Difference between revisions of "Part:BBa K1201000"

Latest revision as of 11:38, 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

Added by Thinker-Shenzhen

We used the titin from Malassezia and Candida albicans for our experiments. At the same time, we prepared colloidal titin due to the poor water-melting properties of common titin.

For the measurement of binding capacity, we took 2 μmol/L of chitinase and 1 mg/mL of colloidal chitin and other fungal chitin 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.

In order to observe the antifungal ability of unmodified titinase, we performed antifungal experiments using the pathogenic fungus Malassezia as the test strain, which was activated by transferring it on PDA medium and incubated at 25°C for 48h.

Binding rates of unmodified titinase to different titins:

Figure 1

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

To observe the antifungal ability of unmodified titinase, we performed antifungal experiments using the pathogenic fungus Malassezia as the test strain, which was transferred to PDA medium for activation and incubated at 25°C for 48 h. After incubation, the discs were punched with a hole punch and inoculated into the central position of a new Petri dish with PDA medium and incubated at 25°C for 24 h. A hole was punched with a hole punch 20 mm from the central perimeter of the disc Add tributylase. The inhibition was observed after 48 h of incubation.

Inhibition of live fungi by chitinase.

Figure 3

Sequence and Features