PDF(792 KB)
Cloning, Expression, Purification and Characterization of L-glutamate Oxidase
LU Chan, ZHENG Pu, SUN Zhi-hao
China Biotechnology ›› 2013, Vol. 33 ›› Issue (6) : 38-44.
PDF(792 KB)
PDF(792 KB)
Cloning, Expression, Purification and Characterization of L-glutamate Oxidase
In order to improve microbial production of L-glutamate oxidase, the L-glutamate oxidase gene (LGOX) from Streptomyces sp.X-119-6 was successfully expressed by connected to the expression vector pET28a and then transformed into E.coli BL21(DE3). The HisTrapTMFF affinity chromatography column was used for the purification of LGOX. Enzymatic properties of the recombinant LGOX were also investigated. Results showed that the recombinant LGOX activity could reach 1.1 U/mg at the IPTG concentration of 0.4 mmol/L at 30℃ for induction 6 h. The optimum reaction temperature and pH were 37℃ and 5.0, respectively. The Km was 2.12 mmol/L and Vmax was 1.06μmol/min·mg. The LGOX had high substrate specificity of L-glutamic acid with application prospect.
L-glutamate oxidase / Cloning / Expressing / Enzymatic properties {{custom_keyword}} /
[1] 毕春元,李玲,李敬龙. L-谷氨酸氧化酶的研究进展. 生命科学,2012, 24(2): 169-173. Bi C Y, Li L, Li J L. Research progress on L-glutamate oxidase. Chinese Bulletin of Life Sciences, 2012, 24(2): 169-173.
[2] Kamei T, Asano K, Nakamura S. Determination of serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase by L-glutamate oxidase. Chemical & Pharmaceutical Bulletin, 1986, 34(1): 409-412.
[3] 郑强,严卫民,徐东,等. L-谷氨酸氧化酶的来源和特性及其在生化检验中的应用. 检验医学教育,2003, 10(2): 32-35. Zheng Q, Yan W M, Xu D, et al. Sources and characteristics of the L-glutamate oxidase and its application in biochemical detection. Chinese Journal of Laboratory Medicine, 2003, 10(2): 32-35.
[4] Rahman M M. Fabrication of a low-detection glutamate biosensor K3Fe(CN)6 mediated on glutamate oxidase using smart-Chips. Journal of Materials Science and Engineering B, 2012, 2(1): 30-39.
[5] Yu Y Y, Liu X Q, Jiang D W, et al.-Au/Pt biosensor for glutamate sensing in vivo integrated with on-line microdialysis system. Biosensors and Bioelectronics, 2011, 26(7): 3227-3232.
[6] Rui C S, Sonomoto K, Ogawa H I, et al. A flow-injection biosensor system for the amperometric determination of creatinine: simultaneous compensation of endogenous interferents. Analytical Biochemistry, 1993, 210(1): 163-171.
[7] 李陈鑫,张芬芬,万巧,等. 中性红掺杂SiO2 纳米粒子修饰的L-谷氨酸生物传感器在Ⅰ型糖尿病模型大鼠脑渗析液分析中的应用. 研究化学传感器,2004, 24(4): 47-52. Li C X, Zhang F F, Wan Q, et al. Amperometric sensor based on neutral red-doped silica(NRDS) nanoparticles as electron transfer mediator for the determination of L-glutamate in the brain of I type diabetic rat. Chemical Sensors, 2004, 24(4): 47-52.
[8] Kamei T, Asano K, Suzuki H, et al. L-glutamate oxidase from Streptomyces violascens.Ⅰ. Production, isolation and some properties. Chemical and Pharmaceutical Bulletin, 1983, 31(4): 1307-1314.
[9] Kusakabe H, Midorikawa Y, Kuninaka K, et al. A occurrence of a new enzyme, L-glutamate oxidase in a wheat bran culture extract of Streptomyces sp.X-119-6. Agricultural Biology and Chemistry, 1983, 47(1): 179-182.
[10] Arima J, Sasaki C, Sakaguchi C, et al. Structural characterization of L-glutamate oxidase from Streptomyces sp. X-119-6. The FEBS Journal, 2009, 276(14): 3894-3903.
[11] Chen C Y, Wu W T, Huang C J, et al. A common precursor for the three subunits of L-glutamate oxidase encoded by gox gene from Streptomyces platensis NTU3304. Canadian Journal of Microbiology, 2001, 47(3): 269-275.
[12] Arima J, Tamura T, Kusakabe H, et al. Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp.X-119-6. The Journal of Biochemistry, 2003, 134(6): 805-812.
[13] 徐水清,李友荣. L-谷氨酸氧化酶产生菌的筛选与产酶条件. 微生物学报,1993, 33(4): 309-312. Xu S Q, Li Y R. Screening of L-glutamate oxidase forming strains and conditions for enzyme production. Acta Microbiologica Sinica, 1993, 33(4): 309-312.
[14] 李青山,李友荣,闵简书. L-谷氨酸氧化酶高产菌株的快速筛选及诱变育种. 华东化工学院学报,1993, 19(2): 148-153. Li Q S, Li Y R, Min J S. Breeding of L-glutamate oxidase producing strains with mutagenesis and a rapid screening method. Journal of East China Institute of Chemical Industry, 1993, 19(2): 148-153.
[15] 沈群. 链霉菌L-谷氨酸氧化酶的发酵条件、纯化及其性质的研究. 北京: 北京农业工程大学,1995. Shen Q. Fermentation conditions, purification and some properties of L-glutamate oxidase from Streptomyces sp.80-5. Beijing: Beijing Agricultural Engineering University, 1995.
[16] 李玲,毕春元,李敬龙. L-谷氨酸氧化酶的分离纯化及酶学性质的研究. 中国酿造,2012, 1: 140-143. Li L, Bi C Y, Li J L. Isolation, purification and characterization of L-glutamate oxidase from Streptomyces sp. China Brewing, 2012, 1: 140-143.
[17] Kusakabe H, Midorikawa Y, Fujishima T, et al. Purification and properties of a new enzyme, L-glutamate oxidase, from Streptomyces sp.X-119-6 grown on wheat bran. Agricultural Biology and Chemistry, 1983, 47(6): 1323-1328.
[18] Utsumi T, Arima J, Sakaguchi C, et al. Arg305 of Streptomyces L-glutamate oxidase plays a crucial role for substrate recognition. Biochemical and Biophysical Research Communications, 2012, 417(3): 951-955.
[19] Bǒhmer A, Müller A, Passarge M, et al. A novel L-glutamate oxidase from Streptomyces endus purification and properties. European Journal of Biochemistry, 1989, 182(2): 327-332.
[20] Wachiratianchai S. Isolation, purification, and characterization of L-glutamate oxidase from Streptomyces sp.18G. Electronic Journal of Biotechnology, 2004, 7(3): 274-281.
[21] Sukhacheva M V, Netrusov A I. Streptomyces sp. Z-11-6, a novel producer of extracellular L-glutamate oxidase. Microbiology, 2000, 69(1): 13-16.
PDF(792 KB)
/
| 〈 |
|
〉 |
