24 92.47 ± 0.20
K+ 90.00 ± 0.91 89.28 ± 0.79
Cu2+ 78.94 ± 1.98 54.65 ± 1.65
Co2+ 4.64 ± 0.20 1.27 ± 0.20
表3  金属离子对重组蛋白酶活性的影响
添加剂 名称 0.1% 0.5%
对照 - 100 100
表面活性剂 SDS 94.46 ± 0.73 89.70 ± 0.97
吐温20 92.05 ± 1.07 79.68 ± 1.02
Trix-100 90.03 ± 0.12 88.07 ± 0.42
洗涤剂 标准配方洗衣液 93.67 ± 0.34 72.83 ± 0.30
汰渍 93.50 ± 0.65 76.19 ± 0.18
超能 92.89 ± 1.01 71.97 ± 0.54
威露士 93.22 ± 0.30 74.06 ± 0.65
表4  表面活性剂和洗涤剂对重组蛋白酶活性的影响
温度/℃ 水+标准配方洗衣液 水+标准配方洗衣液+AH-101 去污比值
F2 F1 Ri F2 F1 Ri Pi
20 25.65 ± 0.43 20.02 ± 0.16 6.63 ± 0.44 31.08 ± 0.77 19.98 ± 0.40 11.10 ± 0.68 1.67
30 24.29 ± 0.47 19.64 ± 0.23 4.65 ± 0.61 34.47 ± 0.46 20.20 ± 0.10 14.28 ± 0.40 3.07
40 26.29 ± 0.84 19.68 ± 0.21 6.61 ± 0.84 36.86 ± 1.22 19.40 ± 0.36 17.46 ± 1.24 2.6
50 24.35 ± 0.47 19.59 ± 0.26 4.76 ± 0.66 41.51 ± 0.88 19.50 ± 0.40 22.01 ± 1.03 4.62
60 24.50 ± 0.38 19.77 ± 0.18 4.73 ± 0.52 40.32 ± 0.54 19.72 ± 0.20 20.59 ± 0.63 4.35
表5  添加AH-101前后洗涤效果对比
图7  添加AH-101前后在50℃条件下的洗涤效果对比 A:JB-02蛋白污布,从上到下依次为未经过洗涤的、经过水+标准洗涤剂洗涤的和经过水+标准洗涤剂+AH-101溶液洗涤的 B:经过水+标准洗涤剂洗涤的JB-02蛋白污布电镜图 C:经过水+标准洗涤剂+AH-101溶液洗涤的JB-02蛋白污布电镜图
样品 洗后白度值 洗前白度值 试片白度差
AH-101 34.47 ± 0.46 20.20 ± 0.10 14.28 ± 0.40
2709 36.89 ± 0.48 23.47 ± 0.29 13.41 ± 0.51
商品酶1 33.21 ± 0.79 19.80 ± 0.15 13.48 ± 0.89
商品酶2 28.54 ± 0.72 18.15 ± 0.32 10.39 ± 0.99
商品酶3 34.47 ± 0.83 22.06 ± 0.22 18.25 ± 0.91
表6  AH-101与常用碱性蛋白酶的洗涤对比实验
[1] 石赟, 张剑. 工业清洗技术的发展现状及酶制剂在清洗剂配方中的应用. 中国洗涤用品工业, 2021(5): 64-69.
Shi Y, Zhang J. The development status of industrial cleaning technology and the application of enzyme preparation in cleaning agent formulation. China Cleaning Industry, 2021(5): 64-69.
[2] Pandey P, Gopal B. Effect of detergents on the growth of two aquatic plants: Azolla pinnata and Hydrilla verticillata. Environment & We: An International Journal of Science and Technology, 2010, 5: 107-114.
[3] Chaturvedi A D, Tiwari K L. Effect of household detergents (surfactants) degraded through aquatic fungi. Recent Research in Science and Technology, 2013, 5: 5.
[4] Cowan-Ellsberry C, Belanger S, Dorn P, et al. Environmental safety of the use of major surfactant classes in North America. Critical Reviews in Environmental Science and Technology, 2014, 44(17): 1893-1993.
pmid: 25170243
[5] Mahmood Q, Shaheen S, Bilal M, et al. Chemical pollutants from an industrial estate in Pakistan: a threat to environmental sustainability. Applied Water Science, 2019, 9(3): 47.
doi: 10.1007/s13201-019-0920-1
[6] Sobrino-Figueroa A. Toxic effect of commercial detergents on organisms from different trophic levels. Environmental Science and Pollution Research, 2018, 25(14): 13283-13291.
doi: 10.1007/s11356-016-7861-0
[7] 李浩, 高楠. 酶领生物洗涤未来——更清洁、更环保、更可持续的商业发展. 中国洗涤用品工业, 2021(2): 49-55.
Li H, Gao N. Enzyme leads the future of biological washing-cleaner, more environmentally friendly and more sustainable business development. China Cleaning Industry, 2021(2): 49-55.
[8] 张天翼. 洗涤用品绿色发展展望. 中国洗涤用品工业, 2020(5): 17-24.
Zhang T Y. Prospects of green chemistry in fabric & home care. China Cleaning Industry, 2020(5): 17-24.
[9] Solanki P, Putatunda C, Kumar A, et al. Microbial proteases: ubiquitous enzymes with innumerable uses. 3 Biotech, 2021, 11(10): 428.
doi: 10.1007/s13205-021-02928-z pmid: 34513551
[10] Gurumallesh P, Alagu K, Ramakrishnan B, et al. A systematic reconsideration on proteases. International Journal of Biological Macromolecules, 2019, 128: 254-267.
doi: S0141-8130(18)35199-7 pmid: 30664968
[11] Barrett A J. Proteolytic enzymes:nomenclature and classification. 2nd ed. Oxford: Oxford University Press, 2001: 1-20.
[12] Kobayashi T, Hakamada Y, Adachi S, et al. Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Applied Microbiology and Biotechnology, 1995, 43(3): 473-481.
pmid: 7632397
[13] Joshi S, Satyanarayana T. Biotechnology of cold-active proteases. Biology, 2013, 2(2): 755-783.
doi: 10.3390/biology2020755
[14] Jablaoui A, Kriaa A, Akermi N, et al. Biotechnological applications of serine proteases: a patent review. Recent Patents on Biotechnology, 2018, 12(4): 280-287.
doi: 10.2174/1872208312666180924112007 pmid: 30246645
[15] Bashir F, Asgher M, Hussain F, et al. Development and characterization of cross-linked enzyme aggregates of thermotolerant alkaline protease from Bacillus licheniformis. International Journal of Biological Macromolecules, 2018, 113: 944-951.
doi: 10.1016/j.ijbiomac.2018.03.009
[16] Konglom N, Chuensangjun C, Pechyen C, et al. Production of poly-γ-glutamic acid by Bacillus licheniformis: synthesis and characterization. Journal of Metals, Materials and Minerals, 2012, 22: 7-11.
[17] Guo J, Cheng G, Gou X Y, et al. Comprehensive transcriptome and improved genome annotation of Bacillus licheniformis WX-02. FEBS Letters, 2015, 589(18): 2372-2381.
doi: 10.1016/j.febslet.2015.07.029
[18] 房红磊. 全能核酸酶NucA的高效分泌表达及分离提取工艺研究. 天津: 天津科技大学, 2022.
Fang H L. The high-effiency secretory expression, separation and purification of benzonase nuclease Nuc A. Tianjin: Tianjin University of Science & Technology, 2022.
[19] Sambrook J, Russell D W. Molecular cloning:laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press, 2001.
[20] 叶棋浓. 现代分子生物学技术及实验技巧. 北京: 化学工业出版社, 2015.
Ye Q N. Current molecular biology techniques and tips. Beijing: Chemical Industry Press, 2015.
[21] Zhou C X, Liu H, Yuan F Y, et al. Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing. International Journal of Biological Macromolecules, 2019, 122: 329-337.
doi: 10.1016/j.ijbiomac.2018.10.170
[22] Zhou C X, Yang G C, Zhang L, et al. Construction of an alkaline protease overproducer strain based on Bacillus licheniformis 2709 using an integrative approach. International Journal of Biological Macromolecules, 2021, 193: 1449-1456.
doi: 10.1016/j.ijbiomac.2021.10.208
[23] 中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会. 中华人民共和国推荐性国家标准: 蛋白酶制剂GB/T 23527-2009. 北京: 中国标准出版社, 2009.
General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China.National standard (recommended) of the People’s Republic of China: proteinase preparations GB/T 23527-2009. Beijing: Standards Press of China, 2009.
[24] Rios P, Bezus B, Cavalitto S, et al. Production and characterization of a new detergent-stable keratinase expressed by Pedobacter sp. 3.14.7, a novel Antarctic psychrotolerant keratin-degrading bacterium. Journal of Genetic Engineering and Biotechnology, 2022, 20(1): 81.
doi: 10.1186/s43141-022-00356-x
[25] Zhang H, Li H, Liu H R, et al. The application of a halotolerant metalloprotease from marine bacterium Vibrio sp. LA-05 in liquid detergent formulations. International Biodeterioration & Biodegradation, 2019, 142: 18-25.
[26] Takami H, Akiba T, Horikoshi K. Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101. Applied Microbiology and Biotechnology, 1989, 30(2): 120-124.
[27] Takami H, Akiba T, Horikoshi K. Characterization of an alkaline protease from Bacillus sp. no. AH-101. Applied Microbiology and Biotechnology, 1990, 33(5): 519-523.
pmid: 1370008
[28] Contesini F J, de Melo R R, Sato H H. An overview of Bacillus proteases: from production to application. Critical Reviews in Biotechnology, 2018, 38(3): 321-334.
doi: 10.1080/07388551.2017.1354354 pmid: 28789570
[29] Baweja M, Nain L, Kawarabayasi Y, et al. Current technological improvements in enzymes toward their biotechnological applications. Frontiers in Microbiology, 2016, 7: 965.
doi: 10.3389/fmicb.2016.00965 pmid: 27379087
[30] 陈坤. 2709碱性蛋白酶的高产工程菌株构建及应用性能分析. 天津: 天津科技大学, 2018.
Chen K. Construction of the engineering bacteria with high yield of alkaline protease 2709 and its application performance. Tianjin: Tianjin Universuty of Science and Technology, 2018.
[31] Li F, Yang L Y, Lv X, et al. Purification and characterization of a novel extracellular alkaline protease from Cellulomonas bogoriensis. Protein Expression and Purification, 2016, 121: 125-132.
doi: 10.1016/j.pep.2016.01.019
[32] Guleria S, Walia A, Chauhan A, et al. Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP 1 isolated from apple rhizosphere. Journal of Basic Microbiology, 2016, 56(2): 138-152.
doi: 10.1002/jobm.v56.2
[33] 国家质量监督检验检疫总局, 国家标准化管理委员会. GB/T 13174-2008, 衣料用洗涤剂去污力及循环洗涤性能的测定. 北京: 中国标准出版社, 2008.
General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration. GB/T 13174-2008, determination of detergency and cycle of washing property for laundry detergents. Beijing: Standarda Press of China, 2008.
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