高灵敏度测定壳聚糖酶活力的新方法及其比较

doi:10.7506/spkx1002-6630-201309056

食品科学 ›› 2013, Vol. 34 ›› Issue (9): 277-281.doi: 10.7506/spkx1002-6630-201309056

高灵敏度测定壳聚糖酶活力的新方法及其比较

张永勤，张杰，常海燕，张坤，刘征东，罗彩华，牟晓凤

青岛科技大学化工学院，山东青岛 266042

收稿日期:2013-03-18 修回日期:2013-04-17 出版日期:2013-05-15 发布日期:2013-05-07
通讯作者: 张永勤 E-mail:zyq0205@qust.edu.cn

A Novel Sensitive Assay for Chitosanolytic Enzyme Activity and Its Comparison with Two Others

ZHANG Yong-qin，ZHANG Jie，CHANG Hai-yan，ZHANG Kun，LIU Zheng-dong，LUO Cai-hua，MOU Xiao-feng

College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China

Received:2013-03-18 Revised:2013-04-17 Online:2013-05-15 Published:2013-05-07
Contact: Zhang Yong-qin E-mail:zyq0205@qust.edu.cn

摘要/Abstract

摘要：

目的：建立一种测定壳聚糖酶活力的新方法——3-甲基-2-苯并噻唑啉酮腙盐酸盐水合物(MBTH)法。方法：以动力学法通过测定水解过程中壳聚糖释放还原端基的速率来计算酶活力，并以测定纤维素酶中壳聚糖酶的微量活力为例，对测定过程中的几个关键参数进行讨论。结果：在37℃、pH6.0酶解条件下，底物壳聚糖溶液的测定质量终浓度需达到2mg/mL以上；酶解反应时间控制在60min内为宜，可以用终点法定量测得壳聚糖酶活力。结论：通过比较MBTH法、DNS法和铁氰化钾法发现MBTH法更准确、更灵敏、检测范围宽、操作简便。其检测限为13mU/mL，而铁氰化钾法是该法的1.6倍，DNS法是该法的116倍。应用本法所测其酶解壳聚糖的Km值为0.12mg/mL，而用DNS法则较难准确测得。

关键词: 壳聚糖酶, 3-甲基-2-苯并噻唑酮, 还原端, 壳聚糖, 3,5-二硝基水杨酸, 铁氰化钾

Abstract:

A novel spectrophotometric method for the quantitative assay of chitosanolytic enzyme activity using 3-methyl-2-
benzothiazolinone (MBTH) was estalished. The proposed method was based on measuring the reducing ends released during the
enzymatic degradation of chitosan with the non-specific enzyme cellulase. The effects of several key hydrolysis parameters on
activity assay were discussed and investigated by multiple-point procedures. The results showed that the substrate concentration
was required to be no less than 2 mg/mL with an initial pH of 6.0, and the hydrolysis was allowed to proceed for no more than
60 min at 37 ℃ before end-point measurement. In comparison with the 3,5-dinitrosalicylic acid (DNS) method and the potassium
ferricyanide method, the MBTH method provided higher accuracy and sensitivity, wider detection range and simpler operation, and
showed a limit of detection of 13 mU/mL, which was 1.6 times smaller than that observed for the potassium ferricyanide method
and 116 times smaller than that observed for the DNS method. The Km for chitosan hydrolysis determined by the MBTH method
was 0.12 mg/mL, whereas accurrate Km measurement was difficult to achieve in the DNS method.

Key words: chitosanolytic enzyme, 3-methyl-2-benzothiazolinone, reducing end, chitosan, 3,5-disalicylic acid, potassium ferricyanide

中图分类号:

Q814.9

张永勤，张杰，常海燕，张坤，刘征东，罗彩华，牟晓凤. 高灵敏度测定壳聚糖酶活力的新方法及其比较[J]. 食品科学, 2013, 34(9): 277-281.

ZHANG Yong-qin，ZHANG Jie，CHANG Hai-yan，ZHANG Kun，LIU Zheng-dong，LUO Cai-hua，MOU Xiao-feng. A Novel Sensitive Assay for Chitosanolytic Enzyme Activity and Its Comparison with Two Others[J]. FOOD SCIENCE, 2013, 34(9): 277-281.

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高灵敏度测定壳聚糖酶活力的新方法及其比较

A Novel Sensitive Assay for Chitosanolytic Enzyme Activity and Its Comparison with Two Others

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