Team：The Fiters/Basic Parts
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BBa_K3584000 is the coding sequence of a polyphenol oxidase (PPO) from Pleurotus ostreatus, which is different from the present enzymes showing the similar activity. PPO is a group of enzyme that are able to oxidize phenol or polyphenol by molecular oxygen to form the corresponding quinone. In a broad sense, polyphenol oxidase can be divided into three categories: monophenol monooxidase (tyrosinase, EC.188.8.131.52), bisphenol oxidase (catechol oxidase, EC. 184.108.40.206) and laccase (laccase, EC.220.127.116.11). Among these three types of polyphenol oxidase, catechol oxidase is mainly distributed in plants, and the polyphenol oxidase in microorganisms mainly includes laccase and tyrosinase. The polyphenol oxidase mentioned in most of the literature is generally the collective name of catechol oxidase and laccase.
Production, purification, and SDS-PAGE analysis of recombinant PPO
In order to present the function of the part, the PPO gene was expressed in E. coli BL21 (DE3) under the control of T7 promoter. Then the bacterial cells are collected and crushed, and the PPO enzyme solution is purified by further confirmation by the SDS-PAGE method, which is found in the corresponding protein band of approximately 57 kDa (Figure 1).
Figure 1. Production, purification, and SDS-PAGE analysis of recombinant PPO. Lane: protein molecular weight standard; lane 8: purified PPO.
Enzymatic activity of the PPO
PPO activity was determined using an analogue of p-cresol (catechol) as a substrate. In proper reaction mixture, PPO and 10 mM catechol was incubated at 50 ℃, pH 7.0 for 3 min, and the change in absorbance at 420 nm was measured spectrophotometrically. One unit of enzyme (PPO) activity was defined as the amount of enzyme that increased absorbance of 0.001 per minute. We found that as the extension of reaction time, reaction liquid of absorbance at 420 nm increased linear (Figure 2), we obtained by catalytic activity of PPO enzyme fluid, and divided by the corresponding protein concentration, the specific activity of PPO (Table 1).
Figure 2. PPO activity was determined using catechol as a substrate. The reaction mixture contained PPO protein and 10 mM catechol which was incubated at 50 ℃, pH 7.0 for 3 min, and the change in absorbance at 420 nm was recorded.
Table 1. Specific activity of recombinant PPO
GFP reporting system to sense the aromatic amino acids
The basic part BBa_K3584005 a part of the composite part BBa_K3584006, which was constructed on the pSU2718-p15A vector. The constructed plasmids were transformed into Escherichia coli Nissle 1917(EcN) receptive state of intestinal probiotics. The recombinant strains were cultured with addition of different concentrations of tyrosine. As shown in Figure 1, the fluorescence intensity of E. coli was obviously seen under UV light.
Figure 1. Fluorescence intensity of E. coli containing part BBa_K3584006 under UV light.
The fluorescence intensity increased with the increase of tyrosine concentration, indicating that the induction system responded well to the tyrosine concentration in E. coli Nissle 1917. When we increased the concentration of tyrosine to 500 μM, the fluorescence intensity was further enhanced, but when the concentration reached 1000 μM, the fluorescence intensity of the strain decreased, so the high concentration of amino acids may have an inhibitory effect on the strain (Figure 2).
Figure 2. Fluorescence intensity of E. coli Nissle 1917 with different tyrosine concentration.
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