铁氨基黏土纳米结构脂肪酶的构筑及催化特性

doi:10.7503/cjcu20190444

摘要/Abstract

摘要：

以铁氨基黏土(Fe-aminoclay)为载体, 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)为共价交联剂, 构筑了铁氨基黏土纳米结构脂肪酶催化剂(Feclay-lipase). 利用X射线衍射(XRD)、透射电子显微镜(TEM)和傅里叶变换红外光谱(FTIR)等技术对Feclay-lipase进行了表征, 并通过酶动力学对比研究了游离脂肪酶和Feclay-lipase的酶学特性. 结果表明, Fe-aminoclay的载酶量为414.4 mg/g, 固定化效率可达82.88%, Feclay-lipase的酶活较游离酶提高了3倍, 最适反应温度提高了10 ℃, 最适反应 pH向碱性偏移, 储存稳定性更好, 在4 ℃下贮存30 d后其酶活无明显减弱.

关键词: 铁氨基黏土, 脂肪酶, 纳米结构载体, 固定化酶, 纳米生物催化剂

Abstract:

Lipase from Aspergillus oryzae was immobilized on Fe-aminoclay support via 1-(3-dimethylami-nopropyl)-3-ethylcarbodiimide hydrochloride(EDC) as covalent crosslinking agent. The nanocomposite structure of immobilized lipase(Feclay-lipase) was characterized by means of X-ray diffraction(XRD), transmission electron microscope(TEM) and Fourier transform infrared spectroscopy(FTIR). The enzymatic properties of free lipase and Feclay-lipase were studied by enzymatic kinetics. The results showed that the optimal immobilization efficiency of Feclay-lipase was 82.88%, immobilization capacity was 414.4 mg/g support, and the enzyme activity was 28.24 U/mg, which was three times higher than that of free lipase(8.21 U/mg). The optimum reaction temperature of Feclay-lipase increased from 45 ℃ to 55 ℃, the optimum pH shifted to alkalinity, and the catalytic activity of Feclay-lipase did not decrease significantly after storage at 4 ℃ for one month.

Key words: Fe-aminoclay, Lipase, Nanostructured support, Enzyme immobilization, Nanobiocatalyst

中图分类号:

O643.3

TrendMD:

范小雨,王可,孙仕勇,马彪彪,吕瑞. 铁氨基黏土纳米结构脂肪酶的构筑及催化特性. 高等学校化学学报, 2019, 40(12): 2512.

Xiaoyu FAN,Ke WANG,Shiyong SUN,Biaobiao MA,Rui LÜ. Construction and Catalytic Performances of Fe-aminoclay Nanostructured Lipase ^†. Chem. J. Chinese Universities, 2019, 40(12): 2512.

图/表 10

Fig.1 XRD patterns(A) and FTIR spectra(B) of Fe-aminoclay and Feclay-lipase with various lipase loadings (A) a. Fe-aminoclay; b. Feclay-10lipase; c. Feclay-50lipase; d. Feclay-100lipase; e. Feclay-150lipase; f. Feclay-200lipase. (B) a. Fe-aminoclay; b. lipase; c. Feclay-10lipase; d. Feclay-50lipase; e. Feclay-100lipase; f. Feclay-150lipase; g. Feclay-200lipase.

Fig.2 Schematic diagram of Fe-aminoclay dispersed in water and lipase immobilized on Fe-aminoclay

Fig.3 Zeta potentials(A) and average particle sizes(B) of Fe-aminoclay, lipase and Feclay-lipase a. Fe-aminoclay; b. lipase; c. Feclay-10lipase; d. Feclay-50lipase; e. Feclay-100lipase; f. Feclay-150lipase; g. Feclay-200lipase.

Fig.4 Photographs of as-prepared Fe-aminoclay powder(A) and Fe-aminoclay(a), Feclay-150lipase(b) and lipase(c) dispersed in water(B)

Fig.5 TEM images of Fe-aminoclay(A) and Feclay-lipase(B), SEM images of Fe-aminoclay(C), Feclay-150lipase(D) and the selected area in (D) image(E) and contact angle images of Fe-aminoclay(F), lipase(G) and Feclay-150lipase(H)

Fig.6 Effect of lipase loading on the catalytic acti-vity and immobilization efficiency for Feclay-lipase

Fig.7 Effect of reaction temperature(A) and pH(B) on catalytic activity of free lipase and Feclay-150lipase

Fig.8 Thermal stability at 60 ℃(A) and storage stability at 4 ℃(B) of free lipase and Feclay-150lipase

Fig.9 Lineweaver-Burk plots of the free lipase and Feclay-lipase(A), absorbance at the peak position for p-NP(410 nm) as a function of time for two different catalytic systems(B)

Table 1 Kinetic parameters of the free and the Feclay-lipase

Sample	R²	K_m/mmol	V_max/(mmol·mL^-1·min^-1)
Free lipase	0.997	5.35	99
Feclay-50lipase	0.996	6.98	217.39
Feclay-150lipase	0.996	7.84	175.42

参考文献 52

[1]	Madhava A., Sindhu R., Binod P., Sukumaran R. K., Pandey A ., Bioresource. Technol., 2017,245, 1304— 1313 doi: 10.1016/j.biortech.2017.05.031 URL
[2]	Kirk O., Borchert T. V., Fuglsang C. C ., Curr. Opin. Biotech., 2002,13, 345— 351 doi: 10.1016/s0958-1669(02)00328-2 URL pmid: 12323357
[3]	Patel N., Rai D., Shivam S., Shahane S., Mishra U., Recent Pat . Biotechnol., 2018,13, 45— 56
[4]	Burton S. G., Cowan D. A., Woodley J. M ., Nat. Biotechnol., 2002,20, 37— 45 doi: 10.1038/nbt0102-37 URL pmid: 11753360
[5]	Adlercreutz P ., Chem. Soc. Rev., 2013,42, 6406— 6436 doi: 10.1039/c3cs35446f URL pmid: 23403895
[6]	Shi J., Wang X., Zhang W., Jiang Z., Liang Y., Zhu Y., Zhang C ., Adv. Funct. Mater., 2013,23, 1450— 1458 doi: 10.1002/adfm.201202068 URL
[7]	Datta K. K. R., Achari A., Eswaramoorthy M ., J. Mater. Chem. A, 2013,1, 6707— 6718 doi: 10.1039/c3ta00100h URL
[8]	Bilal M., Iqbal H. M. N ., Coordin. Chem. Rev., 2019,388, 1— 23 doi: 10.1016/j.ccr.2019.02.024 URL
[9]	Ren S., Li C., Jiao X., Jia S., Jiang Y., Bilal M., Cui J ., Chem. Eng. J., 2019,373, 1254— 1278 doi: 10.1016/j.cej.2019.05.141 URL
[10]	Rodrigues R. C., Virgen Ortiz J. J., Dos Santos J. C. S., Berenguer Murcia A., Alcantara A. R., Barbosa O., Ortiz C., Fernandez Lafuente R ., Biotechnol. Adv., 2019,37, 746— 770 doi: 10.1016/j.biotechadv.2019.04.003 URL pmid: 30974154
[11]	Ramos E. Z., Júnior R. H. M., de Castro P. F., Tardioli P. W., Mendes A. A., Fernandéz Lafuente R., Hirata D. B ., J. Mol. Catal. B:Enzym., 2015,118, 43— 51 doi: 10.1016/j.molcatb.2015.05.009 URL
[12]	Lin S., Sun S., Wang K., Shen K., Ma B., Ren Y., Fan X ., Nanomaterials, 2018,8, 127— 135 doi: 10.3390/nano8020127 URL
[13]	Sun S., Li M., Dong F., Wang S., Tian L., Mann S ., Small, 2016,12, 1920— 1927 doi: 10.1002/smll.201600243 URL pmid: 26923794
[14]	Jiang Y., Liu X., Chen Y., Zhou L., He Y., Ma L., Gao J ., Bioresource. Technol., 2014,153, 278— 283 doi: 10.1016/j.biortech.2013.12.001 URL
[15]	Zhang C., Dong X., Guo Z., Sun Y ., J. Colloid. Interf. Sci., 2018,519, 145— 153 doi: 10.1016/j.jcis.2018.02.039 URL pmid: 29494877
[16]	Jamwal S., Kumar D., Ranote S., Chauhan G. S ., J. Nanosci. Nanotechnol., 2019,19, 7205— 7214 doi: 10.1166/jnn.2019.16667 URL pmid: 31039877
[17]	Xie W. L., Zang X. Z ., Food Chem., 2017,227, 397— 403 doi: 10.1016/j.foodchem.2017.01.082 URL pmid: 28274449
[18]	Zhu J., Sun G ., React. Funct. Polym., 2012,72, 839— 845 doi: 10.1016/j.reactfunctpolym.2012.08.001 URL
[19]	Zdarta J., Meyer A. S., Jesionowski T., Pinelo M ., Catalysts, 2018,8, 92— 118 doi: 10.1186/1471-2148-8-92 URL pmid: 18366743
[20]	Mao C ., Small, 2005,1, 356— 356
[21]	Kim J., Grate J. W., Wang P ., Chem. Eng. Sci., 2006,61, 1017— 1026 doi: 10.1016/j.ces.2005.05.067 URL
[22]	Hong T., Liu W., Li M., Chen C ., Anal. Chim. Acta, 2019,1067, 31— 47 doi: 10.1016/j.aca.2019.02.031 URL pmid: 31047147
[23]	Liu X., Fang Y., Xu Y., Yong L., Wang C ., Chem. Eng. J., 2018,336, 456— 464 doi: 10.1016/j.cej.2017.12.048 URL
[24]	Rege K., Raravikar N. R., Kim D. Y., Schadler L. S., Ajayan P. M., Dordick J. S ., Nano Lett., 2015,3, 829— 832 doi: 10.1021/nl034131k URL
[25]	Wang Z. G., Wan L. S., Liu Z. M., Huang X. J., Xu Z. K ., J. Mol. Catal. B: Enzym., 2009,56, 189— 195 doi: 10.1016/j.molcatb.2008.05.005 URL
[26]	Zhang C., Liu Y., Sun Y ., Biochem. Eng. J., 2019,146, 124— 131 doi: 10.1016/j.bej.2019.03.012 URL
[27]	Xiang X., Wan X., Suo H., Hu Y ., Acta Phys-Chim. Sin., 2018,34, 99— 107
[28]	Tzialla A. A., Pavlidis I. V., Felicissimo M. P., Rudolf P., Gournis D., Stamatis H ., Bioresource Technol., 2010,101, 1587— 1594 doi: 10.1016/j.biortech.2009.10.023 URL pmid: 19910187
[29]	Öztürk H., Pollet E., Phalip V., Güvenilir Y., Avérous L ., Polymers-Basel., 2016,8, 416— 432
[30]	Chen N., Zhang C., Liu Y., Dong X., Sun Y ., Biochem. Eng. J., 2019,145, 137— 144 doi: 10.1016/j.bej.2019.02.018 URL
[31]	Miao C., Yang L., Wang Z., Luo W., Li H., Lv P., Yuan Z ., Fuel, 2018,224, 774— 782 doi: 10.1016/j.fuel.2018.02.149 URL
[32]	Bui V. K. H., Park D., Lee Y. C ., Chem. Eng. J., 2018,336, 757— 772 doi: 10.1016/j.cej.2017.12.052 URL
[33]	Patil A. J., Muthusamy E., Mann S ., J. Mater. Chem., 2005,15, 3838— 3843 doi: 10.1039/b504288g URL
[34]	Lee Y. C., Kim M. I., Woo M. A., Park H. G., Han J. I ., Biosens. Bioelectron., 2013,42, 373— 378 doi: 10.1016/j.bios.2012.10.092 URL pmid: 23211453
[35]	Bradford M. M ., Anal. Biochem., 1976,72, 248— 254 doi: 10.1006/abio.1976.9999 URL pmid: 942051
[36]	Shah E., Mahapatra P., Bedekar A. V., Soni H. P ., RSC Adv., 2015,5, 26291— 26300 doi: 10.1039/C5RA02249E URL
[37]	Jing G., Yun W., Du Y., Zhou L., Ying H., Li M., Yin L., Kong W., Jiang Y ., Chem. Eng. J., 2017,317, 175— 186 doi: 10.1016/j.cej.2017.02.012 URL
[38]	Patil A. J., Mann S ., J. Mater. Chem., 2008,18, 4605— 4615 doi: 10.1039/b805653f URL
[39]	Lebeau B., Brendlé J., Marichal C., Patil A. J., Muthusamy E., Mann S ., J. Nanosci. Nanotechnol., 2006,6, 352— 359 doi: 10.1166/jnn.2006.910 URL pmid: 16573032
[40]	Lee Y. C., Kim E. J., Dong A. K., Yang J. W ., J. Hazard. Mater., 2011,196, 101— 108 doi: 10.1016/j.jhazmat.2011.08.077 URL
[41]	Han H. K., Lee Y. C., Lee M. Y., Patil A. J., Shin H. J ., ACS Appl. Mater. Interfaces, 2011,3, 2564— 2572 doi: 10.1021/am200406k URL pmid: 21609130
[42]	Dong H., Li J., Li Y., Hu L., Luo D ., Chem. Eng. J., 2012,181/182, 590— 596 doi: 10.1016/j.cej.2011.11.095 URL
[43]	Hwang Y., Lee Y. C., Mines P. D., Yun S. H., Andersen H. R ., Appl. Catal. B, 2014,147, 748— 755 doi: 10.1016/j.apcatb.2013.10.017 URL
[44]	Mateo C., Palomo J. M., Fernandez Lorente G., Guisan J. M., Fernandez Lafuente R ., Enzyme. Microb. Tech., 2007,40, 1451— 1463 doi: 10.1016/j.enzmictec.2007.01.018 URL
[45]	Barriuso J., Vaquero M. E., Prieto A., Martínez M.J ., Biotechnol. Adv., 2016,34, 874— 885 doi: 10.1016/j.biotechadv.2016.05.004 URL pmid: 27188926
[46]	Liu J., Guo H., Zhou Q., Wang J., Lin B., Zhang H., Gao Z., Xia C., Zhou X ., J. Mol. Catal. B: Enzym., 2013,96, 96— 102 doi: 10.1016/j.molcatb.2013.06.013 URL
[47]	Miranda M., Urioste D., Andrade Souza L. T., Mendes A. A., de Castro H. F ., Enzyme Res., 2011,2011, 216435 doi: 10.4061/2011/216435 URL pmid: 21876790
[48]	Zou B., Hu Y., Cui F., Jiang L., Yu D., Huang H ., J. Colloid Interf. Sci., 2014,417, 210— 216 doi: 10.1016/j.jcis.2013.11.029 URL pmid: 24407679
[49]	Wang J., Zhao G., Jing L., Peng X., Li Y ., Biochem. Eng. J., 2015,98, 75— 83 doi: 10.1016/j.bej.2014.11.013 URL
[50]	Yong Y., Bai Y. X., Li Y. F., Lin L., Cui Y. J., Xia C. G ., Process Biochem., 2008,43, 1179— 1185 doi: 10.1016/j.procbio.2008.05.019 URL
[51]	Reshmi R., Sugunan S ., J. Mol. Catal. B: Enzym., 2013,97, 36— 44 doi: 10.1016/j.molcatb.2013.04.003 URL
[52]	Mendes A. A., Barbosa B. C. M., Silva M. L. C. P. D., Castro H. F. D ., Biocatalysis, 2009,25, 393— 400