黑曲霉N5-5产单宁酶的酶学性质与固定化

Kinetic Characterization and Immobilization of Tannase Produced by Aspergillus niger N5-5

摘要:

本实验室自主分离的黑曲霉N5-5所产单宁酶已发现对没食子酸丙酯具有良好酶解效果。为了单宁酶的工业化应用，本次研究大批量培养单宁酶，探究其对单宁酸的酶解效果及酶的固定化效果。发酵采用先液体扩培后固体发酵的形式，提酶后利用陶瓷膜过滤技术纯化、浓缩酶液，并对比冷冻干燥和喷雾干燥两种不同干燥方式，还使用树脂载体对酶进行固定化。实验表明，纯化后单宁酶酶活达258.53 U/mL，可水解10%至50%浓度的单宁酸，其中30%以下底物浓度酶解效果较好；冷冻干燥对酶活影响不大而喷雾干燥使酶活降为174.02 U/mL；另外，单宁酶通过树脂载体固定化后，在实验条件下可重复使用至少4次。研究为提高黑曲霉N5-5发酵所产单宁酶的媒介效率、降低酶解反应成本、实现商业化生产建立了理论基础。

Abstract:

Tannase produced by Aspergillus niger N5-5 has been independently isolated in our laboratory, and proven to efficiently hydrolyze propyl gallate. To explore industrial applications for tannase, we cultivated large quantities of tannase and investigated the kinetics of its hydrolysis of tannin, and the effects of immobilization on its enzymatic activity. Fermentation began with growth in liquid media, followed by fermentation on solid media. After enzyme extraction, ceramic membrane filtration was used to purify and concentrate the enzyme. Two different drying methods, (lyophilization and spray drying) were adopted. Activated resins were used to immobilize tannase. The purified tannase solution has an enzymatic activity of 258.53 U/mL. It can hydrolyze 10%~50% tannic acid solutions, and its performance is better when substrate concentrations are less than 30%. Lyophilization has little effect on enzyme activity, while spray drying decreases enzyme activity to 174.02 U/mL. In addition, tannase immobilized on resin can be reused at least 4 times under experimental conditions. This research has established a theoretical basis for improving production efficiency of tannase from Aspergillus niger N5-5, reducing the cost of enzymatic hydrolysis, and realizing commercial enzyme production.