Heterologous expression and its enzymatic properties of β-galactosidase from a <em>Marinomonas</em> strain isolated from the Arctic Ocean

Sun Xi; Liao Li; Ding Haitao; Liu Shuang; Chen Bo

doi:10.3969/j.issn.0253-4193.2015.11.016

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Article Navigation > Haiyang Xuebao > 2015 > 37(11): 165-177

Sun Xi, Liao Li, Ding Haitao, Liu Shuang, Chen Bo. Heterologous expression and its enzymatic properties of β-galactosidase from a Marinomonas strain isolated from the Arctic Ocean[J]. Haiyang Xuebao, 2015, 37(11): 165-177. doi: 10.3969/j.issn.0253-4193.2015.11.016

Citation:

Sun Xi, Liao Li, Ding Haitao, Liu Shuang, Chen Bo. Heterologous expression and its enzymatic properties of β-galactosidase from a Marinomonas strain isolated from the Arctic Ocean[J]. Haiyang Xuebao, 2015, 37(11): 165-177. doi: 10.3969/j.issn.0253-4193.2015.11.016

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Heterologous expression and its enzymatic properties of β-galactosidase from a Marinomonas strain isolated from the Arctic Ocean

doi: 10.3969/j.issn.0253-4193.2015.11.016

1.
College of Bioengineering, East China University of Science and Technology, Shanghai 200237, China;Key Laboratory for Polar Science, State Oceanic Administration, Polar Research Institute of China, Shanghai 200136, China
2.
Key Laboratory for Polar Science, State Oceanic Administration, Polar Research Institute of China, Shanghai 200136, China
3.
College of Bioengineering, East China University of Science and Technology, Shanghai 200237, China

Received Date: 2015-04-10

Abstract

Abstract

A Marinomonas strain isolated from an ice core sample collected from Canada Basin, Arctic Ocean, displayed high β-galactosidase activity in the preliminary screening process. To get its overall understanding towards its enzymatic properties, the galt gene obtained by hiTAIL-PCR amplification was inserted into plasmid pET-28a(+), then transformed into E.coli BL21 (DE3). The recombinant enzyme was purified to electrophoretic homogeneity by one step Ni²⁺ affinity chromatography. The highest yield was achieved with 0.07mM IPTG when cultivated in 20℃ for 22 h. The molecular weight of the monomeric GALT was estimated as about 6.6×10⁴ g/mol and the native GALT was confirmed as homologous trimer through native-PAGE. The optimum temperature of GALT was 35℃ and showed good thermal stability at the temperature of 60℃ below. The optimum pH of GALT was 9.0 and was stable between pH 6.0 and 11.0. GALT showed high tolerance to salinity and the optimum NaCl concentration was 0.5 mol/L. Mineral ions Fe²⁺ and Mn²⁺ were identified as enzyme activators, Mg²⁺, K⁺, DTT and EDTA showed no significant influence towards the enzyme activity, while Zn²⁺ and L-glutathione inhibited the activity strongly. GALT was able to hydrolyse Gal β1-4 GlcNAc but unable to hydrolyze Gal β1-3 GalNAc and Gal β1-3 GlcNAc. In this study, the β-galactosidase gene from Marinomonas sp. BSi20584 was successfully expressed in E.coli system, and a systematic understanding of the enzymatic properties of the recombinant GALT was acquired, which will provide the detailed enzymatic data for further studies on its metabolic adaptation and potential applications.
- the Arctic Ocean,
- β-galactosidase,
- Marinomonas,
- heterologous expression,
- enzymatic properties

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References(31)

References

Khalid A A, Rahim, Byong H L, et al. Specifieity, inhibitory, studies and oligoaseeharide fomartoin by β-Galacotsidaes form psychrotorphie Bacillus subtilis KL88[J]. J Dairy Sci, 1991, 74: 1773-1778.

Cantarel B L, Coutinho P M, Rancurel C. The carbohydrate-active enzymes database(CAZy): an expert resource for glycogenomics[J]. Nucleic Acids Res, 2009, 37: 233-238.

张莉, 李庆章, 田雷. β-半乳糖苷酶研究进展[J]. 东北农业大学学报, 2009, 40(7): 128-131. Zhang Li, Li Qingzhang, Tian Lei. Research advancement of β-Galacotsidaes[J]. Journal of Northeast Agricultural University, 2009, 40(7): 128-131.

林金资, 卢蓉蓉, 李强军, 等. 中性乳糖酶高产菌的选育[J]. 中国乳品工业, 1995(3): 113-116. Lin Jinzi, Lu Rongrong, Li Qiangjun. Screening of high-yielding strains for neutral lactose[J]. China Dairy Industry, 1995(3): 113-116.

Dong C M, Bai X H, Lai Q L, et al. Draft genome sequence of Marinomonas sp. strain D104, a polycyclic aromatic hydrocarbon-degrading bacterium from the deep-sea sediment of the Arctic Ocean[J]. Genome Announcements, 2014, 2(1): 1211-1213.

Lucas-Elío P, Goodwin L, Woyke T, et al. Complete genome sequence of Marinomonas posidonica type strain (IVIA-Po-181(T))[J]. Standards in Genomic Sciences, 2012, 7: 31-43.

Lucas-Elío P, Goodwin L, Woyke T, et al. Complete genome sequence of the melanogenic marine bacterium Marinomonas mediterranea type strain (MMB-1T)[J]. Standards in Genomic Sciences, 2012, 5: 63-73.

周莉莉, 陈瑞琴, 陈秀兰, 等. 应用hiTAIL-PCR技术克隆Marinomonas sp.BSI20584菌株β-半乳糖苷酶基因[J]. 极地研究, 2013, 25(3): 250-256. Zhou Lili, Chen Ruiqin, Chen Xiulan, et al. Cloning of beta-galactosidase genes from Marinomonas sp. BSI20584 by hiTAIL-PCR[J]. Polar Research, 2013, 25(3): 250-256.

郑海学, 刘湘涛, 尚佑军, 等. 猪水泡病病毒结构蛋白基因的克隆及其蛋白二级结构和淋巴细胞表位的预测[J]. 中国兽医科学, 2005, 35(11): 843-850. Zheng Haixue, Liu Xiangtao, Shang Youjun, et al. Cloning of SVDV structural protein gene and predictions of secondary structure and lymphocyte epitopes of structural protein[J]. Chinese Journal of Veterinary Science and Technology, 2005, 35(11): 843-850.

周莉莉. 北极海单胞菌β-半乳糖苷酶的原核表达及其转糖基活性检测[D]. 上海: 华东理工大学, 2012. Zhou Lili. Expression, purification and transgalactosylation of β-galactosidase from the Marinomonas strain isolated from Arctic Ocean[D]. Shanghai: East China University of Science and Technology, 2012.

Vanessa S, Terra K A, Homer S G, et al. Characterization of novel beta-galactosidase activity that contributes to glycoprotein degradation and virulence in Streptococcus pneumoniae[J]. Infection and Immunity, 2010, 78(1): 348-357.

Hidaka M, Fushinobu S, Ohtsu N, et al. Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose[J]. Journal of Biochemistry and Molecμlar Biology, 2002, 322(1): 79-91.

Matthews B W. The structure of E.coli β-galactosidase[J]. Comptes Renndus Biologies, 2005, 328(6): 549-556.

Bultema J B, Kuipers B J H, Dijkhuizen L. Biochemical characterization of mutants in the active site residues of the beta-galactosidase enzyme of Bacillus circμlans ATCC 31382[J]. FebS Open Bio, 2014, 11(4): 354-361.

段文娟, 杨娇艳, 李卓夫, 等. 来源于嗜热古细菌Pyrococcus furiosus乳糖酶基因的原核表达及酶学性质分析[J]. 中国农业科技导报, 2008, 10(2): 76-81. Duan Wenjuan, Yang Yanyan, Li Zhuofu, et al. Expression of beta-galactosidase from Pyrococcus furiosus in E.coli and Analysis of Lactose properities[J]. Journal of Agricultural Science and Technology, 2008, 10(2): 76-81.

Sun X J, Duan X G, Wu D, et al. Characterization of Sulfolobus solfataricus beta-galactosidase mutant F441Y expressed in Pichia pastoris[J]. Journal of the Science of Food and Agriculture, 2013, 94(7): 94-97.

DasSarma S, Capes M D, Karan R, et al. Amino acid substitutions in cold-adapted proteins from Halorubrum lacusprofundi, an extremely halophilic microbe from Antarctica[J]. PLOS ONE, 2013, 8: 82-90.

Wang C, Zhang C W, Yu Q, et al. Beta-galactosidase gene from Lactobacillus delbrueckii subsp. bulgaricus gets non-fusion expression in Escherichia coli[J]. Sichuan Da Xue Xue Bao Yi Xue Ban, 2008, 39: 544-546.

Xu K, Tang X A; Gai Y B, et al. Molecular characterization of cold-inducible beta-galactosidase from Arthrobacter sp. ON14 isolated from Antarctica[J]. Journal of Microbilogy and Biotechnology, 2011, 21: 236-238.

Dabrowski S, Sobiewska G, Maciunska J, et al. Cloning, expression, and purification of the His6-Tagged thermostable β-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme[J]. Protein Expression and Purification, 2000, 19(1): 107-112.

Hildebrandt P, Wanarska M, Kur J. A new cold-adapted beta-D-galactosidase from the Antarctic Arthrobacter sp. 32c-gene cloning, overexpression, purification and properties[J]. BMC Microbiology, 2009, 9(1): 151-152.

Halbmayr E, Mathiesen G, Nguyen T, et al. High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a sakacin P-based expression system[J]. Agricultural and Food Chemistry, 2008, 56(12): 4710-4719.

Maischberger T, Mierau I, Peterbauer C K, et al. High-level expression of Lactobacillus β-galactosidases in Lactococcus lactis using the food-grade. nisin-controlled expression system NICE[J]. Journal of Agricultural and Food Chemistry, 2010, 58 (4): 2279-2287.

Kim C S, Ji E S, Oh D K. Characterization of a thermostable recombinant β-galactosidase from Thermotoga maritima[J]. Journal of Applied Microbiology, 2004, 97 (5): 1006-1014.

Li L, Zhang M, Jiang Z, et al. Characterisation of a thermostable family 42 β-galactosidase from Thermotoga maritima[J]. Food Chemistry, 2009, 112 (4): 844-850.

Kang S K, Cho K K, Ahn J K, et al. Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21; cloning, expression, and enzyme characterization[J]. Journal of biotechnology, 2005, 116 (4): 337-346.

Pessela B C C, Vian A, Mateo C, et al. Overproduction of Thermus sp. Strain T2 β-galactosidase in Escherichia coli and preparation by using tailor-made metal chelate supports[J]. Applied and Environmental Microbiology, 2003, 69(4): 1967-1972.

张凤玉, 胡丹, 龚秀芳, 等. 2型猪链球菌β-D-半乳糖苷酶基因的克隆表达及酶活性测定[J]. 中国生物工程杂志, 2014, 34(2): 39-44. Zhang Fengyu, Hu Dan, Gong Xiufang, et al. Cloning, expression and identification of gene encoding the β-Galactosidases of Streptococcus suis serotype 2[J]. China Biotechnology, 2014, 34(2): 39-44.

聂春明. 乳酸杆菌β-D-半乳糖苷酶重叠基因的克隆、表达及酶学性质分析[D].呼和浩特市:内蒙古农业大学, 2012. Nie Chunming. The overlapping gene cloning, expression and characterization of a β-galactosidases from Lactobacillus crispatus[D]. Hohhot: Inner Mongolia Agricultural University, 2012.

Hung M N, Xia Z, Hu N T, et al. Molecular and biochemical analysis of two β-galactosidases from Bifidobacterium infantis HL96[J]. Applied and Environmental Microbiology, 2001, 67 (9): 4256-4263.

Cummings S P, Black G W. Polymer hydrolysis a cold climate[J]. Extremophiled, 1999, 3(2): 81-87.

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