Preparation and Thermal Kinetic Deactivation of Cross-linked Enzyme Aggregates of Penicillin Acylase†

doi:10.7503/cjcu20131009

Abstract

Abstract:

Cross-linked enzyme aggregates(CLEAs) is an efficient approach to obtain immobilized enzymes without the use of any pre-existing carriers. The preparation of CLEAs usually consisted of two simple steps: precipitation and cross-linking. In this work, CLEAs of penicillin acylase was preparaed using 50% ammo-nium sulphate solution as precipitant and 0.35% glutaraldehyde as cross-linking agent. The resulting CLEAs obtained an activity yield of 30.1% and higher optimal temperature(57 ℃) than free penicillin acylase(47 ℃) as well as an obvious shift of optimal pH from 8.3 to 10.0. Moreover, compared with free enzyme, the thermal stability of CLEAs was largely enhanced which facilitated the application of penicillin acylase in high-temperature reaction systems. Based on the thermal deactivation kinetics study, it was found that the deactivation model of penicillin acylase changed from one-step model to serial model through immobilizing as CLEAs. The higher deactivation energy of CLEAs(549.2 kJ/mol) than free enzyme(248.8 kJ/mol) also explained the excellent stability of CLEAs under high temperature environment. In addition, CLEAs exhibited favorable reusability after using 7 times.

Key words: Cross-linked enzyme aggregate(CLEA), Penicillin acylase, Immobilization, Enzyme stability, Thermal deactivation kinetics

CLC Number:

TrendMD:

MU Yangyang, ZHEN Qiannan, WANG Mengfan, QI Wei, SU Rongxin, HE Zhimin. Preparation and Thermal Kinetic Deactivation of Cross-linked Enzyme Aggregates of Penicillin Acylase^†[J]. Chem. J. Chinese Universities, 2014, 35(6): 1212.

Figures/Tables 9

Fig.1 Effects of ammonium sulfate(A) and glutaraldehyde(B) concentration on the activity yieldActivity yield(%)=Ai/A0×100%; Ai is the activity of enzyme aggregates or CLEAs; A0 is the activity of free enzyme.

Fig.2 Optimal temperatures(A) and pH(B) of free penicillin acylase(a) and its CLEAs(b) Relative activity(%) was calculated by assuming the activity at optimal temperature or pH as 100%.

Fig.3 Thermal stability of free penicillin acylase(a—c) and its CLEAs(d—f)Residual activity(%) was calculated by assuming the initial activity of enzyme samples as 100%. Temperature/K: a, d: 329.15; b, e: 331.15; c, f: 333.15.

Fig.4 Thermal deactivation kinetic curves of free enzyme(A—C) and its CLEAs(A'—C')(A, A') One step model; (B, B') parallel model; (C, C') serial model. ● 329.15 K; ▲ 331.15 K; ■ 333.15 K.

Table 1 One-step deactivation parameters of free penicillin acylase and its CLEAs

Free enzyme			CLEAs
T/K	k_d/min^-1	R²	k_d/min^-1	R²
329.15	0.0083	0.9985	0.0012	0.6480
331.15	0.0264	0.9904	0.0018	0.7587
333.15	0.0343	0.9966	0.0044	0.6548

Table 2 Parallel deactivation parameters of free penicillin acylase and its CLEAs

Free enzyme					CLEAs
T/K	α	k_d1/min^-1	k_d2/min^-1	R²	α	k_d1/min^-1	k_d2/min^-1	R²
329.15	-28.55	0.0109	0.0110	0.9985	0.0372	0.0012	0.0012	0.5308
331.15	12.45	0.0265	0.0265	0.9808	1.320×10^-5	0.0019	0.0019	0.6897
333.15	-33.91	0.0270	0.0267	0.9976	0.6542	0.0372	0.0016	0.9919

Table 3 Serial deactivation parameters of free penicillin acylase and its CLEAs

Free enzyme						CLEAs
T/K	α	β	k_d1/min^-1	k_d2/min^-1	R²	α	β	k_d1/min^-1	k_d2/min^-1	R²
329.15	0.5017	0.5017	0.0083	0.0083	0.9972	0.6531	0.3446	4.024×10^-4	0.0132	0.9935
331.15	0.1048	0.8952	1.1743	0.0240	0.9653	0.7156	0.2837	9.871×10^-4	0.0205	0.9887
333.15						0.6542	0.3461	0.0016	0.0372	0.9892

Fig.5 Activation energy of thermal inactivation reaction

Fig.6 Reusability of CLEAs

References 27

[1]	Zhang Y., Jin W., Yang M. C., Zhang T. Q., Zhou C., Xie F., Song Q., Ren H., Jin Q. H., Mu Y., Chem. Res. Chinese Universities, 2012, 28(5), 792—796
[2]	Mohy E. M. S., Enshasy H. A. E., Hassan M. E., Haroun B., Hassan E. A., J. Appl. Polym. Sci., 2012, 125(5), 3820—3828
[3]	Gotti R., Fiori J., Calleri E., Temporini C., Lubda D., Massolini G., J. Chromatogr. A, 2012, 1234, 45—49
[4]	Pereira S. C., Bussamara R., Marin G., Giordano R. L. C., Dupont J., Giordano R. C., Green Chem., 2012, 14, 3146—3156
[5]	Xue Y. P., Jiang T., Liu X., Zheng Y. G., Biochem. Eng. J., 2013, 74, 88—94
[6]	Li D. C., Cheng S. W., Wei D. Z., Biotechnol. Lett., 2007, 29(12), 1825—1830
[7]	Cabrera Z., Lopez-Gallego F., Fernandez-Lorente G., Palomo J. M., Montes T., Grazu V., Guisán J. M., Fernández-Lafuente R., Enzyme Microb. Tech., 2007, 40(5), 997—1000
[8]	Zhou H. C., Yang L. R., Shou Q. H., Xu P., Li W. S., Wang F. C., Yu P. H., Liu H. Z., Ind. Eng. Chem. Res., 2012, 51(12), 4582—4590
[9]	Chen C. I., Ko Y. M., Lien W. L., Lin Y. H., Li I. T., Chen C. H., Shieh C. J., Liu Y. C., J. Membrane Sci., 2012, 401/402, 33—39
[10]	Cao L., van Rantwijk F., Sheldon R. A., Org. Lett., 2000, 2(10), 1361—1364
[11]	Hormigo D., García-Hidalgo J., Acebal C., Isabel de la M., Arroyo M., Bioresource Technol., 2012, 115, 117—182
[12]	Sinirlioglu A. Z., Sinirlioglu D., Akbas F., Bioresource Technol., 2013, 146, 807—811
[13]	Roessl U., Nahálka J., Nidetzky B., Biotechnol. Lett., 2010, 32, 341—350
[14]	Jaiprakash G. S., Kamalesh K. K., Gangadhar R. A., Biotechnol. Tech., 1987, 1(1), 69—72
[15]	Šulek F., Fernández D. P., Knez Z., Habulin M., Sheldon R. A., Process Biochem., 2011, 46(3), 765—769
[16]	Schoevaart R., Wolbers M. W., Golubovic M., Ottens M., Kieboom A. P. G., van Rantwijk F., van der Wielen L. A. M., Sheldon R. A., Biotechnol. Bioeng., 2004, 87(6), 754—762
[17]	Vafiadia C., Topakasa E., Alissandratosa A., Fauldsb C. B., Christakopoulos P., J. Biotechnol., 2008, 133(4), 497—504
[18]	Barbosa O., Torres R., Ortiz C., Fernandez-Lafuente R., Process Biochem., 2012, 47(8), 1220—1227
[19]	Barbosa O., Torres R., Ortiz C., Fernandez-Lafuente R., Process Biochem., 2012, 47(5), 766—774
[20]	Yu C. Y., Li X. F., Lou W. Y., Zong M. H., J. Biotechnol., 2012, 166(1/2), 12—19
[21]	Yan J. Y., Gui X. H., Wang G. L., Yan Y. J., Appl. Biochem. Biotechnol., 2012, 166(4), 925—932
[22]	Wang M. F., Qi W., He Z. M., Chem. J. Chinese Universities, 2010, 31(9), 1774—1779
	(王梦凡, 齐崴, 何志敏.高等学校化学学报, 2010,31(9), 1774—1779)
[23]	Hu Y., Yang J., Tang S. S., Chu X. M., Zou B., Huang H., Chem. J. Chinese Universities, 2013, 34(5), 1195—1202
	(胡燚, 杨姣, 唐苏苏, 初旭明, 邹彬, 黄和.高等学校化学学报, 2013,34(5), 1195—1202)
[24]	Xie X.Y., Wang C. X., Wang Z. Y., Acta Chim. Sinica, 2006, 64, 2151—2156
	(谢修银, 汪存信, 王志勇. 化学学报, 2006, 64, 2151—5156)
[25]	Ladero M., Ruiz G., Pessela B. C. C., Vian A., Santos A., Garcia-ohoa F., Biochem. Eng. J., 2006, 31, 14—24
[26]	Ladero M., Santos A., Garcia-ohoa F., Enzyme Microb. Tech., 2006, 38(1/2), 1—9
[27]	Grancic P., Illeova V., Polakovic M., Sefcik J., Chem. Eng. Sci., 2012, 70, 14—21