Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (8): 221-232.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1326
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SUN Bao-ting(), QIU Meng-xia, WANG Zi-chen, WANG Zi-yuan, CUI Jian-dong(), JIA Shi-ru
Received:
2020-10-28
Online:
2021-08-26
Published:
2021-09-10
Contact:
CUI Jian-dong
E-mail:sunbaoting@mail.tust.edu.cn;jdcui@tust.edu.cn
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.
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
编号 No. | 因素 Factor/(mmol·L-1) | 水平 Level | ||
---|---|---|---|---|
-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 1 Test factors and level table of response surface
编号 No. | 因素 Factor/(mmol·L-1) | 水平 Level | ||
---|---|---|---|---|
-1 | 0 | 1 | ||
A | 2-甲基咪唑 | 100 | 200 | 300 |
B | 醋酸锌 | 40 | 60 | 80 |
C | Cys | 3 | 4 | 5 |
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 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 |
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 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 |
Mean | 34.74 |
---|---|
R-square | 99.52% |
Adj.R-square | 98.91% |
Std. Dev. | 1.65 |
CV | 4.76% |
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
Enzyme | Km | Vmax |
---|---|---|
PAL | 8.2756 ±0.67% | 0.0269 ±1.08% |
PAL@ZIF-8 | 10.9602 ±0.58% | 0.0171±0.72% |
Table 5 Comparison of apparent kinetic parameters of free PAL and PAL@ZIF-8
Enzyme | Km | Vmax |
---|---|---|
PAL | 8.2756 ±0.67% | 0.0269 ±1.08% |
PAL@ZIF-8 | 10.9602 ±0.58% | 0.0171±0.72% |
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 |
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 |
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