The kit assay is based on the enzymatic hydrolysis of chitinase substrates. This enzymatic hydrolysis releases 4-methylumbelliferone (4MU), which upon ionization in basic pH, can be measured fluorimetrically at an excitation wavelength of 360 nm and an emission wavelength of 450 nm. The use of fluorimetric substrates provides a very sensitive detection system.
The Chitinase Assay Kit provides all the reagents required for efficient and sensitive detection of chitinase activity in fungal and bacterial growth media, macrophage lysates, and purified enzyme preparations. In addition, the kit provides three different substrates for the detection of the various types of the chitinolytic activity:
- 4-Methylumbelliferyl N,N′-diacetyl-β-D-chitobioside - substrate suitable for exochitinase activity detection (chitobiosidase activity)
- 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide - substrate suitable for exochitinase activity detection (β-N-acetylglucosaminidase activity)
- 4-Methylumbelliferyl β-D-N,N′,N′′-triacetylchitotriose - substrate suitable for endochitinase activity detection
Chitinase catalyzes the hydrolytic cleavage of the β-1→4-glycoside bond present in biopolymers of N-acetylglucosamine, primarily in chitin. Chitinases are widely distributed in living organisms and are found in fungi, bacteria, parasites, plants, and animals. They are classified in families based on amino acid sequence similarities.
The chitinolytic enzymes are also categorized based on their enzymatic action on chitin substrates. Endochitinases are defined as the enzymes catalyzing the random cleavage at internal points in the chitin chain. Exochitinases catalyze the progressive release of acetylchitobiose or N-acetylglucosamine from the non-reducing end of chitin, and are referred to as chitobiosidase and β-N-acetylglucosaminidase, respectively.
Chitinases perform different functions in different organisms. In bacteria, they are mainly involved in nutritional processes. In yeast and various fungi, these enzymes participate in morphogenesis. In animals and plants, chitinases primarily play a role in the defense of the organism against pathogen attack.
The kit was tested and found suitable for Trichoderma viride and Streptomyces griseus, along with Hela, Jurkat, CHO, NIH-3T3, U-837 mammalian cell lines, human macropages, rat lung, kidney, liver, and brain tissue.
Use ultrapure water for preparation of reagents.