Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas-phase production of propioin from two propanal molecules by using thiamine diphosphate-dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g(-1) for BAL and BFD, respectively. The half-life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half-life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.
"It is a versatile catalyst for the enantioselective synthesis of a broad variety of α-hydroxy ketones by both C–C bond coupling and kinetic resolution. These chiral compounds are important building blocks for the synthesis of several drugs and natural products       . "
[Show abstract][Hide abstract] ABSTRACT: Benzaldehyde lyase from
Biovar I. (BAL, EC 220.127.116.11) is a versatile catalyst for the organic synthesis of chiral
-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
-hydroxy ketone (R)-2,2′-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.
"Although Schiff-base formation as such is a reversible reaction, several follow-up reactions finally render this modification irreversible . Hence, acetaldehyde affects various enzymatic properties as shown for different enzymes of biotechnological significance such as lipases, 2-deoxy-D-ribose 5-phosphate aldolase (DERA), and thiamine-diphosphate dependent enzymes, respectively [14-17]. Acetaldehyde treatment of commercial lipase preparations from Candida rugosa and Galactomyces geotrichum (formerly Geotrichum candidum) resulted in reduced activity and enantioselectivity, whereas only moderate effects were observed with lipases from Pseudomonas sp. or Rhizopus oryzae [18,19]. "
[Show abstract][Hide abstract] ABSTRACT: Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing.
Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,β-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules.
Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,β-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of the enzymes from buffer at pH 6.0 can provide an easy and effective way to stabilize lipases toward inactivation by acetaldehyde.
[Show abstract][Hide abstract] ABSTRACT: Thiamine diphosphate (ThDP)-dependent enzymes like benzaldehyde lyase from Pseudomonas fluorescens (BAL) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are versatile biocatalysts for the CC bond ligation of aldehydes to form enantiomerically pure 2-hydroxy ketones. However, the large-scale application of this enzyme class is often restricted by the required external addition of the expensive cofactor ThDP, as well as by the common use of dimethyl sulfoxide (DMSO) as a cosolvent, which leads to problems during the work-up procedure. In the present paper we demonstrate that the addition of the excess cofactors, ThDP and magnesium ions (Mg2+), is not required when BAL or BFD are used in Escherichia coli resting cells. Furthermore, the combination of these resting cells with a biphasic reaction medium [methyl tert-butyl ether (MTBE)/aqueous buffer] allows an increase of the substrate concentration up to 1 M, and an efficient extractive work-up. As a practical example, e.g., the synthesis of (R)-2-hydroxy-3,3-dimethoxy-phenylpropanone from benzaldehyde and 2,2-dimethoxyacetaldehyde was optimized, achieving an isolated yield of 78 %, and an enantiomeric excess of 98 % ee in 24 h when operating at a substrate concentration of 0.4 M. The described reaction system in a biphasic medium is suitable for a wide range of aldehydes as substrates. The biphasic reaction medium minimizes also the formation of by-products, which were observed when this reaction was performed in the conventional DMSO/buffer system.
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