Revision as of 12:44, 19 October 2021

Endochitinase enzyme secretion system

This composite part consists of TPS1 promoter, Mating factor alpha prepro-leader (MF alpha), 6 x glycine, chit42 coding sequence, and ADH1 terminator. The product of this system is a secretable endochitinase together with a HA tag. MF alpha enables the enzyme to be secreted. The 6 glycines between MF alpha and chit42 coding sequence avoid the interruption between them, enabling them to form correct spatial structure.

DPPH Assay

The DPPH radical scavenging assay is performed by using DPPH assay, (Chen et al. 2014). Prior to the real test, the chit42 yeast and the wild-type strain are incubated in SC medium overnight until the OD reaches 0.5-0.7. Then, 8% ethanol was added to induce the production, and the yeast was incubated under the same circumstances:25 degrees and rotating speed at 220 rpm. Before the examination, yeast was centrifuged, the supernatant was filtered to do the DPPH test. Briefly, 50ul samples with the addition of 200 ul 51.54mg/ml DPPH solution in 95% ethanol are labeled as As. Besides, Ao is by mixing 50 ul ethanol with 200ul DPPH solution of the same concentration. Additionally, Ar is made by adding 50 ul samples to 200 ul 95% ethanol. During the test, the three groups are triple-examined by using 96 microwell plates. Each group is incubated at 25 degrees in darkness for 30 minutes. After that, all three groups in the same well plate examined the light absorbance are performed by light spectrophotometry at 517 nm. And the DPPH radical scavenging rate of each sample can be deduced by the equation below:

DPPH radical scavenging rate= (1-(As-Ar)/A0)*100%

Figure 1. DPPH function assay of the chit42 strain with ethanol concentration: 8%

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The FY4 strain modified by chit42 containing vectors shows enhanced ability to remove free radicals in solution, as a comparison with the wild-type strain. The result demonstrates that the TPS1 pomoter_chit42_alpha factor part works, the 5% difference of DPPH scavenging rate indicates chit42 strain produces higher levels of chitooligosaccharide than wild type.

Figure 2. DPPH function assay of the wild-type and chit42 strain induced by 4% ethanol

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We also examine the DPPH quenching activity under 4 % ethanol as western blot detects protein deposits when chit42 is induced by 4% ethanol. After induce wild type and chit42 strain with 4% ethanol, the result from figure 24 indicates a higher potency to eliminate radicals compared with chit42 under induction of 8% ethanol, given that DPPH scavenging rate rise by 10% under 4% induction and the rate rises by 5% under 8% ethanol induction. Combining two experiments above, there is a high possibility that the best optimal concentration of ethanol needs to be identified, even the operational concentration of 8% of ethanol according to literature still functions quite well.

Sequence and Features