TY - JOUR

T1 - Synthesis, test and application of chirale fluorescence substrates to evaluate enzymatic processes in different reaction media

AU - Knüttel, Torsten

AU - Meyer, Hartmut

AU - Scheper, Thomas

N1 - Funding information: The authors thank the Deutsche Forschungsgemeinschaft (DFG, as part of the Graduiertenkolleg) for financial support.

PY - 2005/6/14

Y1 - 2005/6/14

N2 - A pseudo-enantiomeric reaction was developed which was monitored on-line via 2D-fluorescence spectroscopy. During enzymatic reactions the enantiomeric excess can be followed on-line. Fluorescence spectroscopic detectable substrates, l-/d-phenylalanine-7-amido-4-methylcoumarine (l-/d-PheAMC) and l-/d-phenylalanine-7-amido-4-trifluoro-methylcoumarine (l-/d-PheAFC) were synthesized and deployed for applications in aqueous systems. Several proteases and esterases were tested to detect suitable biocatalysts for the enzymatic hydrolysis of the coumarine substrates. With α-chymotrypsin and the esterase from porcine liver, every coumarine substrate was hydrolyzed in an aqueous system and the determined enzymatic parameters were compared. The enantioselectivity and the enantiomeric excess of the enzymatic reactions were investigated. In simultaneous applications of the l- and d-substrates, the reactions were monitored on-line and the enantioselectivities, enantiomeric excesses and kinetic parameters were examined. To display the versatility of the on-line method, the enzymatic reactions were transferred to the organic solvent toluene. After the optimisation of the biocatalysis in toluene, the kinetic parameters were measured and the ee-parameters for the substrates and products were examined in off-line and on-line investigations.

AB - A pseudo-enantiomeric reaction was developed which was monitored on-line via 2D-fluorescence spectroscopy. During enzymatic reactions the enantiomeric excess can be followed on-line. Fluorescence spectroscopic detectable substrates, l-/d-phenylalanine-7-amido-4-methylcoumarine (l-/d-PheAMC) and l-/d-phenylalanine-7-amido-4-trifluoro-methylcoumarine (l-/d-PheAFC) were synthesized and deployed for applications in aqueous systems. Several proteases and esterases were tested to detect suitable biocatalysts for the enzymatic hydrolysis of the coumarine substrates. With α-chymotrypsin and the esterase from porcine liver, every coumarine substrate was hydrolyzed in an aqueous system and the determined enzymatic parameters were compared. The enantioselectivity and the enantiomeric excess of the enzymatic reactions were investigated. In simultaneous applications of the l- and d-substrates, the reactions were monitored on-line and the enantioselectivities, enantiomeric excesses and kinetic parameters were examined. To display the versatility of the on-line method, the enzymatic reactions were transferred to the organic solvent toluene. After the optimisation of the biocatalysis in toluene, the kinetic parameters were measured and the ee-parameters for the substrates and products were examined in off-line and on-line investigations.

KW - α-Chymotrypsin

KW - 2D-fluorescence spectroscopy

KW - Coumarines

KW - Enantioselective hydrolysis

KW - Esterase

KW - On-line monitoring

UR - http://www.scopus.com/inward/record.url?scp=25444528238&partnerID=8YFLogxK

U2 - 10.1016/j.enzmictec.2004.04.026

DO - 10.1016/j.enzmictec.2004.04.026

M3 - Article

AN - SCOPUS:25444528238

VL - 37

SP - 673

EP - 686

JO - Enzyme and microbial technology

JF - Enzyme and microbial technology

SN - 0141-0229

IS - 7

ER -