Zymogram technique for detecting enzymatic activity of keratin

Abstract

The invention provides a zymogram technique for detecting enzymatic activity of keratin. The steps of keratin substrate treatment, gel production, electrophoresis and hydrolysis strip color development are modified, and the zymogram technique for detecting the enzymatic activity of the keratin comprises the following steps: (1) adding 10 g of a keratin substrate and 500 ml of an organic solvent into a reflux condensing device, conducting digestion at 80-100 DEG C for 60-100 min, precipitating keratin by means of acetone, with volume twice that of the keratin, precooled at 4 DEG C, conducting centrifugation, collecting keratin precipitate, and conducting airing at 4 DEG C after cleaning is conducted; and (2) producing separation gel: adding a keratin substrate treated in the step (1) whilemaking the mass percentage content of the keratin substrate 0.1%, then conducting stirring at a room temperature and speed of 100 r/min for 1 h, conducting centrifugation, and taking supernate to produce the separation gel with a mass concentration of 12%. According to the zymogram technique for detecting the enzymatic activity of the keratin, the limitations that a traditional zymogram techniqueis low in substrate specificity, low in hydrolysis strip distinguishing degree, low in sensitivity and the like are overcome, and the provided zymogram technique for detecting the enzymatic activity of the keratin is accurate and sensitive.

Description

technical field [0001] The invention belongs to the field of protease detection, and in particular relates to a zymography method for detecting keratinase activity. Background technique [0002] A large amount of keratin waste, such as feathers, fur, horns, hooves, and claws, will be produced in the production of livestock and poultry breeding. Keratin is a kind of rigid fibrous protein, its structure is maintained by the interaction of disulfide bond, hydrogen bond, salt bond and peptide bond, and it is not easy to dissolve and digest. Traditional physical and chemical methods to treat keratin waste not only have a low conversion rate, easily destroy the nutritional content of keratin, but also cause environmental pollution. Keratin degrading microorganisms can produce a class of enzymes with keratin hydrolysis activity. In 1963, Nickerson et al. The enzyme is called keratinase. Most of the keratinases found so far have a wide range of substrates. In addition to degradin...

AI Technical Summary

Problems solved by technology

[0004] However, for the detection of some special proteases (such as: keratinase), there are some technical deficiencies in gel zymography: (1) common substrates Low specificity, unable to detect keratinase with extensive hydrolysis ability; (2) Due to the particularity of keratin substrate, directly adding keratin substrate cannot effectively combine with acrylamide, resulting in gel background, The hydrolysis band has low discrimination and low sensitivity, and cannot accurately analyze the enzyme quantitatively or qualitatively.

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