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

doi:10.7506/spkx1002-6630-201309056

Abstract

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

CLC Number:

Q814.9

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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