Immobilized glutamate decarboxylase by carboxyl magnetic microspheres

doi:10.11949/j.issn.0438-1157.20161301

CIESC Journal ›› 2017, Vol. 68 ›› Issue (4): 1550-1557.DOI: 10.11949/j.issn.0438-1157.20161301

Previous Articles Next Articles

Immobilized glutamate decarboxylase by carboxyl magnetic microspheres

LI Jianan^1,2, XIE Tian³, HU Sheng¹, XIE Dongfang³, FANG Hui³, MEI Lehe^1,4, HUANG Jun³, WANG Jinbo¹, YAO Shanjing⁴

1 School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, Zhejiang, China;
2 College of Pharmacy, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China;
3 School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, Zhejiang, China;
4 College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, Zhejiang, China

Received:2016-09-18 Revised:2016-11-09 Online:2017-04-05 Published:2017-04-05
Supported by:
supported by the National Natural Science Foundation of China (31240054, 31470793, 31670804), the Natural Science Foundation of Zhejiang Province (LZ13B060002) and the Natural Science Foundation of Ningbo (2013A610087).

羧基化磁性微球固定化谷氨酸脱羧酶

李佳男^1,2, 谢湉³, 胡升¹, 谢东芳³, 方卉³, 梅乐和^1,4, 黄俊³, 王进波¹, 姚善泾⁴

1 浙江大学宁波理工学院生物与化学工程学院, 浙江宁波 315100;
2 浙江工业大学药学院, 浙江杭州 310014;
3 浙江科技学院省农产品化学与生物加工技术重点实验室, 浙江杭州 310023;
4 浙江大学化学工程与生物工程学院, 浙江杭州 310027

通讯作者: 梅乐和,黄俊
基金资助:
国家自然科学基金项目(31240054，31470793，31670804)；浙江省自然科学基金重点项目(LZ13B060002)；宁波市自然科学基金项目（2013A610087）。

Abstract

Abstract:

Carboxyl Fe₃O₄ magnetic microspheres were prepared by a chemical co-precipitation method with potassium permanganate oxidation, and glutamate decarboxylase (GAD) was immobilized by the carboxyl magnetic microspheres as a carrier. The magnetic microspheres were characterized by methods of thermogravimetry (TGA), transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). The results indicated that the magnetic microspheres had 95.1% contents of magnetite, homogeneous size and superparamagnetic behavior. GAD was well wrapped up in the magnetic microspheres by Fourier transform infrared (FT-IR) analysis, VSM and X-ray diffraction (XRD). The magnetic microspheres had complete crystal structure, good magnetic response and strong superparamagnetic behaviors before and after GAD immobilization. The enzymatic properties of immobilized and free GAD were analyzed and compared. The results showed that immobilized GAD had better thermostability and pH resistance and retained more than 90% activity after ten repeated batches.

Key words: carboxyl magnetic microspheres, glutamate decarboxylase, immobilization, stability, preparation

摘要：

通过化学共沉淀法结合高锰酸钾氧化制备羧基化Fe₃O₄磁性微球，以该磁性微球作为载体，固定化谷氨酸脱羧酶。利用热重分析（TGA（、透射电镜（TEM（及振动样品磁强计（VSM（对羧基化磁性微球进行表征，结果表明该磁性微球磁含量约为95.1%，粒径均一，呈近似球形且具有超顺磁性。通过对固定化酶进行傅里叶红外光谱（FT-IR（、VSM和X射线衍射（XRD（分析，确定磁性微球载体与谷氨酸脱羧酶分子间形成酰胺键，实现共价结合且固定化酶前后粒子晶形完整，均具有良好的磁响应能力和超顺磁性。与游离谷氨酸脱羧酶相比，固定化酶的热稳定性和酸碱耐受性均有不同程度的提高，且制备的固定化酶重复使用10批后相对酶活力仍大于90%。

关键词: 羧基化磁性微球, 谷氨酸脱羧酶, 固定化, 稳定性, 制备

CLC Number:

TQ028.8

LI Jianan, XIE Tian, HU Sheng, XIE Dongfang, FANG Hui, MEI Lehe, HUANG Jun, WANG Jinbo, YAO Shanjing. Immobilized glutamate decarboxylase by carboxyl magnetic microspheres[J]. CIESC Journal, 2017, 68(4): 1550-1557.

李佳男, 谢湉, 胡升, 谢东芳, 方卉, 梅乐和, 黄俊, 王进波, 姚善泾. 羧基化磁性微球固定化谷氨酸脱羧酶[J]. 化工学报, 2017, 68(4): 1550-1557.

References 28

[1]	KIM H W, KASHIMA Y, ISHIKAWA K, et al. Purification and characterization of the first archaeal glutamate decarboxylase from Pyrococcus horikoshii[J]. Biosci. Biotechnol. Biochem., 2009, 73(1):224-227.
[2]	杨藻宸. 药理学和药物治疗学(上册)[M]. 北京:人民卫生出版社, 2000:487-503.YANG Z C. Pharmacology and Therapeutics[M]. Beijing:People's Medical Publishing House, 2000:487-503.
[3]	SCHULLER H M, AL-WADEI H A, MAJIDI M. Gamma-aminobutyric acid, a potential tumor suppressor forsmall airway-derived lung adenocarcinoma[J]. Carcinogenesis, 2008, 29(10):1979-1985.
[4]	黄俊. 利用短乳杆菌制备γ-氨基丁酸相关过程研究[D]. 杭州:浙江大学, 2006.HUANG J. Process study on the preparation of γ-aminobutyric acid by Lactobacillus brevis[D]. Hangzhou:Zhejiang University, 2006.
[5]	LIANG H Y, DENG LK, LIN H L, et al. Selection of lactic acid bacteria for producting γ-aminobutyric acid and optimization of fermentation[J]. Food Res. Dev., 2008, 24(1):36-40.
[6]	林玲, 胡升, 郁凯, 等. 饱和定点突变拓宽谷氨酸脱羧酶催化pH范围的研究[J]. 高校化学工程学报, 2014, 28(6):1410-1414.LIN L, HU S, YU K, et al. Expanding the active pH range of Lactobacillus brevis glutamate decarboxylase by saturation mutagenesis[J]. J. Chem. Eng. Chin. Univ., 2014, 28(6):1410-1414.
[7]	XIE Z, XIA S, LE G W. Gamma-aminobutyric acid improves oxidative stress and function of the thyroid in high-fat diet fed mice[J]. J. Funct. Foods, 2014, 8C:76-86.
[8]	ABDOU A M, HIGASHIGUCHI S, HORIE K, et al. Relaxation and immunity enhancement effects of γ-aminobutyric acid (GABA) administration in humans[J]. Biofactors, 2006, 26(3):201-208.
[9]	ENNA S J, MC CARSON K E. The role of GABA in the mediation and perception of pain[J]. Adv. Pharmacol., 2006, 54:1-27.
[10]	HUANG J, MEI L H, XIA J. Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA[J]. Biotechnol. Bioeng., 2007, 96(5):924-931.
[11]	刘峥, 林原斌, 吕慧丹. 交联海藻酸钠磁性微球的制备及固定化胰蛋白酶研究[J]. 材料导报, 2006, 20(12):50-53.LIU Z, LIN Y B, LÜ H D. Study on preparation of magnetic particle of crosslinked sodium alginate and immobilization of trypsin[J]. Materials Reviews, 2006, 20(12):50-53.
[12]	王筱婧, 王立, 马晓博, 等. 固定化米糠对谷氨酸脱羧酶活性影响的研究[J]. 食品工业科技, 2009, 30(11):157-159.WANG X J, WANG L, MA X B, et al. Effect of immobilizing the rice bran by sodium alginate on the enzymatic properties of glutamate decarboxylase[J]. Science and Technology of Food Industry, 2009, 30(11):157-159.
[13]	HUANG J, MEI L H, WU H, et al. Biosynthesis of gamma-aminobutyric acid (GABA) using immobilized whole cells of Lactobacillus brevis[J]. World J. Microbiol. Biotechnol., 2007, 23(6):865-871.
[14]	XIE W L, MA N. Immobilized lipase on Fe3O4 nanoparticles as biocatalyst for biodesel production[J]. Energy Fuels, 2009, 23(3):1347-1353.
[15]	丁玲,李曦,张超灿.表面功能化聚苯乙烯磁性微球的制备及表征[J]. 化工新型材料, 2010, 38(10):50-53.DING L, LI X, ZHANG C C. Preparation of polystyrene magnetic microspheres surface-functioned and its characterization[J]. New Chemical Materials, 2010, 38(10):50-53.
[16]	KICKELBICK G, BAUER J, HESING N, et al. Spontaneous vesicle formation of short-chain amphiphilic polysiloxane-b-poly(ethyleneoxide)block copolymers[J]. Langmuir, 2003, 19(8):3198-3201.
[17]	LEE S, AHN J, KIM Y, et al. Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography[J]. Int. J. Mol. Sci., 2013, 14:1728-1739.
[18]	苏鹏飞, 陈国, 赵珺. 表面羧基化Fe3O4磁性纳米粒子的快捷制备及表征[J].高等学校化学学报, 2011, 32(7):1472-1477.SU P F, CHEN G, ZHAO J, et al. Convenient preparation and characterization of surface carboxyl-functioned Fe3O4 magnetic nanoparticles[J]. J. Chem. Eng. Chin. Univ., 2011, 32(7):1472-1477.
[19]	ADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal. Biochem., 1976, 72(1/2):248-254.
[20]	李梦. Fe3O4磁性纳米粒子的制备、表征及应用[D]. 长沙:中南大学, 2012.LI M. Preparation, characterization and application of Fe3O4 magnetic nanoparticles[D]. Changsha:Central South University, 2012.
[21]	LEE J, ISOBE T, SENNA M. Preparation of ultrafine Fe3O4 particles by precipitation in the presence of PVA at high pH[J]. J. Colloid Interface Sci., 1996, 177:490-494.
[22]	杨兆壬. 表面羧基化Fe3O4磁性微球固定化CA酶的制备与研究[D]. 福建:华侨大学, 2014.YANG Z R. Preparation and analysis of carbonic anhydrase immobilized on surface carboxyl-functioned Fe3O4 magnetic Microspheres[D]. Fujian:Huaqiao University, 2014.
[23]	李丹丹, 江连洲, 李杨, 等. 磁性壳聚糖微球固定化碱性蛋白酶的酶学性质[J].食品科学, 2012, 33(21):249-250.LI D D, JIANG L Z, LI Y, et al. Enzymatic properties of magnetic chitosan microspere-immobilized alkaline protease[J]. Food Science, 2012, 33(21):249-250.
[24]	CORR S A, RAKOVICH Y P, GUNKO Y K. Multifunctional magnetic fluorescent nanocomposites for biomedical applications[J]. Nanoscale Res. Lett., 2008, 3(3):87-104.
[25]	PU H T, JIANG F J. Towards high sedimentation stability:magnetorheological fluids based on CNT/Fe3O4 nanocomposites[J]. Nanotechnology, 2005, 16:1486-1489.
[26]	LIU X Y, ZHENG S W, HONG R Y, et al. Preparation of magnetic poly(styrene-co-acrylic acid) microspheres with adsorption of protein[J]. Colloids and Surfaces A:Physicochem. Eng. Aspect, 2014, 443:425-431.
[27]	LIU X Q, GUAN Y P, SHEN R, et al. Immobilization of lipase onto micron size magnetic beads[J]. J. Chromatogr. B, 2005, 822(1/2):91-97.
[28]	ZHANG L M, QIU J H, WU X L, et al. Preparation of magnetic chitosan nanoparticles as support for cellulase immobilization[J]. Industrial & Engineering Chemistry Research, 2014, 53(9):3448-3454.

Immobilized glutamate decarboxylase by carboxyl magnetic microspheres

羧基化磁性微球固定化谷氨酸脱羧酶

PDF (PC)

Knowledge

Cited

Abstract

Cite this article

share this article

References 28

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	He JIANG, Junfei YUAN, Lin WANG, Guyu XING. Experimental study on the effect of flow sharing cavity structure on phase change flow characteristics in microchannels [J]. CIESC Journal, 2023, 74(S1): 235-244.
[2]	Lingding MENG, Ruqing CHONG, Feixue SUN, Zihui MENG, Wenfang LIU. Immobilization of carbonic anhydrase on modified polyethylene membrane and silica [J]. CIESC Journal, 2023, 74(8): 3472-3484.
[3]	Yuyuan ZHENG, Zhiwei GE, Xiangyu HAN, Liang WANG, Haisheng CHEN. Progress and prospect of medium and high temperature thermochemical energy storage of calcium-based materials [J]. CIESC Journal, 2023, 74(8): 3171-3192.
[4]	Yaxin CHEN, Hang YUAN, Guanzhang LIU, Lei MAO, Chun YANG, Ruifang ZHANG, Guangya ZHANG. Advances in enzyme self-immobilization mediated by protein nanocages [J]. CIESC Journal, 2023, 74(7): 2773-2782.
[5]	Wentao WU, Liangyong CHU, Lingjie ZHANG, Weimin TAN, Liming SHEN, Ningzhong BAO. High-efficient preparation of cardanol-based self-healing microcapsules [J]. CIESC Journal, 2023, 74(7): 3103-3115.
[6]	Zhilong WANG, Ye YANG, Zhenzhen ZHAO, Tao TIAN, Tong ZHAO, Yahui CUI. Influence of mixing time and sequence on the dispersion properties of the cathode slurry of lithium-ion battery [J]. CIESC Journal, 2023, 74(7): 3127-3138.
[7]	Lei MAO, Guanzhang LIU, Hang YUAN, Guangya ZHANG. Efficient preparation of carbon anhydrase nanoparticles capable of capturing CO₂ and their characteristics [J]. CIESC Journal, 2023, 74(6): 2589-2598.
[8]	Feng ZHU, Kailin CHEN, Xiaofeng HUANG, Yinzhu BAO, Wenbin LI, Jiaxin LIU, Weiqiang WU, Wangwei GAO. Performance study of KOH modified carbide slag for removal of carbonyl sulfide [J]. CIESC Journal, 2023, 74(6): 2668-2679.
[9]	Bin CAI, Xiaolin ZHANG, Qian LUO, Jiangtao DANG, Liyuan ZUO, Xinmei LIU. Research progress of conductive thin film materials [J]. CIESC Journal, 2023, 74(6): 2308-2321.
[10]	Wenchao XU, Zhigao SUN, Cuimin LI, Juan LI, Haifeng HUANG. Effect of surfactant E-1310 on the formation of HCFC-141b hydrate under static conditions [J]. CIESC Journal, 2023, 74(5): 2179-2185.
[11]	Zijian WANG, Ming KE, Jiahan LI, Shuting LI, Jinru SUN, Yanbing TONG, Zhiping ZHAO, Jiaying LIU, Lu REN. Progress in preparation and application of short b-axis ZSM-5 molecular sieve [J]. CIESC Journal, 2023, 74(4): 1457-1473.
[12]	Runzhu LIU, Tiantian CHU, Xiaoa ZHANG, Chengzhong WANG, Junying ZHANG. Synthesis and properties of phenylene-containing α,ω-hydroxy-terminated fluorosilicone polymers [J]. CIESC Journal, 2023, 74(3): 1360-1369.
[13]	Xueting ZHANG, Jijiang HU, Jing ZHAO, Bogeng LI. Preparation of high molecular weight polypropylene glycol in microchannel reactor [J]. CIESC Journal, 2023, 74(3): 1343-1351.
[14]	Xiangshang CHEN, Zhenjie MA, Xihua REN, Yue JIA, Xiaolong LYU, Huayan CHEN. Preparation and mass transfer efficiency of three-dimensional network extraction membrane [J]. CIESC Journal, 2023, 74(3): 1126-1133.
[15]	Zhiyuan JIN, Guorong SHAN, Pengju PAN. Preparation and heat and salt resistance of AM/AMPS/SSS terpolymer [J]. CIESC Journal, 2023, 74(2): 916-923.