%0 Journal Article
%T Direct Spectrophotometric Assay for Benzaldehyde Lyase Activity
%A Dessy Natalia
%A Christina Kohlmann
%A Marion B. Ansorge-Schumacher
%A Lasse Greiner
%J Biotechnology Research International
%D 2011
%I Hindawi Publishing Corporation
%R 10.4061/2011/478925
%X Benzaldehyde lyase from Pseudomonas fluorescens Biovar I. (BAL, EC 4.1.2.38) is a versatile catalyst for the organic synthesis of chiral ¦Á-hydroxy ketones. To allow fast assessment of enzyme activity, a direct spectrophotometric assay is desirable. Here, a new robust and easy-to-handle assay based on UV absorption is presented. The assay developed is based on the ligation of the ¦Á-hydroxy ketone (R)-2,2¡ä-furoin from 2-furaldehyde. A robust assay with direct monitoring of the product is facilitated with a convenient concentration working range minimising experimental associated with low concentrations. 1. Introduction Benzaldehyde lyase (BAL; EC 4.1.2.38) from Pseudomonas fluorescens Biovar I (BAL, EC 4.1.2.38) belongs to the group of thiamine diphosphate- (ThDP-) dependent enzymes [1]. It is a versatile catalyst for the enantioselective synthesis of a broad variety of ¦Á-hydroxy ketones by both C¨CC bond coupling and kinetic resolution. These chiral compounds are important building blocks for the synthesis of several drugs and natural products [2¨C8]. According to its synthetic importance and application prospects, several methods for the determination of BAL activity have already been established. Most prominent are the uses of high pressure liquid chromatography (HPLC) or a combination of gas chromatography and mass spectroscopy (GC-MS) [3, 9]. However, these methods are material- and time-consuming when it comes to sample preparation and performance of measurements, and are therefore hardly applicable to the monitoring of fast reaction courses or large sample batches. For these purposes, establishment of spectrometric methods is highly desirable. In fact, the BAL-catalysed carboligation of benzaldehyde into benzoin has been determined via spectrophotometry at a wavelength of 250£¿nm. However, both benzaldehyde and benzoin revealed a considerable absorption at this wavelength resulting in an overlap for which a correction had to be carried out [1]. The same applies for the spectrophotometric determination of the condensation of furaldehyde into furoin at a wavelength of 277£¿nm. Fluorescence spectroscopy at an excitation wavelength of 360£¿nm and an emission wavelength of 470£¿nm was used for the investigation of the carboligation of 3,5-dimethylbenzaldehyde (DMBA) into 3,3¡ä,5,5¡ä-tetramethoxybenzoin (TMB) [10]. However, the linear range of this assay strongly depends on the pathway of the excitation light [11]. Furthermore, the method is restricted to this substrate-product pair suffering from the same limited solubility as benzoin (see below). Generally,
%U http://www.hindawi.com/journals/btri/2011/478925/