半胱氨酸辅助的酶@ZIF-8固定化酶制备及其特性研究

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

摘要/Abstract

摘要：

以共沉淀包埋的策略将酶固定在ZIF-8金属有机骨架中能显著提高酶的催化稳定性,但是,所制备的酶@ZIF-8固定化酶的固定化效率和酶活回收率却很低。为了解决这一问题,本研究以半胱氨酸（cysteine,Cys）为辅助剂,采用共沉淀包埋的策略将苯丙氨酸解氨酶（PAL）固定在ZIF-8载体中,制备出PAL@ZIF-8固定化酶。利用单因素实验及响应面实验优化了PAL@ZIF-8制备条件,并对其pH耐受性、温度耐受性和重复使用性等催化性能进行研究。响应面优化结果表明,2-甲基咪唑浓度、醋酸锌浓度和Cys浓度对酶活回收率有显著影响,醋酸锌浓度和Cys浓度对酶活回收率存在交互影响,在200 mmol/L 2-甲基咪唑、60 mmol/L醋酸锌和4 mg Cys,2 mg聚乙烯吡咯烷酮（polyvinylpyrrolidone,PVP）的优化条件下,PAL@ZIF-8最大酶活回收率为（56.15±0.94）%。与不添加Cys制备的PAL@ZIF-8相比,酶活回收率提高了近40%,而且PAL@ZIF-8比游离PAL表现出更好的温度耐受性和pH耐受性。重复使用7次后,PAL@ZIF-8仍能保持初始酶活的60%左右,表现出较好的重复使用稳定性。以上结果表明,这种以Cys为辅助剂将酶共沉淀包埋在ZIF-8中的方法是一种高效的酶固定化策略。

关键词: 苯丙氨酸解氨酶, 金属有机框架物, 半胱氨酸, 酶固定化

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

孙宝婷, 邱萌霞, 王子辰, 王梓源, 崔建东, 贾士儒. 半胱氨酸辅助的酶@ZIF-8固定化酶制备及其特性研究[J]. 生物技术通报, 2021, 37(8): 221-232.

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.

图/表 15

图1 半胱氨酸辅助的PAL@ZIF-8制备过程

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

图2 反式肉桂酸标准曲线

Fig.2 Standard curve of trans-cinnamic acid

图3 各因素对PAL@ZIF-8酶活回收率的影响 A：2-甲基咪唑浓度对PAL@ZIF-8酶活回收率的影响;B：醋酸锌浓度对PAL@ZIF-8酶活回收率的影响;C：Cys对PAL@ZIF-8酶活回收率的影响;D：PVP对PAL@ZIF-8酶活回收率的影响

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

表1 响应面实验因素及水平表

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

表2 Box-Behnken实验设计及响应值

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

表3 Box-Behnken实验设计方差分析表

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

表4 模型的可信度分析

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%

图4 各因素交互作用对苯丙氨酸解氨酶酶活回收率影响的响应面图和等高线图 a：Cys添加量对酶活回收率影响;b：醋酸锌浓度对酶活回收率影响;c：2-甲基咪唑浓度对酶活回收率影响

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

图5 PAL@ZIF-8扫描电镜图 A：传统ZIF-8扫描电镜图;B：Cys辅助合成PAL@ZIF-8扫描电镜图

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

图6 PAL@ZIF-8激光扫描共聚焦显微镜图

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

图7 ZIF-8、PAL、L-Cys和PAL@ZIF-8的能谱图

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

图8 PAL@ZIF-8的催化性能 A：最适催化温度;B：温度耐受性;C：最适催化pH;D：pH耐受性

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

表5 PAL与PAL@ZIF-8的表观动力学常数

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%

表6 负载率及酶活回收率对比

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

图9 PAL@ZIF-8重复使用性及储存稳定性 A：重复使用性;B：储存稳定性

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

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