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Background: Global resource depletion poses a dramatic threat to our society and creates a strong demand for alternative resources that do not compete with the production of food. Meeting this challenge requires a thorough rethinking of all steps of the value chain regarding their sustainability resource demand and the possibility to substitute current, petrol-based supply-chains with renewable resources. This regards also the production of catalysts for chemical synthesis. Phototrophic microorganisms have attracted considerable attention as a biomanufacturing platform for the sustainable production of chemicals and biofuels. They allow the direct utilization of carbon dioxide and do not compete with food production. Photosynthetic enzyme production of catalysts would be a sustainable supply of these important components of the biotechnological and chemical industries. This paper focuses on the usefulness of recombinant cyanobacteria for the photosynthetic expression of enantioselective catalysts. As a proof of concept, we used the cyanobacterium Synechocystis sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes., Results: We investigated the expression yield and the usefulness of cyanobacterial cell extracts for conducting stereoselective reactions. The cyanobacterial enzyme expression achieved protein yields of 3% of total soluble protein (%TSP) while the expression in E. coli yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism Sulfolobus tokodai ST0071 and arylmalonate decarboxylase from Bordetella bronchiseptica showed excellent enantioselectivity (>99% ee) and yield (>91%) in the desymmetrisation of prochiral malonates., Conclusions: We were able to present the proof-of-concept of photoautotrophic enzyme expression as a viable alternative to heterotrophic expression hosts. Our results show that the introduction of foreign genes is straightforward. Cell components from Synechocystis did not interfere with the stereoselective transformations, underlining the usability of photoautotrophic organisms for the production of enzymes. Given the considerable commercial value of recombinant biocatalysts, cyanobacterial enzyme expression has thus the potential to complement existing approaches to use phototrophic organisms for the production of chemicals and biofuels.

Arylmalonate decarboxylase (AMDase) catalyzes the cofactor‐free asymmetric decarboxylation of prochiral arylmalonic acids and produces the corresponding monoacids with rigorous R selectivity. Alteration of catalytic cysteine residues and of the hydrophobic environment in the active site by protein engineering has previously resulted in the generation of variants with opposite enantioselectivity and improved catalytic performance. The substrate spectrum of AMDase allows it to catalyze the asymmetric decarboxylation of small methylvinylmalonic acid derivatives, implying the possibility to produce short‐chain 2‐methylalkanoic acids with high optical purity after reduction of the nonactivated C=C double bond. Use of diimide as the reductant proved to be a simple strategy to avoid racemization of the stereocenter during reduction. The developed chemoenzymatic sequential cascade with use of R‐ and S‐selective AMDase variants produced optically pure short‐chain 2‐methylalkanoic acids in moderate to full conversion and gave both enantiomers in excellent enantiopurity (up to 83 % isolated yield and 98 % ee). A chemoenzymatic cascade: By combining enzymatic decarboxylation and subsequent diimide‐mediated C=C double bond reduction optically pure short‐chain 2‐methylalkanoic acids are formed (see Scheme). This route exploits the outstanding enantioselectivity and capacity of bacterial arylmalonate mutants to produce both enantiomers in pure form. [ABSTRACT FROM AUTHOR]

Bacterial arylmalonate decarboxylase (AMDase) shows high enantioselectivity and a broad substrate spectrum in the asymmetric synthesis of optically pure arylaliphatic carboxylic acids. The determination of the structure of AMDase has greatly extended the understanding of the catalytic mechanism of this unique cofactor-free decarboxylase and allowed the generation of tailor-made enzyme variants with improved catalytic properties. Despite this increase in knowledge and applicability, the natural role of the enzyme remains unknown. This mini-review summarizes the recent findings on the molecular mechanism and the synthetic application of the enzyme. [ABSTRACT FROM AUTHOR]

The profen family belongs to the most important non-steroidal anti-inflammatory drugs (NSAIDs). A considerable number of biocatalytic processes for the synthesis of optically pure (S)-profens have been proposed. Despite of the excellent enantioselectivity and the large advantages that enzyme catalysis offers in terms of sustainability, biocatalytic processes have failed so far to meet the technical and economic challenges of commercialization. This critical review outlines recent trends and developments of novel applications that appear very promising in terms of enantioselectivity, efficiency, sustainability and yield. Special emphasis is placed on the contribution of protein engineering in overcoming the limitations of these enzymes for technical applications, and thus providing promising biocatalysts for the preparation of pharmaceutical products. The natural catalytic diversity, assisted by modern methods of protein engineering, provides novel concepts and leads for the environmentally friendly synthesis of pharmacologically important drugs. Considerable progress can be expected in the coming decades. Furthermore, aspects regarding ecological footprints and the impact of each biocatalytic route are critically addressed, considering aspects like the type of solvent, waste produced, availability of substrate, etc. When possible, suggestions for combining efficiency with more sustainable synthetic approaches are also given. [ABSTRACT FROM AUTHOR]

Arylmalonate decarboxylase (EC 4.1.1.76, from Alcaligenes bronchisepticus KU 1201) is an enzyme that catalyzes asymmetric decarboxylation of arylmalonate. The enzyme accepts as substrates compounds that have a sterically small hydrophobic α-substituent, such as H, F, and methyl. To widen the applicability of this enzyme and to obtain information on the interaction between the enzyme and substrates, we tried the reaction of compounds with a hydrophilic substituent. Although the relative reactivities were lower compared to that of phenylmalonate, α-hydroxy- and α-aminophenylmalonate were decarboxylated to give optically active monobasic acids. [ABSTRACT FROM AUTHOR]

We have already isolated, purified, and characterized arylmalonate decarboxylases (AMDase; EC. 4.1.1.76) from Alcaligenes bronchisepticus KU1201 and Achromobacter sp. KU1311. These are unique enzymes that give optically pure α-arylpropionates from the corresponding α-aryl-α-methylmalonates. Recently, we have further screened novel AMDase producers from soil samples under acidic conditions and succeeded in isolating Enterobacter cloacae KU1313. The gene encoding the enzyme was cloned by polymerase chain reaction and sequenced. The AMDase gene consists of 720 nucleotides, which specifies a 240-amino-acid protein. The recombinant enzyme was purified and shown that the pH-activity profiles were quite different from those of known AMDases. [ABSTRACT FROM AUTHOR]

We have isolated, purified and characterized arylmalonate decarboxylase (AMDase; EC 4.1.1.76). This is an unique enzyme that gives optically pure arylpropionates from the corresponding arylmalonates. Recently, we have screened similar enzyme producers from soil samples and succeeded in isolating Achromobacter sp. KU1311. The gene encoding the enzyme was cloned and sequenced. The AMDase gene consists of 720 nucleotides, which specifies a 240 amino acid protein with a relative molecular mass of 24,735. This enzyme was purified and its characteristics were compared with those of the hitherto known enzyme from Alcaligenes bronchisepticus KU1201. [Copyright &y& Elsevier]

Phenylacetaldehyde dehydrogenase (PADH) was purified and characterized from Brevibacterium sp. KU1309, which can grow on the medium containing 2-phenylethanol as the sole carbon source. This enzyme was a homotetrameric protein with a subunit of 61 kDa. The enzyme catalyzed the oxidation of aryl (benzaldehyde, phenylacetaldehyde, 3-phenylpropionaldehyde) and aliphatic (hexanal, octanal, decanal) aldehydes to the corresponding carboxylic acids using NAD+ as the electron acceptor. The PADH activity was enhanced by several divalent cationic ions such as Mg2+, Ca2+, and Mn2+. On the other hand, it was inhibited by SH reagents (Hg2+, p-chloromercuribenzoate, iodoacetamide, and N-ethylmaleinimide). The substrate specificity of the enzyme is compared with those of various aldehyde dehydrogenases. [ABSTRACT FROM AUTHOR]

We have developed a method for the preparation of l-4-chlorophenylalanine from its racemate with Escherichia coli cells expressing a single foreign gene. l-4-Chlorophenylalanine was obtained in a high optical yield by the inversion of configuration of its d-form via the tandem reactions catalyzed by d-amino acid dehydrogenase (DadA) and branched-chain amino acid aminotransferase (BCAAT). While we constructed a plasmid for BCAAT utilizing the gene from Sinorhizobium meliloti ATCC 51124, the first enzyme DadA was the dadA -gene product from E. coli host cell itself, which was activated by the addition of l-alanine in the growth medium. [ABSTRACT FROM AUTHOR]

A novel 2-phenylethanol dehydrogenase has been purified from a soil bacterium Brevibacterium sp. KU 1309. The enzyme was purified about 1400-fold to homogeneity, and found to be a monomeric enzyme of apparent 39 kDa. The enzyme had broad substrate specificity and catalyzes a reversible oxidation of various primary alcohols to aldehydes. The enzyme required NAD+, but not NADP+ as a cofactor. Thus, the enzyme was classified into a group of NAD+-dependent primary alcohol dehydrogenase. The activity was inhibited by Cu2+, Ni2+, Ba2+, Hg2+ and p-chloromercuribenzoate. The enzyme is expected to be applicable as an effective biocatalyst in the oxidation of various alcohols. [Copyright &y& Elsevier]

Abstract: We screened new microorganisms having deracemization activity of α-amino acids and isolated some active strains. Whole cells of these strains were capable of inverting the chirality of 4-chlorophenylalanine from d- to l-configuration. In particular, Nocardia diaphanozonaria JCM 3208 exhibits the deracemization activity towards wide variety of α-amino acids, such as phenylglycine and 2-aminoheptanoic acid. Examination of the time course of the reaction revealed that α-keto acid was produced as the key intermediate. In addition, mechanistic studies using cell-free extract suggested that deracemization process is realized by two enzymatic reactions; d-stereoselective oxidative deamination reaction and l-selective transamination reaction. Finally, we could establish the reaction conditions utilizing the cell-free system to proceed the deracemization of phenylalanine, which was degraded when the whole cells were used as the biocatalyst. [Copyright &y& Elsevier]

A novel decarboxylase which catalyzes an enantioselective decarboxylation of α-aryl-αmethylmalonates to α-arylpropionates has been purified from a soil bacterium Alcaligenes bronchisepticus KU 1201. The enzyme was purified 300-fold to homogeneity, judged from the analysis of N-terminal amino acid sequence, and found to be a monomeric enzyme of apparent 24 kDa. The enzyme catalyzes a decarboxylation giving α-arylalkanoates from substituted malonates such as αarylmalonate and α-alkyl-α-arylmalonates. The decarboxylase is not a biotin containing enzyme because avidin have no influence on the enzyme activity. In addition, the enzyme does not require known co-factors (ATP, ADP and coenzyme A) for maximum activity. The enzyme activity was inhibited by sulfhydryl agents. The electronic effect of the substituents on kcat for the enzymic decarboxylation of arylmalonates has been studied. The logarithm of relative value of kcat gave a linear correlation to Hammett's σ with a ... value of +1.9, for substituted phenylmalonates. Comparing the relative activities, it is clear that the enzyme prefers α-arylmalonates to α-aryl-α-methylmalonates. Thus, the enzyme was tentatively named as arylmalonate decarboxylase. [ABSTRACT FROM AUTHOR]

Using three rounds of structure-guided directed evolution, the catalytic activity of the (S)-selective arylmalonate decarboxylase variant G74C/C188S could be increased up to 920-fold. The best variant had a 220-fold improved activity in the production of (S)-naproxen with excellent enantioselectivity (>99% ee). [ABSTRACT FROM AUTHOR]

The enantiotopos differentiating selectivity of the mutant arylmalonate decarboxylase (S36N/G74C/C188S), which catalyzes asymmetric decarboxylation of arylmethylmalonates to give the corresponding arylpropionates, was revealed to be the same as that of the wild type enzyme, in spite of the fact that two enzymes gave the opposite enantiomer with each other. [ABSTRACT FROM AUTHOR]

The catalytic promiscuity of AMDase was demonstrated using a well-designed substrate based on the consideration of the reaction mechanism. As the reaction catalyzed by AMDase is supposed to proceed via an enolate intermediate, we expected that the enzyme promotes the aldol type reaction when an acceptor is properly arranged, which turned out to be true. [ABSTRACT FROM AUTHOR]

Abstract: Arylmalonate decarboxylase (AMDase, EC. 4.1.1.76) catalyzes asymmetric decarboxylation of α-arylmalonic acid to give optically pure (R)-α-arylpropionic acid. This enzyme has four cysteine residues, one of which, Cys188, is estimated to be located in the active site. It is believed that it delivers a proton from the si-face of the intermediate enolate to give (R)-product. Based on database searches, it has been revealed that AMDase has some homology with glutamate racemase and other isomerases. Glutamate racemase has a pair of Cys residues in the active site, at positions 73 and 184, which are believed to abstract and deliver a proton from both sides of the substrate. On the other hand, AMDase has only one Cys at 188, and this is considered to be the reason why this enzyme gives optically pure products. The estimated 3D-structure of AMDase suggests that Gly74 is located in the opposite side of Cys188. Then, it was expected that introduction of one Cys around the region of Gly74, i.e., from 68 to 77 in addition to the replacement of Cys188 with less acidic Ser would invert the enantioselectivity of the enzyme. Thus, 10 mutants were prepared and their activities as well as their enantioselectivities were examined. As expected, two of them, S71C/C188S and G74C/C188S, exhibited decarboxylation activity and gave the opposite enantiomer to that formed by the wild type enzyme. [Copyright &y& Elsevier]