A rapid and sensitive method for screening of chitinase inhibitors using Ostazin Brilliant Red labelled chitin as a substrate for chitinase assay.

Abstract:

AIMS:The development of a simple, rapid and sensitive method for the screening of chitinase inhibitors using Ostazin Brilliant Red labelled chitin as a substrate for chitinase assay. METHODS AND RESULTS:The colloidal substrate prepared from the Ostazin Brilliant Red labelled chitin was used in the estimation of chitinase activity with and without the addition of potential chitinase inhibitor. The dye labelled N-acetylglucosamine (GlcNAc) released because of hydrolysis of the substrate was measured spectrophotometrically at 530 nm. The release of dye labelled GlcNAc from the colloidal Red chitin was proportional to the chitinase activity measured using other methods. The activities measured using acid swollen chitin and -Red chitin were comparable in the range 0.1-0.5 U ml(-1). The addition of chitinase inhibitor(s) (1000 ppm) proportionally reduced the release of dye labelled GlcNAc from the substrate in the reaction mixture. CONCLUSIONS:The assay for chitinase activity measuring the release of dye-labelled GlcNAc was simple, rapid and sensitive. There was no need to measure GlcNAc using a separate colorimetric method. SIGNIFICANCE AND IMPACT OF THE STUDY:The method provides a simple, one-step procedure for quantitative estimation of total chitinolytic activity and for screening of endochitinase and/or N-acetylglucosaminidase inhibitors acting singly or in combination. The method has a great potential in developing medium throughput procedure for the screening of chitinase inhibitors using 96-well plate reader.

journal_name

Lett Appl Microbiol

authors

Shirazi F,Kulkarni M,Deshpande MV

doi

10.1111/j.1472-765X.2007.02117.x

subject

Has Abstract

pub_date

2007-06-01 00:00:00

pages

660-5

issue

6

eissn

0266-8254

issn

1472-765X

pii

LAM2117

journal_volume

44

pub_type

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