高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (9): 1888.doi: 10.7503/cjcu20190185
收稿日期:
2019-03-27
出版日期:
2019-09-10
发布日期:
2019-07-29
通讯作者:
苏二正
E-mail:ezhsu@njfu.edu.cn
基金资助:
WU Rong1,2,DONG Qihui1,2,SUN Yiyi1,SU Erzheng1,2,*()
Received:
2019-03-27
Online:
2019-09-10
Published:
2019-07-29
Contact:
SU Erzheng
E-mail:ezhsu@njfu.edu.cn
Supported by:
摘要:
以食品工业中常用的木瓜蛋白酶为模式酶, 建立了吸附-纤维素覆膜联合固定化酶方法. 通过对吸附载体类别、 纤维素种类及溶剂、 保护剂种类及其浓度、 干燥方式及时间等的优化, 得到最佳的吸附-纤维素覆膜联合固定化酶工艺. 以硅藻土或HPD-417(大孔树脂)作为吸附载体, 甲基纤维素(分子量40000~50000)丙酮溶液作为覆膜溶液, 加入6%(质量分数)的聚乙二醇或麦芽糖作为覆膜保护剂, 于4 ℃干燥9 h, 制得固定化木瓜蛋白酶, 硅藻土吸附-纤维素覆膜固定化酶酶活回收率达到96.50%, HPD-417吸附-纤维素覆膜固定化酶酶活回收率达到93.92%. 对吸附-纤维素覆膜固定化酶的性质进行了研究, 发现纤维素覆膜后固定化酶具有良好的热稳定性, 于80 ℃下保存12 h后, 固定化酶活残余率仍然能保持90%左右; 在pH=4.5~9.5的范围内, 固定化酶的稳定性较好; 连续使用9次后, 固定化酶活残余率仍能保持95%左右.
中图分类号:
TrendMD:
吴蓉, 董其惠, 孙伊伊, 苏二正. 吸附-纤维素覆膜联合固定化酶. 高等学校化学学报, 2019, 40(9): 1888.
WU Rong, DONG Qihui, SUN Yiyi, SU Erzheng. Efficient Enzyme Immobilization by Combining Adsorption and Cellulose Membrane Coating †. Chem. J. Chinese Universities, 2019, 40(9): 1888.
Adsorption material | Protein immobilization yield(%) | Immobilized enzyme activity/(U·g-1) | Enzyme activity recovery rate(%) |
---|---|---|---|
D130 | 94.91±0.07 | 11.68±0.040 | 41.91±1.57 |
HPD-450 | 99.01±0.06 | 12.18±0.016 | 40.49±0.27 |
ADS-17 | 95.95±0.18 | 25.68±0.054 | 66.51±0.59 |
HPD-417 | 95.11±0.07 | 28.57±0.032 | 60.33±0.46 |
D101 | 96.21±0.33 | 9.38±0.042 | 30.38±0.34 |
AB-8 | 95.65±0.03 | 10.34±0.12 | 39.63±0.90 |
Activated carbon(30 mesh) | 34.12±0.49 | 6.01±0.012 | 43.29±1.34 |
Activated carbon(40 mesh) | 35.49±0.33 | 6.09±0.022 | 37.64±0.46 |
Zeolite | 38.69±0.25 | 9.44±0.026 | 42.96±0.61 |
Kaolin | 55.55±0.25 | 29.07±0.016 | 98.13±1.12 |
Imvite | 100.00 | 32.76±0.026 | 99.47±1.28 |
Attapulgite | 58.92±0.40 | 13.05±0.032 | 53.08±1.76 |
Diatomite | 51.47±0.09 | 30.61±0.022 | 98.38±2.62 |
Table 1 Immobilization of papain on different carriers by adsorbing method
Adsorption material | Protein immobilization yield(%) | Immobilized enzyme activity/(U·g-1) | Enzyme activity recovery rate(%) |
---|---|---|---|
D130 | 94.91±0.07 | 11.68±0.040 | 41.91±1.57 |
HPD-450 | 99.01±0.06 | 12.18±0.016 | 40.49±0.27 |
ADS-17 | 95.95±0.18 | 25.68±0.054 | 66.51±0.59 |
HPD-417 | 95.11±0.07 | 28.57±0.032 | 60.33±0.46 |
D101 | 96.21±0.33 | 9.38±0.042 | 30.38±0.34 |
AB-8 | 95.65±0.03 | 10.34±0.12 | 39.63±0.90 |
Activated carbon(30 mesh) | 34.12±0.49 | 6.01±0.012 | 43.29±1.34 |
Activated carbon(40 mesh) | 35.49±0.33 | 6.09±0.022 | 37.64±0.46 |
Zeolite | 38.69±0.25 | 9.44±0.026 | 42.96±0.61 |
Kaolin | 55.55±0.25 | 29.07±0.016 | 98.13±1.12 |
Imvite | 100.00 | 32.76±0.026 | 99.47±1.28 |
Attapulgite | 58.92±0.40 | 13.05±0.032 | 53.08±1.76 |
Diatomite | 51.47±0.09 | 30.61±0.022 | 98.38±2.62 |
Cellulose | Adsorption material | Immobilized enzyme activity by adsorption/(U·g-1) | Immobilized enzyme activity after coating/(U·g-1) | Residual enzyme activity after coating(%) |
---|---|---|---|---|
Cellulose acetate | Diatomite | 30.94±0.27 | 18.68±0.88 | 60.37±0.73 |
ADS-17 | 25.56±1.92 | 17.60±0.93 | 68.88±0.39 | |
HPD-417 | 27.66±0.83 | 19.91±1.21 | 71.99±1.38 | |
Diacetate cellulose | Diatomite | 30.94±0.27 | 20.90±0.96 | 67.54±1.91 |
ADS-17 | 25.56±1.92 | 18.76±0.77 | 73.41±1.28 | |
HPD-417 | 27.66±0.83 | 20.78±0.35 | 75.11±0.93 | |
Methyl cellulose | Diatomite | 30.94±0.27 | 21.03±0.93 | 67.97±1.36 |
40000—50000 | ADS-17 | 25.56±1.92 | 19.26±1.24 | 75.34±1.21 |
HPD-417 | 27.66±0.83 | 21.55±0.82 | 77.91±0.63 | |
Methyl cellulose | Diatomite | 30.94±0.27 | 21.47±0.69 | 69.38±1.91 |
12000—13000 | ADS-17 | 25.56±1.92 | 20.36±0.79 | 79.67±0.86 |
HPD-417 | 27.66±0.83 | 21.18±0.83 | 76.56±1.22 |
Table 2 Immobilization of papain by adsorbing-cellulose membrane coating method with cellulose dissolved in acetone
Cellulose | Adsorption material | Immobilized enzyme activity by adsorption/(U·g-1) | Immobilized enzyme activity after coating/(U·g-1) | Residual enzyme activity after coating(%) |
---|---|---|---|---|
Cellulose acetate | Diatomite | 30.94±0.27 | 18.68±0.88 | 60.37±0.73 |
ADS-17 | 25.56±1.92 | 17.60±0.93 | 68.88±0.39 | |
HPD-417 | 27.66±0.83 | 19.91±1.21 | 71.99±1.38 | |
Diacetate cellulose | Diatomite | 30.94±0.27 | 20.90±0.96 | 67.54±1.91 |
ADS-17 | 25.56±1.92 | 18.76±0.77 | 73.41±1.28 | |
HPD-417 | 27.66±0.83 | 20.78±0.35 | 75.11±0.93 | |
Methyl cellulose | Diatomite | 30.94±0.27 | 21.03±0.93 | 67.97±1.36 |
40000—50000 | ADS-17 | 25.56±1.92 | 19.26±1.24 | 75.34±1.21 |
HPD-417 | 27.66±0.83 | 21.55±0.82 | 77.91±0.63 | |
Methyl cellulose | Diatomite | 30.94±0.27 | 21.47±0.69 | 69.38±1.91 |
12000—13000 | ADS-17 | 25.56±1.92 | 20.36±0.79 | 79.67±0.86 |
HPD-417 | 27.66±0.83 | 21.18±0.83 | 76.56±1.22 |
Cellulose | Adsorption material | Immobilized enzyme activity by adsorption/(U·g-1) | Immobilized enzyme activity after coating/(U·g-1) | Residual enzyme activity after coating(%) |
---|---|---|---|---|
Cellulose acetate | Diatomite | 30.94±0.27 | 19.64±0.91 | 63.47±0.72 |
ADS-17 | 25.56±1.92 | 17.24±1.21 | 67.47±0.69 | |
HPD-417 | 27.66±0.83 | 19.29±0.66 | 69.74±1.35 | |
Diacetate cellulose | Diatomite | 30.94±0.27 | 21.02±0.71 | 67.93±1.22 |
ADS-17 | 25.56±1.92 | 17.30±1.92 | 67.69±0.82 | |
HPD-417 | 27.66±0.83 | 19.89±1.25 | 71.89±0.69 | |
Methylcellulose | Diatomite | 30.94±0.27 | 21.44±0.52 | 69.35±0.77 |
40000—50000 | ADS-17 | 25.56±1.92 | 18.79±1.12 | 73.51±0.96 |
HPD-417 | 27.66±0.83 | 20.31±0.57 | 73.42±1.02 | |
Methylcellulose | Diatomite | 30.94±0.27 | 21.60±0.82 | 69.81±1.28 |
12000—13000 | ADS-17 | 25.56±1.92 | 18.56±0.72 | 72.61±0.65 |
HPD-417 | 27.66±0.83 | 21.04±0.91 | 76.07±0.74 |
Table 3 Immobilization of papain by adsorbing-cellulose membrane coating method with cellulose dissolved in ethyl acetate
Cellulose | Adsorption material | Immobilized enzyme activity by adsorption/(U·g-1) | Immobilized enzyme activity after coating/(U·g-1) | Residual enzyme activity after coating(%) |
---|---|---|---|---|
Cellulose acetate | Diatomite | 30.94±0.27 | 19.64±0.91 | 63.47±0.72 |
ADS-17 | 25.56±1.92 | 17.24±1.21 | 67.47±0.69 | |
HPD-417 | 27.66±0.83 | 19.29±0.66 | 69.74±1.35 | |
Diacetate cellulose | Diatomite | 30.94±0.27 | 21.02±0.71 | 67.93±1.22 |
ADS-17 | 25.56±1.92 | 17.30±1.92 | 67.69±0.82 | |
HPD-417 | 27.66±0.83 | 19.89±1.25 | 71.89±0.69 | |
Methylcellulose | Diatomite | 30.94±0.27 | 21.44±0.52 | 69.35±0.77 |
40000—50000 | ADS-17 | 25.56±1.92 | 18.79±1.12 | 73.51±0.96 |
HPD-417 | 27.66±0.83 | 20.31±0.57 | 73.42±1.02 | |
Methylcellulose | Diatomite | 30.94±0.27 | 21.60±0.82 | 69.81±1.28 |
12000—13000 | ADS-17 | 25.56±1.92 | 18.56±0.72 | 72.61±0.65 |
HPD-417 | 27.66±0.83 | 21.04±0.91 | 76.07±0.74 |
Fig.1 Effects of polyethylene glycol(A1, A2), sorbitol(B1, B2) and maltose(C1, C2) as protectants on immobilized papain with methyl cellulose(12000—13000)(A1—C1) and methyl cellulose(40000—50000)(A2—C2) dissolved in acetone as coating solution a. Diatomite; b. HPD-417.
Fig.3 Effect of drying time on the immobilization of papain (A) Polyethylene glycol as the protectant; (B) maltose as the protectant. a. Diatomite; b. HPD-417.
Fig.4 Optimal pH(A) and pH stability(B) of free and immobilized papain by adsorbing-cellulose membrane coating method a. Diatomite; b. HPD-417; c. free enzyme.
Fig.5 Optimum temperature(A) and thermal stability(B) of free and immobilized papain by adsorbing-cellulose membrane coating method a. Diatomite; b. HPD-417; c. free enzyme.
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