Introduction: Biological reactions are catalyzed by enzymes. Many enzymes show a highly selective be — Physical Chemistry — Thermodynamics Chemistry Question
Enzymatic hydrolysis of N-acetyl alanine as an example of an environmentally benign process
Introduction:
Biological reactions are catalyzed by enzymes. Many enzymes show a highly selective behaviour and often are able to selectively catalyze reactions with one enantiomer of a racemate. In modern chemistry enzymes are used for many processes in vitro, especially for the synthesis of enantiomerically pure products. In this experiment the hydrolysis of N-acyl-alanine with the enzyme acylase I is investigated.
The process of the reaction can be monitored by following the formation of alanine using the reaction with ninhydrin, as shown below.
[VISUAL]
List of chemicals needed:
- rac. N-acetyl-alanine, 265 mg
- acylase I, 10 mg
- lithium hydroxide, 84 mg
- ninhydrine solution (Sigma N 1632), 2 cm3
- dimethyl sulfoxide, ca. 70 cm3
- lithium acetate buffer, pH 5.2.
Procedure:
Racemic N-acetyl-alanine (262 mg, 2.0 mmol) is dissolved in water (10 cm3). A solution of lithium hydroxide (84 mg, 2.0 mmol) in water (4 cm3) is then gradually added with gentle stirring. The pH is monitored with pH-paper until pH = 7 is reached. A solution of acylase I (10 mg) in water is added with vigorous stirring for 2 minutes. (The above enzyme solution is prepared by adding the enzyme to 5 cm3 of water and filtration using a small glass filter covered with diatomaceous earth.) Subsequently, water is added to reach the total volume of exactly 20.0 cm3. The reaction mixture is kept at a temperature of 37 °C in a water bath for 60 minutes. Then an accurate volume of 0.25 cm3 (use a syringe or a fine measuring pipette) is transferred into a test tube and ninhydrin (Sigma N 1632), (1.25 cm3) is added. This mixture is heated in boiling water during 20 minutes whereby a deep purple color is developed. After cooling, this mixture is accurately added to buffer solution consisting of 4 M lithium acetate aqueous buffer (pH = 5.2) and dimethyl sulfoxide in the ratio 1 : 3 in a volumetric flask of 250 cm3. Adjust the volume to 250 cm3. Then measure the absorbance of the solution at λ = 592 nm using a spectrophotometer. Use as a reference ninhydrin in the same lithium acetate buffer in dimethyl sulfoxide. ε (purple complex) = 13350 dm3 mol-1 cm-1.
Record the following data:
1. The initial concentration of racemic N.
2. The absorbance at λ = 592 nm.
3. Calculate the amount of substance (mmol) of alanine formed in the enzymatic reaction. Use the Lambert-Beer’s law.
4. Calculate the percentage of conversion.
Special note:
If time permits you may decide to stop the reaction after 10, 25, 40 and 60 minutes and determine the concentration of alanine in each case. Then construct a graph of the concentration of alanine vs. time and estimate the optimal reaction time.
Will the alanine formed be optically active? (Yes or no.)
Model Answer
Yes.
Will the N-acetyl-alanine that remains, be optically inactive, optically enriched or optically pure when the conversion is smaller than 50 %?
Model Answer
Optically enriched.
Idem when the conversion is 50 % exactly?
Model Answer
Optically pure.
Is the conversion larger than 50 % possible? (Yes or no.)
Model Answer
When the enzyme is highly selective: no.
When the enzyme is not highly selective: yes. In this case the preferred enantiomer will be hydrolyzed very fast and the other enantiomer will be converted more slowly.