A single nucleotide polymorphism in BCAT1 gene is associated with type 2 diabetes mellitus

The level of circulatory branched chain amino acids (BCAAs) is often increased in type 2 diabetes mellitus (T2DM). Catabolism of BCAAs involves a transamination reaction mediated by the branched chain amino acid aminotransferase (BCAT1) enzyme. Differences in the level of BCAT1 were found to be linked with hypertension, obesity, and cancer. Herein, using a case control design, we tested the association of rs9668920 and rs12321766 polymorphisms in BCAT1 gene with T2DM. Three hundred subjects were recruited in the study. Genotyping of the indicated polymorphisms was achieved using restriction fragment length polymorphism technique after amplification of the target sequences. The results showed that, under a recessive inheritance model, the GG genotype of rs9668920 increased the risk of T2DM (P=0.026; OR 2.60; 95% CI 1.119–6.048). This effect was independent of the age, body mass index, waist circumference, serum glucose, cholesterol, triglycerides, and BCAAs (P>0.05). In conclusion, The GG genotype of BCAT1 rs9668920 SNP might be a risk factor of T2DM. More studies are required to confirm this finding.

Chiral Synthesis of 3-Amino-1-phenylbutane by a Multi-Enzymatic Cascade System

Asymmetric synthesis of chiral amines from prochiral ketones using transaminases is an attractive biocatalytic strategy. Nevertheless, it is hampered by its unfavorable thermodynamic equilibrium. In the present work, an insitu by-product removal strategy was applied for the synthesis of 3-amino-1-phenylbutane (3-APB) by coupling a transaminase with a pyruvate decarboxylase (PDC), which does not require the use of any expensive additional cofactor. Using this strategy, the pyruvate obtained in the transamination reaction is transformed by PDC into acetaldehyde and CO2 which are of high volatility. Two different transaminases from Chromobacterium violaceum (CviTA) and Vibrio fluvialis (VflTA) were characterized to find out the appropriate pH conditions. In both cases, the addition of PDC dramatically enhanced 3-APB synthesis. Afterwards, different reaction conditions were tested to improve reaction conversion and yield. It was concluded that 30 °C and a 20-fold alanine excess lead to the best process metrics. Under the mentioned conditions, yields higher than 60% were reached with nearly 90% selectivity using both CviTA and VflTA. Moreover, high stereoselectivity for (S)-3-APB was obtained and ee of around 90% was achieved in both cases. For the first time, the asymmetric synthesis of 3-APB using PDC as by-product removal system using CviTA is reported.

Structural Evidence of Active Site Adaptability towards Different Sized Substrates of Aromatic Amino Acid Aminotransferase from Psychrobacter Sp. B6

Aromatic amino acid aminotransferases present a special potential in the production of drugs and synthons, thanks to their ability to accommodate a wider range of substrates in their active site, in contrast to aliphatic amino acid aminotransferases. The mechanism of active site adjustment toward substrates of psychrophilic aromatic amino acid aminotransferase (PsyArAT) from Psychrobacter sp. B6 is discussed based on crystal structures of complexes with four hydroxy-analogs of substrates: phenylalanine, tyrosine, tryptophan and aspartic acid. These competitive inhibitors are bound in the active center of PsyArAT but do not undergo transamination reaction, which makes them an outstanding tool for examination of the enzyme catalytic center. The use of hydroxy-acids enabled insight into substrate binding by native PsyArAT, without mutating the catalytic lysine and modifying cofactor interactions. Thus, the binding mode of substrates and the resulting analysis of the volume of the catalytic site is close to a native condition. Observation of these inhibitors’ binding allows for explanation of the enzyme’s adaptability to process various sizes of substrates and to gain knowledge about its potential biotechnological application. Depending on the character and size of the used inhibitors, the enzyme crystallized in different space groups and showed conformational changes of the active site upon ligand binding.

Multi-enzyme pyruvate removal system to enhance (R)-selective reductive amination of ketones

The ArR-ωTA/TdcE/FDH/LDH system is an efficient system for increasing the conversion in the transamination reaction.

Para-(aza,thio)xanthenylated anilines in the pereaminination reaction

It is known that the biochemical enzymatic reaction of the reversible transfer of an amino group from an amino-acid to a keto-acid is called a transamination reaction. However, the transamination reaction is applicable not only for biochemical enzymatic reactions, but is also often used in organic synthesis to produce aromatic azomethines. As objects of study in the transamination reaction, we selected substituted N-benzylidenanilines (imines, Schiff bases) and anilines, containing a biologically active heterocyclic fragment in the para-position of the aniline ring. We have shown the feasibility of transamination of substituted N-benzylidenanilines (N-benzylidenaniline, N-benzyliden-4-(5H-benzopyrano[2,3-b]pyridin-5-yl)aniline, N-benzyliden-4-methoxyaniline), heterocyclic anilines (4-(9H-xanthen-9-yl)aniline, 4-(9H-thioxanthen-9-yl)aniline or 4-(5H-benzopyrano[2,3-b]pyridin-5-yl)aniline). It was found that the interaction of 4-(9H-xanthen-9-yl)aniline, 4-(9H-thioxanthen-9-yl)aniline or 4- (5H-benzopyrano[2,3-b]pyridin-5-yl)aniline with N-benzylidenanilines, the imine aniline cycle is replaced by the corresponding fragment of heterylated aniline, with the formation of new N-benzylidenanilines, the structure of which is proved by a breakdown of mixed melting and H1 NMR spectroscopy. However, the transamination reaction does not proceed with the use of N-benzyliden-4-methoxyaniline. This, apparently, is associated with the presence of an electron-donating substituent at the para-position of the aniline imines fragment. Thus, a series of activity of the studied compounds in the transamination reaction of substituted anilines was experimentally established. The most active of these is 4-methoxyaniline, followed by 4-(9H-xanthen-9-yl)aniline, 4-(9H-thioxanthen-9-yl) aniline, 4-(5H-benzopyrano[2,3-b]pyridin-5-yl)aniline, and closes the series of the least active, unsubstituted aniline. The synthesis method proposed in this work allows one to obtain new substituted N-benzylidenanilines, and the studied series of activity allows one to predict the behavior of anilines containing various electron-donating and electron-withdrawing substituents in the transamination reaction with N-benzylidenanilines.

Continuous Flow Mode Biocatalytic Transamination Using Macrocellular Silica Monoliths: Optimizing Support Functionalisation and Enzyme Grafting

<div>Transaminases are immobilized onto macrocellular silica monoliths and used for carrying a continuous flow mode transamination reaction. Monoliths were prepared via an emulsion-templated sol-gel method and functionalized by amino-moieties (APTES) in order to covalently immobilize the enzymes, using glutaraldehyde as a cross-linking agent. In order to obtain higher performance and improved reproducibility, we investigate the key parameters of APTES functionalization and of enzyme grafting. Four functionalization protocols were studied. It is shown that controlling the moisture levels in monolith and in the functionalisation solution led to a 3-fold increase in activity as compared to the previously reported data, and greatly improved the reproducibility. Additionally, we report a strong beneficial effect of running the enzyme immobilization at room temperature instead of 4°C, further enhancing the obtained activity. Finally, the popular method which consists in stabilizing the covalent attachment of the enzyme by reducing the imine bonds formed between the enzyme and the functionalized surface was investigated. We highlight a strong enzyme deactivation caused by cyanoborohydride, making this strategy irrelevant in this case. All in all, the improvements presented here for enzyme immobilization in macrocellular silica monoliths, lead to the preparation of more active materials for continuous flow mode biocatalysis.<br></div>

Continuous Flow Mode Biocatalytic Transamination Using Macrocellular Silica Monoliths: Optimizing Support Functionalisation and Enzyme Grafting

<div>Transaminases are immobilized onto macrocellular silica monoliths and used for carrying a continuous flow mode transamination reaction. Monoliths were prepared via an emulsion-templated sol-gel method and functionalized by amino-moieties (APTES) in order to covalently immobilize the enzymes, using glutaraldehyde as a cross-linking agent. In order to obtain higher performance and improved reproducibility, we investigate the key parameters of APTES functionalization and of enzyme grafting. Four functionalization protocols were studied. It is shown that controlling the moisture levels in monolith and in the functionalisation solution led to a 3-fold increase in activity as compared to the previously reported data, and greatly improved the reproducibility. Additionally, we report a strong beneficial effect of running the enzyme immobilization at room temperature instead of 4°C, further enhancing the obtained activity. Finally, the popular method which consists in stabilizing the covalent attachment of the enzyme by reducing the imine bonds formed between the enzyme and the functionalized surface was investigated. We highlight a strong enzyme deactivation caused by cyanoborohydride, making this strategy irrelevant in this case. All in all, the improvements presented here for enzyme immobilization in macrocellular silica monoliths, lead to the preparation of more active materials for continuous flow mode biocatalysis.<br></div>