The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds.

An object of the present invention is to provide a method of industrially producing a high-purity L-cyclic amino acid more inexpensively and with a high efficiency, from a cyclic amino acid having a double bond at the 1-position. The present invention provides a method in which an L-cyclic amino acid is produced by allowing a cyclic amino acid having a double bond at the 1-position to react with a specific enzyme having a catalytic ability to reduce a cyclic amino acid having a double bond at the 1-position to produce an L-cyclic amino acid.

The invention relates to a process to produce a (1r,4r)-4-substituted cyclohexane-1-amine [further referred as trans-4-substituted cyclohexane-1-amine] of formula (T), starting from a diastereomeric mixture of 4-substituted cyclohexane-1-amines (formula (C)+formula (T)) or any salt of them by using a single transaminase biocatalyst in whole-cell, soluble or immobilized form in the presence of an amine acceptor used in sub-equimolar up to equimolar quantities in batch mode or in continuous-flow mode. In the first aspect of the present invention 2-(trans-4-aminocyclohexyl)acetic acid esters, more preferably a C.sub.1-6 alkyl esters, particularly 2-(trans-4-aminocyclohexyl)acetic acid ethyl ester may be produced. In the second aspect of the present invention hydroxyl-protected or protective group-free trans-4-(2-hydroxyethyl)cyclohexan-1-amines, particularly trans-4-(2-hydroxyethyl)cyclohexan-1-amine may be produced. In the third aspect of the present invention protected 2-(trans-4-aminocyclohexyl)acetaldehydes, particularly trans-4-((1,3-dioxolan-2-yl)methyl)cyclohexan-1-amine may be produced.

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An omega-transaminase of R-configuration is provided. The omega-transaminase of R-configuration has the amino acid sequence as shown in SEQ ID NO: 2, or has at least 80% identity to the amino acid sequence as shown in SEQ ID NO: 2, or has the amino acid sequence of proteins which have substituted, deleted or added one or more amino acids and have the activity of an omega-transaminase with a high stereoselectivity R-configuration catalytic activity; and does not have the amino acid sequence encoded by the nucleotide sequence as shown in SEQ ID NO: 4. The high stereoselectivity refers to the content of one of the stereoisomers being at least about 1.1 times that of the other. A use of the omega-transaminase of R-configuration is provided.

An object of the present invention is to provide a method of industrially producing a high-purity L-cyclic amino acid more inexpensively and with a high efficiency, from a cyclic amino acid having a double bond at the 1-position. The present invention provides a method in which an L-cyclic amino acid is produced by allowing a cyclic amino acid having a double bond at the 1-position to react with a specific enzyme having a catalytic ability to reduce a cyclic amino acid having a double bond at the 1-position to produce an L-cyclic amino acid.

A method for synthesizing a brivaracetam chiral intermediate (R)-3-cyanohexanoic acid by catalyzing the hydrolysis of 3-cyanohexanitile using an enzyme with nitile hydrolysis activity, and the enzyme with nitrile hydrolysis activity is obtained by carrying out a single mutation or a double mutation on an amino acid at position 140 or an amino acid at position 175 in an amino acid sequence as set forth in SEQ ID NO.2. Compared with a wild type, the nitrilase mutant has the activity increased by 10 times, an ee value increased to 300 or more from 39, a substrate conversion rate of 45%, and a product ee which can reach 98.5%, and the yield of (R)-3-aminomethyl-hexanoic acid by catalytic hydrogenation synthesis using (R)-3-cyanohexanoic acid reaches 85% or more. This features a short synthesis route, mild reaction conditions, and high atom economy, and can be applied to the industrial synthesis of the brivaracetam intermediate.

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds.

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds.