Direct Enzymatic Synthesis Research Articles

Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome

L-Carnosine is a natural biologically active dipeptide with critical physiological functions, such as antioxidant, antiglycation, and cytoplasmic buffering properties. Direct enzymatic synthesis is a promising way for L-carnosine production. In this study, a new aminopeptidase (gene_236976) with synthetic activity toward L-carnosine was identified by a metagenome mining approach from deep-sea sediment and functionally expressed in Escherichia coli. The enzyme shared a low identity of 14.3% with reported L-carnosine dipeptidase (SmPepD) from Serratia marcescens. β-Alanine methyl ester was proven to be the best substrate for the synthesis, and no ATP was needed for the enzymatic reaction. The enzyme activity was increased by structure-guided rational design. Only the mutant of G310 site gave positive results, and G310A mutant showed the best performance among the site-direct saturation mutagenesis, indicating that the additional CH3 group of mutant G310A was the main factor affecting the enzymatic activity. The engineered enzyme produced about 10 mM L-carnosine was produced from substrates of 50 mM β-alanine methyl ester and 50 mM L-histidine, under a tentatively optimized condition. This study enriched the enzyme resources for developing the microbial synthesis process of L-carnosine production.

Direct Enzymatic Synthesis of Fatty Amines from Renewable Triglycerides and Oils.

Fatty amines represent an important class of commodity chemicals which have broad applicability in different industries. The synthesis of fatty amines starts from renewable sources such as vegetable oils or animal fats, but the process has multiple drawbacks that compromise the overall effectiveness and efficiency of the synthesis. Herein, we report a proof-of-concept biocatalytic alternative towards the synthesis of primary fatty amines from renewable triglycerides and oils. By coupling a lipase with a carboxylic acid reductase (CAR) and a transaminase (TA), we have accomplished the direct synthesis of multiple medium and long chain primary fatty amines in one pot with analytical yields as high as 97 %. We have also performed a 75 mL preparative scale reaction for the synthesis of laurylamine from trilaurin, obtaining 73 % isolated yield.

Engineering of l-threonine aldolase for the preparation of 4-(methylsulfonyl)phenylserine, an important intermediate for the synthesis of florfenicol and thiamphenicol

l-Threonine aldolases (l-TAs) catalyze the aldol condensation of aldehyde and glycine, offering direct enzymatic synthesis of β-hydroxy-α-amino acids under mild conditions. However, this method suffers from moderate yield and low stereoselectivity at the β-carbon. Given the importance of 4-(methylsulfonyl)phenylserine for the synthesis of florfenicol and thiamphenicol, mutations of a l-threonine aldolase from Pseudomonas sp. (l-PsTA) were performed in this study by error-prone PCR and combinatorial mutation. Some beneficial mutants were obtained by screening the mutant library using a stepwise visual method. 4-(Methylsulfonyl)phenylserine was synthesized in up to 71 % diastereomeric excess (de), which are much higher than the previously reported 2 % de value, by using the newly identified mutants. The mutants V200I and C187S/V200I were found to improve the product yield and stereoselectivity for the aldol condensation of various benzaldehydes with glycine. These results show that the amino acid residues outside of the substrate-binding cavity of l-PsTA play an important role in determining its Cβ-stereoseletivity.

Direct Enzymatic Synthesis of a Deep-Blue Fluorescent Noncanonical Amino Acid from Azulene and Serine.

We report a simple, one-step enzymatic synthesis of the blue fluorescent noncanonical amino acid β-(1-azulenyl)-l-alanine (AzAla). By using an engineered tryptophan synthase β-subunit (TrpB), stereochemically pure AzAla can be synthesized at scale starting from commercially available azulene and l-serine. Mutation of a universally conserved catalytic glutamate in the active site to glycine has only a modest effect on native activity with indole but abolishes activity on azulene, suggesting that this glutamate activates azulene for nucleophilic attack by stabilization of the aromatic ion.

Direct Enzymatic Synthesis of 1‐Phosphatidyl‐β‐D‐glucose by Engineered Phospholipase D

AbstractA simple, direct method for the synthesis of 1‐phosphatidyl‐β‐D‐glucose (1‐PGlc) was established, being the first example of direct enzymatic synthesis of this recently discovered, highly important bioactive phospholipid. The method employs phospholipase D (PLD, E.C. 3.1.4.4)‐catalyzed transphosphatidylation, in which the polar head group of phosphatidylcholine is exchanged to glucose. Although wild‐type PLD can catalyze the transfer reaction, it provides only 6‐phosphatidyl‐glucose, a positional isomer of 1‐PGlc, due to its strong preference towards the primary hydroxyl group. To synthesize 1‐PGlc, engineered PLD variants, previously isolated as the ones active on secondary hydroxyls of inositol, were screened for the ability to catalyze the reaction. One of the variants, 187K/191W/385Y (KWY), was identified as the best‐performing in 1‐PGlc synthesis, however, in addition to 1‐PGlc the reaction with KWY also afforded undesired positional isomers as byproducts. To facilitate the isolation of 1‐PGlc, the isomers were converted into the corresponding amines by reductive amination. Following the column chromatography, the desired product was isolated with the overall yield of 12.5 %. The structure of the product was confirmed by 1H‐NMR analysis.

Blue-light dependent reactive oxygen species formation by Arabidopsis cryptochrome may define a novel evolutionarily conserved signaling mechanism.

Cryptochromes are widespread blue-light absorbing flavoproteins with important signaling roles. In plants they mediate de-etiolation, developmental and stress responses resulting from interaction with downstream signaling partners such as transcription factors and components of the proteasome. Recently, it has been shown that Arabidopsis cry1 activation by blue light also results in direct enzymatic conversion of molecular oxygen (O2 ) to reactive oxygen species (ROS) and hydrogen peroxide (H2 O2 ) in vitro. Here we explored whether direct enzymatic synthesis of ROS by Arabidopsis cry1 can play a physiological role in vivo. ROS formation resulting from cry1 expression was measured by fluorescence assay in insect cell cultures and in Arabidopsis protoplasts from cryptochrome mutant seedlings. Cell death was determined by colorimetric assay. We found that ROS formation results from cry1 activation and induces cell death in insect cell cultures. In plant protoplasts, cryptochrome activation results in rapid increase in ROS formation and cell death. We conclude that ROS formation by cryptochromes may indeed be of physiological relevance and could represent a novel paradigm for cryptochrome signaling.

Facile and direct synthesis of long-chain chitin from chitobiose via proton-assisted nonaqueous biocatalysis

Facile and direct enzymatic synthesis of long-chain chitin from chitobiose without activation of an anomeric carbon via traditional organic chemistry was first achieved in a one-step and one-pot reaction by the combined use of a surfactant-enveloped enzyme (SEE) and an organic protic acid co-catalyst in the aprotic nonaqueous solvent, N,N-dimethylacetamide. The SEE biocatalyst was successfully prepared by enveloping lysozyme, having chitinase activity, with sucrose oleate surfactant. When o-nitrobenzenesulfonic acid was used as a co-catalyst, a degree of polymerization of synthesized chitin reached ca. 210 in ca. 79% yield by proton-assisted SEE-mediated biocatalysis. The conversion ratios and average chain lengths depended on the types of organic protic acids, and high catalytic efficiencies were maintained in each case, resulting in high yields of ca. 40–80%. The SEE biocatalyst, which has enzymatic activity even in aprotic organic media containing strong organic acids, allowed the facile and high-yielding efficient synthesis of long-chain chitin from chitobiose in its original form. Therefore, proton-assisted SEE-mediated biocatalysis should prove useful for the glycosynthesis of many glycomaterials.

Direct Enzymatic Synthesis of a Polyester with Free Pendant Mercapto Groups

An aliphatic polyester with free pendant mercapto groups was prepared by direct lipase-catalyzed polycondensation of hexane-1,6-diol and dimethyl 2-mercaptosuccinate. The polycondensation reaction using immobilized lipase from Candida antarctica (lipase CA) was carried out in bulk at 70 degrees C without any formation of disulfide or thioester linkages to produce the corresponding polyester with Mw = 14000 g/mol in high yield. The content of free mercapto groups in the polyester could be controlled by copolymerization with other monomers. The obtained polyester was easily cross-linked to form gels by the air oxidation of the pendant thiols to disulfides in dimethyl sulfoxide.

Template-free enzymatic synthesis of electrically conducting polyaniline using soybean peroxidase

Synthesis of polyaniline (PANI) catalyzed by soybean peroxidase at 1 °C in either aqueous or partially organic media, was studied as a function of pH and reaction media. Kinetic studies indicated that, unlike chemical polymerization, enzymatic polymerization of aniline showed neither induction period nor auto acceleration. The redox reversibility and chemical structure of the synthesized PANI was strongly dependent on the starting pH of the reaction medium. UV–vis, FT-IR, WAXD and TGA analysis are used to explain how the enzymatic reaction conditions influence both the chemical structure and physical properties of the PANI. Optimal reaction conditions are outlined for the direct enzymatic synthesis of electrically conductive emeraldine salt with yield as high as 71%.

Enzymatic Synthesis and Polymerization of Cyclic Trimethylene Carbonate Monomer with/without Methyl Substituent

AbstractThe direct enzymatic synthesis of a cyclic trimethylene carbonate (1,3‐dioxane‐2‐one) monomer with/without a methyl substituent was carried out using dimethyl or diethyl carbonate and 1,3‐diol with the objective of producing aliphatic poly(trimethylene carbonate), a typical biodegradable synthetic plastic. The lipase‐catalyzed condensation of dimethyl or diethyl carbonate with aliphatic 1,3‐diols using immobilized Candida antarctica lipase (lipase CA) in an organic solvent at 70 °C afforded the corresponding methyl‐substituted and unsubstituted cyclic trimethylene carbonates. The cyclic trimethylene carbonates obtained by the reaction of dimethyl or diethyl carbonates with 1,3‐propanediol and 2‐methyl‐1,3‐propanediol were polymerized by lipase to produce the corresponding polycarbonates.Total TMC yield as a function of the reaction time.magnified imageTotal TMC yield as a function of the reaction time.

Protease-catalyzed peptide synthesis on solid support.

The direct enzymatic synthesis of peptides from amino acids is widely used as a useful alternative to chemical synthesis. However, good yields of such enzyme-catalyzed reactions require altered reaction conditions to overcome the bias for hydrolysis in aqueous medium. We argue that the synthesis/hydrolysis equilibrium can be shifted toward synthesis in aqueous medium by immobilizing the amine on solid support. In this report, we show the first examples of solid-phase peptide synthesis catalyzed by a protease in bulk aqueous buffer.

Thermodynamically Controlled Synthesis Of Cefamandole

The direct enzymatic synthesis of the antibiotic cefamandole is presented as an alternative for the current processes based on either chemical synthesis or kinetic enzymatic synthesis. The influence of pH and temperature on the apparent equilibrium constant was measured; from these data, the maximum cefamandole concentration was predicted. In spite of a beneficial apparent equilibrium constant at pH 3.75, less product was formed as compared to the concentration at the optimum pH (4.25). This effect is caused by the low solubility of one of the substrates at pH 3.75.The maximum product concentration (at the optimum pH of 4.25) was found to be 22 mM which is a significant concentration. The thermodynamically controlled synthesis may prove to be an alternative for the currently used processes if it can be combined with an effective in-situ cefamandole removal step.

A direct enzymatic synthesis of β- d-galactopyranosyl- d-xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo

By enzymatic β- d-galactosylation of d-xylose a mixture of 4-, 3-, and 2-O-β- d-galactopyranosyl- d-xyloses ( 1, 4, and 7, respectively) was obtained in 50% isolated yield. Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K m values of 250.0, 4.5, and 14.0 mM, respectively. The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning. The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.

A Direct Enzymatic Synthesis of Laurencin from Laurediol

Abstract Deacetyllaurencin was synthesized in an enzymatic manner from (3E,6R,7R)-laurediol with lactoperoxidase in the presence of hydrogen peroxide and sodium bromide.

IV. Acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum: Studies on substrate specificity using phenylacetyl-CoA variants.

Two different penicillins (p- and m-methylbenzylpenicillin) were obtained "in vitro" by direct enzymatic synthesis, using homogeneously pure acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase from Penicillium chrysogenum, 6-APA and p- or m-tolylacetyl-CoA. The Km for these substrates were 6 and 15 mM, respectively, indicating that the affinity of the enzyme for these two molecules is much lower that shown by phenylacetyl-CoA (0.55 mM). Furthermore, acyltransferase does not recognize o-tolylacetyl-CoA as a substrate suggesting that the position of the methyl group on the aromatic moiety may have a very important role in the formation of the enzyme-substrate complex.

Direct Enzymatic Synthesis of Aspartamea

La synthese de l'aspartame a partir du L acide aspartique et de la L phenylalanine methyl ester peut etre effectuee par une enzyme extraite de Micrococcus caseolyticus, dans des conditions definies (pH, temperature, solvant, concentration en enzymes et substrat)

Direct enzymatic synthesis of natural penicillins using phenylacetyl-CoA: 6-APA phenylacetyl transferase of Penicillium chrysogenum: minimal and maximal side chain length requirements.

In vitro synthesis of different natural penicillins (hexanoyl, heptanoyl and octanoyl-penicillin) have been carried out by direct acylation of 6-aminopenicillanic acid (6-APA) with several fatty acid-CoA derivatives (hexanoyl-CoA, heptanoyl-CoA and octanoyl-CoA). The reactions were catalyzed by the enzyme Acyl-CoA: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum AS-P-78. This enzyme only accepts as substrate, aliphatic side chain precursors whose carbon length is between 6 and 8 atoms. Although the enzymatic synthesis of octanoylpenicillin has been previously reported the in vitro synthesis of hexanoyl and heptanoyl penicillins is described here for the first time.

Direct Enzymatic Synthesis of Penicillin G Using Cyclases of Penicillium chrysogenum and Acremonium chrysogenum

Direct enzymatic synthesis of penicillin G has been obtained by in vitro cyclization of the linear tripeptide–like molecule phenyl–acetyl–L–cysteinyl–D–valine (PCV) using partially purified cyclases of Penicillium chrysogenum and Acremonium chrysogenum. The reaction product shows an antibiotic spectrum similar to that of penicilin G, is sensitive to β–lactamase and penicillin acylase, and has been identified by chromatography, electrophoresis and HPLC as penicillin G.