Enzymatic properties of sarcosine oxidase SOX
Sarcosine oxidase (SOX) enzymatic number EC18.104.22.168, it is the third enzyme in the degradation of muscle metabolite creatinine, creatinine is finally degraded into glycine, formaldehyde and hydrogen peroxide. In the field of IVD, SOX is used as an important diagnostic enzyme preparation.
The application of sarcosine oxidase in the field of in vitro diagnostics is based on its catalytic principle, which can be coupled with creatinase (creatinine iminohydrolase) and creatinase (creatine amidinohydrolase CRH) to degrade creatinine into glycine and formaldehyde And hydrogen peroxide. The level of creatinine is an important reference index for judging kidney function, so it also has a good application prospect in the field of medicine.
Biochemical detection of raw material sarcosine oxidase
The enzymatic activity of sarcosine oxidase:
Using sarcosine as the substrate, take 50μL of SOX enzyme solution of different concentrations, and add 3mL of coloring solution to 1mmo1/L 4-aminoantipyrine, 6mmo1/L phenol, and 7000U/L horseradish. Oxidase, 20mmo1/L Tris-HC1 buffer (pH8.0), add 150μL sarcosine (0.2mo1/L) after mixing, shake and mix well, react in 37℃ water bath for 5min, then boil water after completion The reaction was terminated by boiling in the bath for 3min, cooling, and measuring the absorbance at 500nm. The unit of enzyme activity is defined as: at 37℃, the amount of enzyme required to convert 1μmoL of sarcosine to produce formaldehyde per minute is defined as 1 unit of enzyme activity (U).
Purification of sarcosine oxidase:
Pick a single colony from the solid plate, inoculate the seed medium, and cultivate overnight at 37°C. Inoculated into the fermentation medium according to the volume fraction of 5% inoculum, cultured to 0D6000.6-0.8 at 37℃, 200r/min, and induced with 1mmo1/L IPTG for 8h. After the bacterial solution was broken and centrifuged, the enzyme supernatant was taken for purification, and the purified enzyme solution was stored at 4°C for later use.
The optimal reaction temperature of sarcosine oxidase:
In the pH8.0, 20mmo1/LTris-HC1 buffer system, the enzyme activity at different temperatures is measured in the range of 20-80℃, and the influence of temperature on the enzyme activity is observed to determine the optimal reaction temperature of the enzyme;
The optimum pH value of sarcosine oxidase:
Measure the enzyme activity of s0x in different pH reaction systems (pH4.0-11.0). The highest enzyme activity is recorded as 100%, and the relative enzyme activity under other pH conditions is calculated to determine the optimal pH for the enzymatic reaction. The buffer system of different pH is citric acid-sodium citrate buffer (pH4.0-5.0), phosphoric acid-potassium phosphate buffer (pH6.0-7.0), Tris-HC1 buffer (pH8.0-9.0), Sodium bicarbonate-NaOH buffer (pH 10.0-11.0).
Sarcosine oxidase can be used with chromogen substrate TOPS, peroxidase and other reagents to determine serum creatinine level. Desheng provides enzyme preparations including SOX, CRH, chromogen substrate, buffer, etc. A variety of kit raw materials included.
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