Difference between revisions of "Part:BBa K1201000"

Revision as of 11:10, 9 October 2022

Derived from serratia marcescens

Chitinase gene; ChiA. Is a processive enzyme which degrades chitin from the reducing end. Suitable for projects which require an enzyme that degrades chitin.

Characterization We add 1 mL 200 m g / L of substrate to a clean test tube, 3mL 0.05 mol / L phosphate buffer , 3 min 50 ° C water bath temperature protection Min, and add 1 mL enzyme solution, mix and heat in 50 degrees water 15 min. After that, we used boiling water to terminate the enzymatic reaction, and add water to a volume of 10 mL. Evenly, the solution was centrifuged at 3000r /min for 10 min. Measure the OD value of the upper clear. Definition of enzyme unit: The amount of enzyme required to produce 1 gram of p-nitroaniline per hour under the above reaction conditions is defined as 1 enzyme activity ( U /m L ).

The calculation formula is: (A-A0)*Enzyme dilution factor/kt. In the formula: A: The absorbed value of the enzymatic hydrolyzate sample after constant volume A is fixed

A0: The absorbance of the post-enzymatic solution blank

T : Enzymatic reaction time, unit: h

k, linear coefficient, 0.0648

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For the measurement of binding capacity

we took 2 μmol/L of chitinase in 50 mmol/L phosphate buffer (pH=8) and mixed thoroughly at 4°C (assuming no degradation), reacted for 1 h on a rotary mixer, 10,000 r/min for 5 min and collected the supernatant, which was the unbound protein, by Protein concentration was measured by the BCA method.

Figure 1 Binding rates

For the measurement of chitinase activity, chitinase is able to hydrolyse chitin to produce N-acetylglucosamine, which further reacts with 35-dinitrosalicylic acid to produce a brownish-red compound with a characteristic absorption peak at 540 nm, and the activity of chitinase can be characterised by the change in absorbance value.

Figure 2  Chitinase activity

Sequence and Features