Gene modification and expression of α cold-adapted amylase in Aeromonas

doi:10.6048/j.issn.1001-4330.2024.02.025

Xinjiang Agricultural Sciences ›› 2024, Vol. 61 ›› Issue (2): 479-484.DOI: 10.6048/j.issn.1001-4330.2024.02.025

• Microbes·Physiology and Biochemistry·Animal Husbandry Veterinarian • Previous Articles Next Articles

Gene modification and expression of α cold-adapted amylase in Aeromonas

CHU Min(), SHI Yingwu, GU Meiying, YANG Hongmei, HUO Xiangdong, ZHANG Zhidong()

Xinjiang Laboratory of Special Environmental Microbiology/Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2023-06-21 Online:2024-02-20 Published:2024-03-19
Correspondence author: ZHANG Zhidong(1977-), male, from Urumqi, Xinjiang, Professor, Dr.research field:Microbial ecology,(E-mail)zhangzheddong@sohu.com
Supported by:
Key R & D Project in Xinjiang Uygur Autonomous Region "Xinjiang Agricultural Microbial Fermentation and Biological Enzyme Preparation Key Technology Research and Development(2022B02053-3);The Third Comprehensive Scientific Expedition In Xinjiang "Investigation of Stress-Resistant Microbial Resources in Tuha Basin and Its Application Potential Assessment"(2022xjkk1204);The Independent Cultivation Project Team Construction Special Project of Xinjiang Academy of Agricutural Sciences "Xinjiang Special Environment Microbial Resources Digging and Utilization Innovation Team"(nkyzztd-001)

气单胞菌属低温淀粉酶基因改造与原核表达

楚敏(), 史应武, 顾美英, 杨红梅, 霍向东, 张志东()

新疆农业科学院微生物应用研究所/新疆特殊环境微生物实验室,乌鲁木齐 830091

通讯作者: 张志东(1977-),男,新疆乌鲁木齐人,研究员,博士,研究方向为微生物生态, (E-mail)zhangzheddong@sohu.com
作者简介:楚敏(1977-),女,新疆乌鲁木齐人,副研究员,硕士,研究方向为微生物学与分子生物学,(E-mail)chuliu2002@163.com
基金资助:
新疆维吾尔自治区重点研发任务专项(2022B02053-3);第三次新疆综合科学考察(2022xjkk1204);新疆农业科学院自主培育项目团队建设专项(nkyzztd-001)

Abstract

Abstract:

【Objective】 In order to obtain the low-temperature amylase gene and its related functions, and better apply it in industrial production.【Methods】 Cloning of the low temperature ɑ-amylase gene from the starting strain LA7 to BL-21 (DE3). Other known low temperature ɑ-amylase gene was homology analysis,design specific primers,and C13 fragment was obtained by PCR amplification.Then clone it to pMAL-2X,Convert it to BL-21 (DE3),and screening blue and white spots.The ɑ-amylase gene was verified by PCR and double enzyme digestion of EcoRⅠ and Hind Ⅲ,the highly expressed bioengineering strain pMAL-2X- C13was obtained.The site directed mutagenic primers was design,take pMAL-2X- C13 as the template,three mutant strains of low temperature amylase gene C19, C29 and C43 were obtained.【Results】 SDS-PAGE test result display that the molecular size of the protein is 114 kD and the protein expression of mutant strain C19 was higher than that of strain pMAL-2X- C13.【Conclusion】 The protein expressed of mutant strain C19 more than the original strain pMAL-2X- C13.It provides a scientific and theoretical basis for the future application of the engineering bacteria in industrial production.

Key words: Aeromonas; α cold-adapted amylase; genetically engineered; procaryotic expression

摘要：

【目的】获取低温淀粉酶基因,分析其相关功能,为工业生产上的应用提供参考。【方法】以气单胞菌(Aeromonas)LA77为出发菌株克隆低温α-淀粉酶基因,以BL-21(DE3)为宿主菌进行克隆。分析其它已知气单胞菌属低温α-淀粉酶基因同源性,设计特异引物, PCR扩增获得C13片段,将其克隆到pMAL-2X,转化到BL-21(DE3)中,筛选蓝白斑,验证PCR及EcoRⅠ和HindⅢ的双酶切,获得高效表达生物工程菌株pMAL-2X-C13。设计定点突变引物,以pMAL-2X-C13为模板,获得低温淀粉酶基因突变株三株C19、C29、C43。【结果】表达蛋白的分子大小为114kD,突变菌株C19蛋白表达量均高于pMAL-2X-C13。【结论】低温淀粉酶基因突变株C19比原始菌株pMAL-2X-C13淀粉酶基因蛋白表达量更高。

关键词: 气单胞菌属, α-低温淀粉酶, 基因改造, 原核表达

CLC Number:

S188

CHU Min, SHI Yingwu, GU Meiying, YANG Hongmei, HUO Xiangdong, ZHANG Zhidong. Gene modification and expression of α cold-adapted amylase in Aeromonas[J]. Xinjiang Agricultural Sciences, 2024, 61(2): 479-484.

楚敏, 史应武, 顾美英, 杨红梅, 霍向东, 张志东. 气单胞菌属低温淀粉酶基因改造与原核表达[J]. 新疆农业科学, 2024, 61(2): 479-484.

Figures/Tables 8

Fig.1 DNA of cold-adapted amylase gene Note:1-5:the purpose of bands M: 2 000 bp DNA mark:2 kb、1.6 kb、1 kb、750 bp、500 bp、250 bp

Fig.2 PCR product of cold-adapted amylase gene Note:1:the purpose of band

Fig.3 Amino acid phylogenetic tree of the different source of amylase

Fig.4 EcoR I and Hind Ⅲ restriction enzyme electrophoresis Note:1-4:Positive clone DL2000 Marker:2 000 bp、1 600 bp、1 000 bp、750 bp、500 bp、250 bp

Fig.5 Clony PCR Note:1-4:Positive clone DL2000 Marker:2 000 bp、1 600 bp、1 000 bp、750 bp、500 bp、250 bp

Fig.6 PCR product of mutant 19#,29# Note:1-4: product of mutant 19# 5-8:product of mutant 29# 9-12:product of mutant 43# M:DL2000 2 kb、1.6 kb、1 kb、750 bp、500 bp、250 bp、100 bp

Fig.7 Protein expression of 13# gene Note:1:empty vector P2X before induction; 2:after induction of empty vector P2X; 3:13 #before induction;4:13 #after induction;5:the Supernatant after induction;6:the precipitation after induction Marker:97 KD、66 KD、53 KD、36 KD、24 KD

Fig.8 Induced expression of different mutants Note:1:13 #before induction 2:13 # supernatant after induction 3:13 # precipitation after induction;4:19 # supernatant after induction 5:19 # precipitation after induction 6:29 # supernatant after induction;7:29# precipitation after induction 8:43 # supernatant after induction 9:43 # precipitation after induction Marker: 97 KD、66 KD、53 KD、36 KD、24 KD

References 12

[1]	Tucke G A,Woods L F J著,李雁群,肖功年译. 酶在食品加工中的应用(第二版)[M]. 北京: 中国轻工业出版社, 2002.
	Tucke G A, Woods L F J. Enzymes in food processing (2rd Edition translated by LI Yanqun,XIAO Gongnian)[M]. Beijing: China Light Industry Press, 2002.
[2]	Charles G, Mohamed A, Mostafa B, et al. Cold-adapted enzymes:from fundamentals to biotechnology[J]. Trends in Biotechnology, 2000, 18(3):103-107. DOI URL
[3]	Feller G, D’Amico D, Gerday C. Thermodynamic stability of a cold active amylase from the psych rophile Alteromonas haloplanctzs[J]. Biochemistry, 1999, 38(14):4613-4619. PMID
[4]	Cotârleţ M, Negoiţâ T G, Bahrim G E, et al. Partial characterization of cold active amylases and proteases of Streptomyces sp.from Antarctica[J]. Brazilian Journal of Microbiology, 2011, 42(3)868-877. DOI PMID
[5]	Zhang J W, Zeng R Y. Purification and Characterization of a Cold-Adapted α-Amylase Produced by Nocardiopsis sp.7326 Isolated from Prydz Bay, Antarctic[J]. Marine Biotechnology, 2008, 10(1):75-82. DOI URL
[6]	王晓红, 茆军, 傅力, 等, 低温淀粉酶产生菌的筛选及酶学性质研究[J], 农产品加工, 2007, (1):7-9.
	WANG Xiaohong, MAO Jun, FU Li, et al. Filtration and Enzymology Property of cold-active Amyase-producing Fungus[J]. Farm Products Processing, 2007,(1):7-9.
[7]	Gobius K S, Pemberton J M. Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila.[J]. Journal of Bacteriology, 1988, 170(3):1325-1332. PMID
[8]	Lu M S, Fang Y W, Li H Z, et al. Isolation of a novel cold-adapted amylase-producing bacterium and study of its enzyme production conditions[J]. Annals of Microbiology, 2010, 60(3):557-563. DOI URL
[9]	Alikhajeh J, Khajeh K, Ranjbar B, et al. Structure of Bacillus amyloliquefaciens-amylase at high res o lution:i mplications for thermal stability[J]. Acta Cryst, 2010, 66(2):121-129.
[10]	Lu M S, Wang S J, Fang Y W, et al. Cloning, expression, purification, and characterization of cold-adapted α-amylase from Pseudoalteromonas arctica GS230.[J]. Protein Journal, 2010, 29(8):591-597. DOI PMID
[11]	闫亚茹. 理性设计提高低温α-淀粉酶稳定性的研究[D]. 保定: 河北农业大学, 2018.
	YAN Yaru. Improvement of the stability of cold-adaption α-amylase through rational design[D]. Baoding: Hebei Agricultural University, 2018.
[12]	王翔, 沈盼盼, 周玉玲, 等. 低温淀粉酶菌株分离及其酶学性质研究[J]. 湖北大学学报(自然科学版), 2018, 40(5):448-451,456.
	WANG Xiang, SHEN Panpan, ZHOU Yuling, et al. The producing strain isolation and enzymatic properties of a cold-active amylase[J]. Journal of Hubei University(Natural Science Edition), 2018, 40(5):448-451,456.

Gene modification and expression of α cold-adapted amylase in Aeromonas

气单胞菌属低温淀粉酶基因改造与原核表达

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