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Expression, Purification and Enzymatic Properties of β-glucosidase from Lactobacillus paracasei |
Yu-feng XIE1,2,Xue-mei HAN1,Fu-ping LU1,**() |
1 State Key Laboratory of Food Nutrition and Safety, College of Biotechnology,Tianjin University of Science & Technology, Tianjin 300457, China 2 College of Food Science and Engineering, Harbin University, Harbin 150086,China |
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Abstract To improve the conversion efficiency of glucoside to small molecule compounds, the gene encoded β-glucosidase from Lactobacillus paracasei TK1501 was inserted into pET28a(+), and further transformed into E. coli BL21(DE3) for heterologous expression. The recombinant enzyme, which purified by nickel affinity chromatography, is conferred with the high specific activity of 675.56U/mg, and the molecular weight of 86.63kDa.The biochemical characterization of this recombinant enzyme shows that it exhibit the highest bio-activity in 30℃ and pH of 6.5, and the β-glucosidase activity was barely inhibited by Mg 2+ and Ca 2+, but largely by Cu 2+ with even no catalytic activity. It was also found that this enzyme possess a broad substrates specificity toward genistin, daidzin, daidzein, geniposide, salicin, heptosporin, polydatin and arbutin. Finally, The kinetics characteristics shows that Km and Vmax of this enzyme are 1.44mmol/L and 58.32mmol/(L·s), respectively, and the catalytic coefficient (kcat) is 3 982/s using β-pNPG as the substrate. The all results above show that the β-Glucosidases from Lactobacillus paracasei TK1501 play important roles in the process of hydrolysis of soybean isoflavone and synthesis of glycosides.
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Received: 15 October 2018
Published: 04 June 2019
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Corresponding Authors:
Fu-ping LU
E-mail: lfp@tust.edu.cn
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[1] |
James R, Bancha M, Supaporn B , et al. β-Glucosidases: Multitasking,moonlighting or simply misunderstood. Plant Science, 2015,24(1):246-259.
|
|
|
[2] |
郑芳芳, 王金佩, 林宇, 等. 链霉菌GXT6 β-葡萄糖苷酶的酶学性质及葡萄糖耐受性分子改造. 微生物学报, 2018,58(10):1839-1852.
|
|
|
[2] |
Zheng F F, Wang J P, Lin Y , et al. Characterization of β-glucosidase from Streptomyces sp. GXT6 and its molecular modification of glucose tolerance. Acta Microbiologica Sinica, 2018,58(10):1839-1852.
|
|
|
[3] |
王锐丽 . 嗜热菌β-葡萄糖苷酶 A水解大豆异黄酮的研究. 安徽农学通报, 2016,22(18):26-28.
|
|
|
[3] |
Wang R L . Hydrolysis of soybean isoflavone by β-glucosidases A from Thermoanaerobacter etholicus. Anhui Agricultural Science Bulletin, 2016,22(18):26-28.
|
|
|
[4] |
Messina M, Erdman J, Setchell K D . Introduction to and perspectives from the Fifth International Symposium on the role of soy in preventing and treating chronic disease. The Journal of Nutrition, 2004,134(5):1205S-1206S.
doi: 10.1093/jn/134.5.1205S
|
|
|
[5] |
Tsangalis D, Ashton J, McGill A , et al.Biotransformation of isoflavones by bifidobacteria in fermented soymilk supplemented with D-glucose and L-Cysteine. Journal of Food Science, 2003,68(2):623-631.
doi: 10.1111/jfds.2003.68.issue-2
|
|
|
[6] |
Messina M . Soy foods, isoflavones, and the health of postmenopausal women. The American Journal of Clinical Nutrition, 2014,100(suppl):423S-430S.
doi: 10.3945/ajcn.113.071464
|
|
|
[7] |
Chun J, Kim G M, Lee K W , et al. Conversion of isoflavone glucosides to aglycones in soymilk by fermentation with lactic acid bacteria. Journal of Food Science, 2007,72(2):M39-M44.
doi: 10.1111/jfds.2007.72.issue-2
|
|
|
[8] |
Hati S, Vij S, Singh B P , et al. β-Glucosidase activity and bioconversion of isoflavones during fermentation of soymilk. Food Research International, 2015,95(1):216-220.
|
|
|
[9] |
孙国祥, 吴美仙 . β-葡萄糖苷酶水解结合型大豆异黄酮参数相关性研究. 浙江海洋学院学报(自然科学版), 2014,33(3):284-289.
|
|
|
[9] |
Sun G X, Wu M X . Correlation study on parameters of hydrolysis soy isoflavones and β-glucosidases. Journal of Zhejiang Ocean University (Natural Science), 2014,33(3):284-289.
|
|
|
[10] |
Mergulhaoa F J, Summersb D K, Monteiro G A . Recombinant protein secretion in Escherichia coli. Biotechnology Advances, 2005,23(3):177-202.
doi: 10.1016/j.biotechadv.2004.11.003
|
|
|
[11] |
Sorensen H P, Mortensen K K . Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology, 2005,115(2):113-128.
doi: 10.1016/j.jbiotec.2004.08.004
|
|
|
[12] |
商永梅, 包永红 . 类芽孢杆菌属β-葡萄糖苷酶在大肠杆菌中可溶性重组表达的优化. 食品工业科技, 2014,35(23):186-190.
|
|
|
[12] |
Shang Y M, Bao Y H . Optimization of soluble expression of recombinant Paenibacillus sp. β-glucosidase in Escherichia coli. Science and Technology of Food Industy, 2014,35(23):186-190.
|
|
|
[13] |
江民华, 林厚民, 尹金阳 , 等. 差异柠檬酸杆菌GXW-1β-葡萄糖苷酶的酶学性质及分子改造. 微生物学报, 2017,57(3):363-374.
|
|
|
[13] |
Jiang M H, Lin H M, Yin J Y , et al. Characterization and molecular modification of β-glucosidase from Citrobacter koser GXW-1. Acta Microbiologica Sinica, 2017,57(3):363-374.
|
|
|
[14] |
李娟, 汤斌, 李松 , 等. 匍枝根霉β-葡萄糖苷酶 BGLIII 关键位点的结构功能. 食品与发酵工业, 2015,41(2):1-6.
|
|
|
[14] |
Li J, Tang B, Li S , et al. Structure and function of the key site of β-glucosidase BGLIII from Rhizopus stolonifer. Food and Fermentation Industries, 2015,41(2):1-6.
|
|
|
[15] |
闫青 . 黑曲霉耐热β-葡萄糖苷酶的分离纯化及结构分析. 秦皇岛:河北科技师范学院, 2016.
|
|
|
[15] |
Yan Q . Isolation,Purification and structure characterization of thermal resistant β-glucosidase of Aspergillus niger. Qinghuangdao:Hebei Normal University of Science and Technology, 2016.
|
|
|
[16] |
钱利纯 . 高效水解大豆异黄酮β-葡萄糖苷酶及其对肉公鸡生产性能的影响研究. 杭州:浙江大学, 2008.
|
|
|
[16] |
Qian L C . The hydrolyzing efficiency of isoflavone by β-glucosidase and effect of the enzyme on the growth performance of male broiler. Hangzhou:Zhejiang University, 2008.
|
|
|
[17] |
赵云, 刘伟丰, 毛爱军 , 等. 多粘芽孢杆菌(Bacillus polymyxa)-葡萄糖苷酶基因在大肠杆菌中的表达,纯化及酶学性质分析. 生物工程学报, 2004,20(5):741-744.
|
|
|
[17] |
Zhao Y, Liu W F, Mao A J , et al. Expression,purification and enzymatic characterization of Bacillus polymyxa β-glucosidase gene(bglA)in Escherichia coli. Chinese Journal of Biotechnology, 2004,20(5):741-744.
|
|
|
[18] |
Maekawa A, Hayase M, Yubisui T , et al. A cDNA cloned from Physarum polycephalum,encodes new type of family 3 β-glucosidase that is a fusion protein containing a calx-β motif. The International Journal of Biochemistry and Cell Biology, 2006,38(12):2164-2172.
doi: 10.1016/j.biocel.2006.06.010
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