Highly Enantioselective Hydrolysis of (R,S)-Phenylalanine Isopropyl Ester by Subtilisin Carlsberg. Continuous Synthesis of (S) -Phenylalanine in a Hollow Fiber/Liquid Membrane Reactor

• Published in: Biotechnology progress Vol. 8; no. 3; pp. 197 - 203
• Main Authors: Ricks, Edith E., Estrada-Valdés, Maria C., Mclean, Thomas L., Iacobucci, Guillermo A.
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 01.05.1992; American Institute of Chemical Engineers
• Subjects: (R,S)-phenylalanine isopropyl ester; Bioconversions. Hemisynthesis; Biological and medical sciences; bioreactors; Biotechnology; Fundamental and applied biological sciences. Psychology; hydrolysis; immobilized enzymes; Methods. Procedures. Technologies; phenylalanine; subtilisin; synthesis; Continuous process; Hydrolysis; Enzymatic synthesis; Membrane reactor; Bioreactor; Enantioselectivity; Phenylalanine; Enzyme; Hollow fiber; Subtilisin
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp00015a004

The resolution of racemic phenylalanine esters with esterases has been reexamined in relation to the development of a continuous process based on the use of hollow fiber/liquid membrane (SLM) reactors. The requirement for high enantioselectivity was met by phenylalanine isopropyl ester, whose (R)‐enantiomer was found refractory to the action of subtilisin Carlsberg, in water, pH 7.5, at 25 °C. The continuous feeding of (R, S)‐Phe‐O‐iPr‐HCl at 2 mm/h to 0.7 g/L subtilisin resulted in steady‐state reaction conditions that gave (S)‐Phe‐OH of 95% enantiomeric excess (ee) at the theoretical rate. The unreacted (R)‐Phe‐O‐iPr, which permeated out continuously, had 80% ee and was racemized quantitatively by heating with a salicylaldehyde catalyst in refluxing toluene. Doubling the feeding rate (4 mm/h) created excessive accumulation of (R)‐Phe‐O‐iPr in the reactor, which inhibited the enzyme through nonproductive binding, lowering the (S)‐Phe‐OH resolution rate to 68% of the theoretical rate. Similar experiments with (R, S)‐Phe‐OMe‐HCl gave (S)‐Phe‐OH of 9–48% ee, due to the enzymatic hydrolysis of (R)‐Phe‐OMe experienced during the runs. When α‐chymotrypsin was used for the continuous hydrolysis of (R, S)‐Phe‐O‐iPr, the isolated (S)‐Phe‐OH showed 67% ee, indicating that substantial hydrolysis of the (‐R)‐ester had occurred.