Chitodextrinase
Cloned chitinase (EC 3.2.1.14)
Source - Vibrio parahemolyticus gene
expressed in E. coli. Secreted enzyme. (Patent pending)
Specificity - The enzyme shows endochitinase
activity on chitin or swollen chitin but not chitosan. The enzyme releases chitin
oligomers with a limit digestion product of N,N’-diacetyl-chitobiose. Chitin
oligomers are quickly hydrolyzed beginning with a chitotetramer. Chitotriose is slowly
hydrolyzed and the enzyme shows transglycosylase activity with high concentrations of
small oligomers optimized at pH 8.8.
Description - The chitinase functions at an
unusually broad pH range of 4.5 to 10, has a degree of salt tolerance and temperature
tolerance to 50°C. With these durable properties, this chitinase will function
efficiently in a broad range of environmental conditions, which makes it suitable for
practical applications. In a fermentative degradation scheme for chitin biomass, a
continuous or batch enzyme reaction could be conducted at pH 9, 45°C in 1 M salt, which
would deter most contaminating organisms. (Patent pending) Also, the enzyme can be handled
as a stable, lyophilized powder or frozen solution, owing to its stability. No other
detectable glycosyl hydrolase activities are present. Enzyme is supplied either in
practical grade in 2.5 M ammonium sulfate or chromatographically purified as a lyophilized
powder. Molecular weight approximately 97,000.
Specific Activity - One unit of enzyme releases 1
mg (5 micromoles) of soluble product from insoluble swollen chitin per hour at 45°C.
Enzyme Assay - Ten microliter aliquots of
chitinase solution are incubated in a shaker bath for 10 minutes at 50°C with 190 µl of
tritium labeled chitin suspension at pH 6 to 9. The reaction is stopped with 200µl TCA
(20%), followed by centrifugation and determination of radioactivity in the supernatant
fraction. Chitooligosaccharide activity was measured by estimation of sulfuric acid
charred spots on a silica gel G thin layer chromatogram; acetonitrile:water/4:1.
Stability - The enzyme retained 100% activity in
pH 6 or 9 phosphate buffers with 1M NaCl and 25% activity at 4M NaCl. It is stable at pH 7
in solution at room temperature or 4°C and is stable to freezing and thawing.
Heat Stability - Chitinase optimal activity occurs
at 50°C (stable for 30-60 minutes), and inactivation at 70°C. The enzyme was stable for
3 days at 45°C. The enzyme should be stored at 4°C.
pH range - The enzyme is active from pH 5 to 9.5
with a broad optimum from 6 to 9.
| Catalog Number |
Quantity |
| R-1020-A Purified grade |
10 units |
| R-1021-B Practical grade |
10 units |
|
