That amount of enzyme which catalyzes the hydrolysis of one micromole of N-Acetyl-L-Methionine per hour at 25° C, pH 7.0.
Assay Method
The assay is based on the reaction described by Mitz and Schulueter, Biochim. The enzyme catalysis is followed by measuring absorbance at 238 nm.
Applications
Acylase 1 (EC 3.5.1.14) is an extremely stable enzyme in the dry state. In solution, the enzyme is stable at high temperatures (70° C) at pH 7, but below pH 5 it is rapidly and irreversibly inactivated. The enzyme shows a high degree of optical specificity toward its substrates. For this reason, it has been used extensively for the resolution of racemic amino acids.
Reagents
0.1 M Potassium phosphate buffer, pH 7.0.
0.015 M N-Acetyl-L-Methionine (2.87 mg/ml). Dissolve in 0.01 M potassium phosphate buffer. Adjust pH to 7.0 with 2 M NaOH if necessary.
Acylase 1 (enzyme) solution. Dilute in 0.01 M potassium phosphate buffer, pH 7.0 to yield a concentration of 1000-2000 U/ml. Prepare fresh prior to assay.
Procedure
Set the spectrophotometer (equipped with strip chart recorder and temperature control) at 238 nm and 25° C.
In a cuvette place 2.9 ml of 0.015 M N-Acetyl-L-Methionine (substrate). Incubate cuvette in spectrophotometer at 25°C for 5 minutes.
Record absorbance at 238 nm (blank).
Initiate the reaction by adding 0.1 ml enzyme solution to the cuvette. Follow the reaction by measuring the absorbance at 238 nm for 5-8 minutes.
