Stable industrial protease catalyzed peptide bond formation in organic solvent.

Chen, Shui‐Tein; Hsiao, Shu-Chyong; Wang, Kung‐Tsung

doi:10.1016/s0960-894x(01)81103-3

Cited by 17 publications

(3 citation statements)

References 27 publications

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“…Amidation would be of particular interest because many natural amide synthetases, such as NRPS and ATP‐grasp enzymes, suffer from very narrow substrate specificity, which limits their applications in biocatalysis. Alternatively, hydrolases (such as lipases and proteases), which are commonly used for amide formation, require systems in which very little water is present, such as organic solvents, to drive the reaction towards amide bond formation …”

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confidence: 99%

“…Amidation would be of particular interest because many natural amide synthetases,s uch as NRPS [14] and ATP-grasp enzymes [15,16] ,s uffer from very narrow substrate specificity,w hich limits their applications in biocatalysis.A lternatively,h ydrolases (such as lipases [17] and proteases [18] ), which are commonly used for amide formation, [2] require systems in which very little water is present, such as organic solvents,todrive the reaction towards amide bond formation. [19][20][21][22] Four CARc andidates (CARmm from Mycobacterium marinum, [3] CARni from Nocardia iowensis, [4] CARtp from Tsukamurella paurometabola, [10] and CARse from Segniliparus rotundus, [23] )were produced recombinantly in Escherichia coli,w ith the coexpressed gene for the Bacillus subtilis phosphopantetheinyl transferase (PPTase;S fp), [24] which is required for post-translational addition of the PPant group. [4] These CARs were purified (Supporting Information, Figure S1) and the amidation of carboxylic acid substrates 1-6 was tested.…”

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Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

et al. 2017

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Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

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Section: Figurementioning

confidence: 99%

mentioning

confidence: 99%

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

et al. 2017

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show abstract

Section: Figurementioning

confidence: 99%

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

et al. 2017

View full text Add to dashboard Cite

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site‐directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

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Kinetic resolution of esters of amino acids in t‐butanol containing 5% water catalyzed by a stable industrial alkaline protease

1994

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We developed a procedure for the resolution of esters of amino acids in 95% t-butanol, followed by saponification of the unreacted esters to afford both enantiomers with high yield and optical purity. The hydrolysis, catalyzed by alkaline protease, was conducted in a mixture of t-butanol(95%) and water (5%) at 25"C, with a pH controlled at pH 8.5 by the addition of NaOH (2 M). The hydrolyzed L-amino acid, which was insoluble under these conditions, precipitated during the course of hydrolysis. After separation of the precipitate, the pH of the filtrate was adjusted to 11.5 to sapomfy the unreacted ester. The D-antipode precipitated at pH 6.2-6.5. Both optically pure antipodes were obtained with high enantiomeric excesses and yields by simple filtration. o 1994 Wiey-Liss, Inc. KEY WORDS: resolution, D,L-&Oacid esters, alkaline protease, simple separation, high enantiomeric excess and yieldsThe search for proteases for peptide synthesis that are stable in organic solvents is the subject of extensive investigation. 's2 Several studies have demonstrated the possibility of using proteases to catalyze peptide synthesis in organic solvents, but a drawback of those reactions is the poor stability of the enzyme in organic solvents. Many immobilization processes have been developed to overcome this. Here we describe the use of an inexpensive enzyme, alcalase, as a catalyst for resolution of amino acids, some of which have uncommon side chains and are found in many biologically and pharmaceutically important peptides. 35 Most of these unnatural amino acids cannot be obtained by fermentation or recombinant DNA technology. Resolution is an effective way to produce optically pure unnatural amino acids.Alcalase is a proteolytic enzyme prepared from a selective strain of Bacillus lichenifomis. The major enzyme component in alcalase is subtilisin Carlsberg (alkaline protease A), which is a serine protease and which is widely used as additives in detergents as a digesting enzyme. This alcalase can maintain enzymatic activity and stability in organic solvents.6v7 Application of alcalase in organic synthesis has been limited, although it is a potent and inexpensive catalyst.'*' Scheme 1 shows the reaction sequence of the resolution. The hydrolysis catalyzed by alkaline protease was conducted in a mixture of t-butanol (95%) and water (5%) at 25°C with a pH controller at pH 8.5. The hydrolyzed amino acid was insoluble under these conditions and precipitated during the course of hydrolysis. The precipitate was separated by filtration, and the pH of the filtrate was adjusted to 11.5 to saponify the unreacted ester of the D-&O acid.

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Stable industrial protease catalyzed peptide bond formation in organic solvent.

Cited by 17 publications

References 27 publications

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Kinetic resolution of esters of amino acids in t‐butanol containing 5% water catalyzed by a stable industrial alkaline protease

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