Study on the expression, purification and properties of cystathionine β-lyase
Homocysteine (Hcy), also known as homocysteine, is an amino acid containing sulfhydryl groups and is an important intermediate product in the metabolism of methionine and cysteine. Studies have shown that Hcy is responsible for the heart and brain Risk factors for vascular diseases, diabetes and neurological diseases. Therefore, the establishment of a method for rapid and accurate detection of Hcy content is of great significance to basic research and clinical diagnosis.
The principle of the determination of Hcy by cystathionine β-lyase (CBL)
This laboratory intends to establish a method for the determination of Hcy by enzyme catalysis. The method is fast, convenient, sensitive and accurate. The reaction principle is as follows: Hcy and L-serine are catalyzed by cystathionine synthase (CBS) to generate L -Cysthionine, L-cystathionine is catalyzed by cystathionine β-lyase (CBL) to produce pyruvate, which reacts with 2,4-dinitrophenylhydrazine under alkaline conditions to produce a brownish-red product. The pyruvate content can be obtained by measuring the absorbance value with a spectrophotometer at 520nm, thereby calculating the concentration of Hcy in the sample. Let's take a look at the enzymatic properties of CBL.
Separation and purification of cystathionine β-lyase (CBL)
The supernatant after sonication of the bacterial cells was centrifuged and loaded onto the pre-equilibrated HisTrapFastFlow column. The target protein was directly adsorbed on the column and eluted with the elution buffer solution. The CBL was purified in one step and the enzyme activity recovery rate was 71%. The purity of the purified CBL reached 949/6, and the unit enzyme activity was 134.6U.mg. The study found that the purified CBL protein solution almost all aggregated and precipitated after freezing and thawing. This may be caused by the ice crystals produced during the freezing and thawing of the protein solution. Caused by the tertiary structure of the protein.
The stability of CBL enzyme activity at different temperatures
CBL is relatively stable at 2O～35℃. As the temperature increases, the stability gradually decreases. When the temperature exceeds 40℃, the enzyme inactivation speed increases. When the temperature rises to 50℃, the relative enzyme activity drops to 32.2%. Therefore, It is most effective to use CBL enzyme to catalyze the reaction at 35℃, and it is not suitable to keep the enzyme solution in an environment above 30℃ for a long time. The CBL obtained by fermentation is more stable below 40℃. The CBL purified from Lacto-COCCUSlactis is at 60℃. It is relatively stable below ℃, the enzyme activity drops sharply above 60℃, and is almost completely inactivated at 70℃.
Optimal pH and pH stability of CBL
The recombinase has higher activity under alkaline conditions (pH value 8.0～1O.O), the highest reactivity under pH value 8.0, and lower activity in acidic solution (pH value < 7.0), at pH value 5.0 The activity is almost zero under the conditions of pH 5.0-5.5, only 10-15% of the activity is between pH 5.0 and 5.5, the reaction activity is the highest between pH 7.5 and 8.5, and the activity drops sharply when the pH value is> 8.5.
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