Preparation of @ZIF-8 Immobilized Enzyme by Using Cysteine as Auxiliary Reagent and Its Characterization

doi:10.13560/j.cnki.biotech.bull.1985.2020-1326

Abstract

Abstract:

Immobilization of enzyme in ZIF-8 metal-organic framework by co-precipitation embedding strategy may significantly improve the catalytic stability of the enzyme. However,immobilization efficiency and enzyme activity recovery of the prepared enzymes@ZIF-8 composites are very low. For solving this problem,phenylalanine ammonialyase（PAL）was encapsulated into the ZIF-8 using cysteine（Cys）as an auxiliary reagent by “co-precipitation” approach to prepare PAL@ZIF-8 immobilized enzymes. The preparation conditions of PAL@ZIF-8 immobilized enzymes were optimized by single factor experiment and response surface experiment. Moreover,catalytic properties of PAL@ZIF-8 immobilized enzymes including pH tolerance,temperature tolerance and reusability were investigated. The results of the response surface experiment showed that 2-methylimidazole concentration,zinc acetate concentration,and Cys concentration significantly affected the activity recovery of the enzyme,and there was an interaction between zinc acetate concentration and Cys concentration on activity recovery. The maximum activity recovery of the PAL@ZIF-8 immobilized enzymes was（56.15±0.94）% under 200 mmol/L 2-methylimidazole,60 mmol/L zinc acetate,4 mg Cys,and 2 mg polyvinylpyrrolidone（PVP）. Compared with the PAL@ZIF-8 without Cys,the activity recovery of the PAL@ZIF-8 immobilized enzymes increased about 40%. Furthermore,the PAL@ZIF-8 immobilized enzymes presented better temperature and pH tolerance than free PAL,and retained 60% of the initial activity after 7 cycles,showing good reusability. Above results indicate that this method of embedding enzyme co-precipitation in ZIF-8 using Cys as auxiliary reagent is an efficient strategy for enzyme immobilization.

Key words: phenylalanine ammonialyase, metal organic frameworks, cysteine, enzyme immobilization

SUN Bao-ting, QIU Meng-xia, WANG Zi-chen, WANG Zi-yuan, CUI Jian-dong, JIA Shi-ru. Preparation of @ZIF-8 Immobilized Enzyme by Using Cysteine as Auxiliary Reagent and Its Characterization[J]. Biotechnology Bulletin, 2021, 37(8): 221-232.

Figures/Tables 15

Fig.1 Cysteine-assisted preparation of PAL@ZIF-8

Fig.2 Standard curve of trans-cinnamic acid

Fig.3 Influence of various factors on the recovery of PAL@ZIF-8 activity A: Effect of 2-methylimidazole concentration on the recovery of PAL@ZIF-8 activity. B: Effect of zinc acetate concentration on the recovery of PAL@ZIF-8 activity. C: Effect of addition of Cys on the recovery of PAL@ZIF-8 activity. D: Effect of addition of PVP on the recovery of PAL@ZIF-8 activity

Table 1 Test factors and level table of response surface

编号 No.	因素 Factor/（mmol·L^-1）	水平 Level
编号 No.	因素 Factor/（mmol·L^-1）	-1	0	1
A	2-甲基咪唑	100	200	300
B	醋酸锌	40	60	80
C	Cys	3	4	5

Table 2 Box-Behnken experimental design and response values

编号 No.	A	B	C	酶活回收率 Recovery of PAL activity /%
1	200	60	4	55.21
2	300	80	4	23.86
3	200	40	5	34.83
4	200	60	4	55.47
5	200	80	3	30.15
6	100	80	4	19.98
7	300	60	3	26.83
8	100	60	5	18.52
9	100	40	4	26.71
10	200	80	5	25.49
11	200	60	4	55.81
12	200	60	4	55.97
13	300	60	5	22.67
14	200	60	4	55.62
15	200	40	3	40.32
16	300	40	4	31.89
17	100	60	3	20.47

Table 3 Analysis for regression equation of Box-Behnken design

方差来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方差 Mean square	F	P	显著性 Significance
Model	3983.91	9	442.66	161.95	< 0.0001	**
A	47.78	1	47.78	17.48	0.0041	**
B	107.24	1	107.24	39.23	0.0004	**
C	21.58	1	21.58	7.90	0.0261	*
AB	4.02	1	4.02	1.47	0.2646
AC	14.59	1	14.59	5.34	0.0541
BC	23.23	1	23.23	8.50	0.0225	*
A²	2247.70	1	2247.70	822.32	< 0.0001	**
B²	352.11	1	352.11	128.82	< 0.0001	**
C²	832.62	1	832.62	304.61	< 0.0001	**
残差误差	19.13	7	2.73
失拟	15.65	3	5.22	6.00	0.0581
纯误差	3.48	4	0.8702
合计	4003.04	16

Table 4 Reliability analysis of the model

Mean	34.74
R-square	99.52%
Adj.R-square	98.91%
Std. Dev.	1.65
CV	4.76%

Fig.4 Response surface plot and contour plot of the effect of interaction of various factors on the recovery of PAL activity a：The effect of Cys addition on the recovery of PAL activity. b：Effect of zinc acetate concentration on the recovery of PAL activity. c：Effect of 2-methylimidazole concentration on the recovery of PAL activity

Fig.5 Scanning electron microscope(SEM)of PAL@ZIF-8 A: SEM of traditional ZIF-8; B: SEM of cysteine-assisted PAL@ZIF-8

Fig.6 Laser scanning confocal microscopy of PAL@ZIF-8

Fig.7 Energy disperse spectroscopy spectrum of ZIF-8,PAL,L-Cys and PAL@ZIF-8

Fig. 8 Catalytic performance of PAL@ZIF-8 A: Optimal temperature. B: Temperature tolerance. C: Optimal pH. D: pH tolerance

Table 5 Comparison of apparent kinetic parameters of free PAL and PAL@ZIF-8

Enzyme	K_m	V_max
PAL	8.2756 ±0.67%	0.0269 ±1.08%
PAL@ZIF-8	10.9602 ±0.58%	0.0171±0.72%

Table 6 Comparison of load rate and recovery of PAL activity

分组 Group	酶蛋白负载率 Enzyme load rate/%	酶活回收率 Recovery of PAL activity/%
传统PAL@ZIF-8 Traditional PAL@ZIF-8	82.11±0.27	13.38±0.12
Cys辅助的PAL@ZIF-8 Cysteine-assisted PAL@ZIF-8	94.07±0.14	56.15±0.94

Fig. 9 Reusability and storage stability of PAL@ZIF-8 A: Reusability. B: Stability

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