JPWO2008129873A1 - S-selective phenylalanine aminomutase, DNA encoding the same, method for producing (S) -β-phenylalanine, and method for producing (S) -β-phenylalanine analogues - Google Patents

Abstract

According to the present invention, DNA encoding S-selective phenylalanine aminomutase is obtained, and (S) -β-phenylalanine is produced from L-phenylalanine or cinnamic acid and ammonia using a transformant containing the DNA. To do. In addition, according to the present invention, DNA encoding S-selective phenylalanine aminomutase is obtained, and a corresponding (S) -β-phenylalanine analog is obtained from an L-phenylalanine analog using a transformant containing the DNA. Manufacturing.

Description

  The present invention has an S-isomer-selective phenylalanine aminomutase having an enzyme activity for synthesizing (S) -β-phenylalanine, a DNA encoding the same, a recombinant DNA obtained by incorporating the DNA into a vector, and the recombinant DNA The present invention relates to a transformant, a method for producing (S) -β-phenylalanine, and a method for producing an (S) -β-phenylalanine analog.

  In recent years, β-phenylalanine has been a useful compound as a pharmaceutical intermediate. Since β-phenylalanine has one asymmetric carbon and has a stereoisomer, β-phenylalanine having high stereochemical purity (optical purity) as well as chemical purity is demanded.

  Conventionally, in order to obtain β-phenylalanine stereoselectively, a method of isolating and purifying the target stereoisomer by optical resolution of racemic β-phenylalanine has been used. However, according to this method, the theoretical yield is as low as 50%, and the production process becomes complicated in multiple steps, so that the obtained β-phenylalanine is also expensive. Therefore, development of a stereoselective β-phenylalanine production method for realizing high yield and low price is expected.

(R) -β-phenylalanine is an intermediate of paclitaxel (taxol), which is a kind of anticancer agent. Walker et al. Show that phenylalanine aminomutase extracted from Taxus brevifolia, a plant of the Taxus genus, is an aminomutase that converts (S) -α-phenylalanine (L-phenylalanine) to (R) -β-phenylalanine. Clarified (Non-Patent Document 1). Walker et al. Also reported that phenylalanine aminomutase derived from Taxus cuspidata converts L-phenylalanine to (R) -β-phenylalanine, and converts both R-form and S-form β-phenylalanine to L-phenylalanine as a reverse reaction. (Non-Patent Document 2). Therefore, it can be said that these phenylalanine aminomutases derived from Taxus are phenylalanine aminomutases having high R-isomer selectivity.
J. Am. Chem. Soc. 1998, Vol. 120, 5333-5334 J. Bio. Chem. 2004, Vol. 279, No. 52, 53947-53954 J. Am. Chem. Soc. 2006, Vol. 128, 10660-10661 Biochemistry 2003, Vol. 42, 12708-12718

  On the other hand, (S) -β-phenylalanine is also in increasing demand as an intermediate for pharmaceuticals. However, phenylalanine aminomutase having S-isomer selectivity is not known. Therefore, in order to obtain (S) -β-phenylalanine having high optical purity, it is necessary to rely on the complicated optical resolution method described above.

  Clardy et al. Found an andrimid biosynthetic gene cluster from a microorganism that produces andrimid, which is a kind of antibiotic containing the (S) -β-phenylalanine skeleton, deciphered its DNA sequence, and known deduced amino acid sequence of each gene product. The andrimid biosynthetic pathway was predicted by analyzing homology with the amino acid sequence of the enzyme. And since AdmH which is one of the gene products has homology with SgcC4 (tyrosine aminomutase), it is speculated that it is an aminomutase having an activity of converting α-phenylalanine into β-phenylalanine (non-patented). Reference 3). However, whether or not the AdmH gene is expressed and the gene product is produced, and even if the gene product is produced, whether or not the AdmH has phenylalanine aminomutase activity has been verified. Absent. Furthermore, even if it is phenylalanine aminomutase based on AdmH, there are high and low stereoselectivity in homologous proteins of AdmH, so AdmH has the characteristics of industrially useful high stereoselectivity. Whether or not is completely unknown. Taxanes-derived phenylalanine aminomutase, which has 20% homology with AdmH at the amino acid level, has high stereoselectivity in β-phenylalanine synthesis reaction (Non-patent Document 2), whereas it is 36% at the amino acid level with AdmH. It is known that SgcC4 showing the homology of is low stereoselectivity in the β-tyrosine synthesis reaction (Non-patent Document 4).

  The present invention has been made in view of the above circumstances, and its object is to provide S-selective phenylalanine aminomutase, DNA encoding the same, recombinant DNA having the DNA, and trait having the recombinant DNA. It is in providing the manufacturing method of a converter and (S)-(beta) -phenylalanine, and the manufacturing method of (S)-(beta) -phenylalanine analog.

  According to the present invention, an S-isomer-selective phenylalanine aminomutase having an enzyme activity for synthesizing (S) -β-phenylalanine from L-phenylalanine or an enzyme activity for synthesizing (S) -β-phenylalanine from cinnamic acid and ammonia. Is provided.

  In this invention, it can be set as said S body selective phenylalanine aminomutase which has the enzyme activity which synthesize | combines (S)-(beta) -phenylalanine from L-phenylalanine.

  Moreover, in this invention, it can be set as said S body selection phenylalanine aminomutase which has the enzyme activity which synthesize | combines (S)-(beta) -phenylalanine from cinnamic acid and ammonia.

Moreover, according to the present invention,
S-selective phenylalanine aminomutase having the following physicochemical properties is provided.
(1) Action: Synthesizes (S) -β-phenylalanine from L-phenylalanine or (S) -β-phenylalanine from cinnamic acid and ammonia.
(2) Optimal pH: pH 8.0 to 9.5
(3) Molecular weight: 57,000 ± 5,000 (SDS-polyacrylamide gel electrophoresis)

  In addition, according to the present invention, an S-isomer-selective phenylalanine aminomutase having the amino acid sequence represented by SEQ ID NO: 1 is provided.

  The present invention also provides S-selective phenylalanine aminomutase having an amino acid sequence in which one to a plurality of amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1.

  According to the present invention, a DNA encoding the above S-isomer-selective phenylalanine aminomutase is provided.

  In addition, according to the present invention, there is provided DNA encoding a protein having the base sequence represented by SEQ ID NO: 3 and having S-selective phenylalanine aminomutase activity.

  In addition, according to the present invention, the base sequence represented by SEQ ID NO: 3 has a base sequence in which one to a plurality of bases are substituted, deleted, inserted or added, and S-selective phenylalanine aminomutase. DNA encoding a protein having activity is provided.

  In addition, according to the present invention, there is provided DNA that hybridizes with a DNA having the base sequence represented by SEQ ID NO: 3 under a stringent condition and encodes a protein having S-selective phenylalanine aminomutase activity. The

  The present invention also provides a recombinant DNA obtained by incorporating the above DNA into a vector.

  Moreover, according to this invention, the transformant which has said DNA or said recombinant DNA is provided.

  In the present invention, the above transformant whose host organism is Escherichia coli can be used.

  Moreover, according to the present invention, (S) -β-phenylalanine is produced from L-phenylalanine in the presence of the above S-isomer-selective phenylalanine aminomutase. Is provided.

  In addition, according to the present invention, (S) -β-phenylalanine is produced from L-phenylalanine in the presence of the transformant or a processed product thereof. Is provided.

  According to the present invention, (S) -β-phenylalanine is produced from cinnamic acid and ammonia in the presence of the above S-isomer-selective phenylalanine aminomutase. A manufacturing method is provided.

  In addition, according to the present invention, (S) -β-phenylalanine is produced from cinnamic acid and ammonia in the presence of the transformant or a processed product thereof. A manufacturing method is provided.

  Moreover, according to the present invention, in the presence of the S-isomer-selective phenylalanine aminomutase described above, in formula (1) (in formula (1), Q is an optionally substituted phenyl group, pyridyl group, thienyl. Represents a group or a furyl group, X, Y and Z each independently represent a substituent substituted with Q, a hydrogen atom, a fluoro group, a chloro group, a bromo group, a nitro group, a hydroxyl group, or an alkyl having 1 to 3 carbon atoms. An amino group which may be substituted with a group, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms). There is provided a method for producing an (S) -β-phenylalanine analog, characterized by producing a phenylalanine analog.

  Moreover, according to the present invention, in the presence of the S-isomer-selective phenylalanine aminomutase described above, in formula (2) (in formula (2), Q is an optionally substituted phenyl group, pyridyl group, thienyl. X and Y each independently represents a substituent substituted with Q, and represents a hydrogen atom, a fluoro group, a chloro group, a bromo group, a nitro group, a hydroxyl group, an amino group, a methyl group or a methoxy group. And a corresponding (S) -β-phenylalanine analog is produced from the L-phenylalanine analog represented by the formula (1).

  Moreover, according to the present invention, in the presence of the S-isomer-selective phenylalanine aminomutase described above, in the general formula (3) (in the formula (3), Q represents a phenyl group, a 2-fluorophenyl group, a 3-fluorophenyl group. Group, 4-fluorophenyl group, 2-chlorophenyl group, 3-chlorophenyl group, 4-chlorophenyl group, 2,4-dichlorophenyl group, 4-bromophenyl group, 4-nitrophenyl group, 4-hydroxyphenyl group, 2- A methylphenyl group, a 3-methylphenyl group, a 4-methylphenyl group, a 4-methoxyphenyl group, a 3-pyridyl group, a 2-thienyl group, or a 2-furyl group. Provided is a method for producing a (S) -β-phenylalanine analog, characterized in that the corresponding (S) -β-phenylalanine analog is produced. That.

  According to this invention, (S) -β-phenylalanine can be synthesized from L-phenylalanine. Also, (S) -β-phenylalanine can be synthesized from cinnamic acid and ammonia. Thereby, (S) -β-phenylalanine can be synthesized stereoselectively in one step. The theoretical yield of (S) -β-phenylalanine synthesis using this enzyme is 100%. Therefore, (S) -β-phenylalanine can be obtained inexpensively and efficiently.

  According to this invention, (S) -β-phenylalanine can be synthesized stereoselectively in one step. Therefore, (S) -β-phenylalanine can be produced inexpensively and efficiently.

  The above-described object and other objects, features, and advantages will become more apparent from the preferred embodiments described below and the accompanying drawings.

It is a figure which shows the relative activity of S body selection phenylalanine aminomutase in each pH. The enzyme activity in Tris-HCl buffer (pH 9.0) was set to 100%.

(First embodiment)
The first embodiment of the present invention is an S-selective phenylalanine aminomutase.
This S-selective phenylalanine aminomutase has an enzyme activity for synthesizing (S) -β-phenylalanine from L-phenylalanine and / or an enzyme activity for synthesizing (S) -β-phenylalanine from cinnamic acid and ammonia.

  Said S body selection phenylalanine aminomutase has the enzyme activity which synthesize | combines (S)-(beta) -phenylalanine from L-phenylalanine. This reaction formula (4) is shown below.

  Moreover, said S body selection phenylalanine aminomutase has the enzyme activity which synthesize | combines (S) -beta-phenylalanine from cinnamic acid and ammonia. This reaction formula (5) is shown below.

  Moreover, said S body selective phenylalanine aminomutase has the enzyme activity which synthesize | combines a corresponding (S) -beta-phenylalanine analog from a L-phenylalanine analog. The reaction formulas (6) to (8) are shown below.

  Q represents an optionally substituted phenyl group, pyridyl group, thienyl group or furyl group. X, Y, and Z each independently represent a substituent that substitutes for Q. X, Y and Z groups are a hydrogen atom, a fluoro group, a chloro group, a bromo group, a nitro group, a hydroxyl group, an amino group optionally substituted with an alkyl group having 1 to 3 carbon atoms, and an alkyl group having 1 to 3 carbon atoms. Represents a group or an alkoxy group having 1 to 3 carbon atoms.

  Specifically, L-phenylalanine analogs include L-monohalogenophenylalanine, L-dihalogenophenylalanine, L-trihalogenophenylalanine, L-mononitrophenylalanine, L-dinitrophenylalanine, L-trinitrophenylalanine, L- Monohydroxyphenylalanine, L-dihydroxyphenylalanine, L-trihydroxyphenylalanine, L-monoaminophenylalanine, L-diaminophenylalanine, L-triaminophenylalanine, L-monoalkylphenylalanine, L-dialkylphenylalanine, L-trialkylphenylalanine, L -Monoalkoxyphenylalanine, L-dialkoxyphenylalanine, L-trialkoxyphenylalanine, L-monoalkoxymono Rukylphenylalanine, L-monoalkoxymonoaminophenylalanine, L-monoalkoxymonohalogenophenylalanine, L-monoalkoxymonohydroxyphenylalanine, L-monoalkoxymononitrophenylalanine, L-monoalkylmonoaminophenylalanine, L-monoalkylmonohalogenophenylalanine L-monoalkylmonohydroxyphenylalanine, L-monoalkylmononitrophenylalanine, L-monoaminomonohalogenophenylalanine, L-monoaminomonohydroxyphenylalanine, L-monoaminomononitrophenylalanine, L-monohalogenomonohydroxyphenylalanine, L -Monohalogenomononitrophenylalanine, L-monohydroxymononitrophenylalanine L-2-pyridylalanine, L-monohalogeno-2-pyridylalanine, L-dihalogeno-2-pyridylalanine, L-mononitro-2-pyridylalanine, L-dinitro-2-pyridylalanine, L-monohydroxy-2- Pyridylalanine, L-dihydroxy-2-pyridylalanine, L-monoamino-2-pyridylalanine, L-diamino-2-pyridylalanine, L-monoalkyl-2-pyridylalanine, L-dialkyl-2-pyridylalanine, L -Monoalkoxy-2-pyridylalanine, L-dialkoxy-2-pyridylalanine, L-monoalkoxymonoalkyl-2-pyridylalanine, L-monoalkoxymonoamino-2-pyridylalanine, L-monoalkoxymonohalogeno- 2-pyridylalanine, L-monoal Coxymonohydroxy-2-pyridylalanine, L-monoalkoxymononitro-2-pyridylalanine, L-monoalkylmonoamino-2-pyridylalanine, L-monoalkylmonohalogeno-2-pyridylalanine, L-monoalkylmono Hydroxy-2-pyridylalanine, L-monoalkylmononitro-2-pyridylalanine, L-monoaminomonohalogeno-2-pyridylalanine, L-monoaminomonohydroxy-2-pyridylalanine, L-monoaminomononitro- 2-pyridylalanine, L-monohalogenomonohydroxy-2-pyridylalanine, L-monohalogenomononitro-2-pyridylalanine, L-monohydroxymononitro-2-pyridylalanine, L-3-pyridylalanine, L- Monohalogeno-3-pyridyla Nin, L-dihalogeno-3-pyridylalanine, L-mononitro-3-pyridylalanine, L-dinitro-3-pyridylalanine, L-monohydroxy-3-pyridylalanine, L-dihydroxy-3-pyridylalanine, L- Monoamino-3-pyridylalanine, L-diamino-3-pyridylalanine, L-monoalkyl-3-pyridylalanine, L-dialkyl-3-pyridylalanine, L-monoalkoxy-3-pyridylalanine, L-dialkoxy- 3-pyridylalanine, L-monoalkoxymonoalkyl-3-pyridylalanine, L-monoalkoxymonoamino-3-pyridylalanine, L-monoalkoxymonohalogeno-3-pyridylalanine, L-monoalkoxymonohydroxy-3- Pyridylalanine, L-monoarco Cymononitro-3-pyridylalanine, L-monoalkylmonoamino-3-pyridylalanine, L-monoalkylmonohalogeno-3-pyridylalanine, L-monoalkylmonohydroxy-3-pyridylalanine, L-monoalkylmononitro- 3-pyridylalanine, L-monoaminomonohalogeno-3-pyridylalanine, L-monoaminomonohydroxy-3-pyridylalanine, L-monoaminomononitro-3-pyridylalanine, L-monohalogenomonohydroxy-3- Pyridylalanine, L-monohalogenomononitro-3-pyridylalanine, L-monohydroxymononitro-3-pyridylalanine, L-4-pyridylalanine, L-monohalogeno-4-pyridylalanine, L-dihalogeno-4-pyridyl Alanine, L-mononitro -4-pyridylalanine, L-dinitro-4-pyridylalanine, L-monohydroxy-4-pyridylalanine, L-dihydroxy-4-pyridylalanine, L-monoamino-4-pyridylalanine, L-diamino-4-pyridyl Alanine, L-monoalkyl-4-pyridylalanine, L-dialkyl-4-pyridylalanine, L-monoalkoxy-4-pyridylalanine, L-dialkoxy-4-pyridylalanine, L-monoalkoxymonoalkyl-4- Pyridylalanine, L-monoalkoxymonoamino-4-pyridylalanine, L-monoalkoxymonohalogeno-4-pyridylalanine, L-monoalkoxymonohydroxy-4-pyridylalanine, L-monoalkoxymononitro-4-pyridylalanine , L-monoalkyl monoa No-4-pyridylalanine, L-monoalkylmonohalogeno-4-pyridylalanine, L-monoalkylmonohydroxy-4-pyridylalanine, L-monoalkylmononitro-4-pyridylalanine, L-monoaminomonohalogeno- 4-pyridylalanine, L-monoaminomonohydroxy-4-pyridylalanine, L-monoaminomononitro-4-pyridylalanine, L-monohalogenomonohydroxy-4-pyridylalanine, L-monohalogenomononitro-4- Pyridylalanine, L-monohydroxymononitro-4-pyridylalanine, L-2-thienylalanine, L-monohalogeno-2-thienylalanine, L-dihalogeno-2-thienylalanine, L-mononitro-2-thienylalanine, L -Dinitro-2-thienylalanine L-monohydroxy-2-thienylalanine, L-dihydroxy-2-thienylalanine, L-monoamino-2-thienylalanine, L-diamino-2-thienylalanine, L-monoalkyl-2-thienylalanine, L-dialkyl -2-thienylalanine, L-monoalkoxy-2-thienylalanine, L-dialkoxy-2-thienylalanine, L-monoalkoxymonoalkyl-2-thienylalanine, L-monoalkoxymonoamino-2-thienylalanine, L-monoalkoxymonohalogeno-2-thienylalanine, L-monoalkoxymonohydroxy-2-thienylalanine, L-monoalkoxymononitro-2-thienylalanine, L-monoalkylmonoamino-2-thienylalanine, L- Monoalkyl monohalogeno-2 -Thienylalanine, L-monoalkylmonohydroxy-2-thienylalanine, L-monoalkylmononitro-2-thienylalanine, L-monoaminomonohalogeno-2-thienylalanine, L-monoaminomonohydroxy-2-thienyl Alanine, L-monoaminomononitro-2-thienylalanine, L-monohalogenomonohydroxy-2-thienylalanine, L-monohalogenomononitro-2-thienylalanine, L-monohydroxymononitro-2-thienylalanine, L-3-thienylalanine, L-monohalogeno-3-thienylalanine, L-dihalogeno-3-thienylalanine, L-mononitro-3-thienylalanine, L-dinitro-3-thienylalanine, L-monohydroxy-3- Thienylalanine, L-dihydroxy 3-thienylalanine, L-monoamino-3-thienylalanine, L-diamino-3-thienylalanine, L-monoalkyl-3-thienylalanine, L-dialkyl-3-thienylalanine, L-monoalkoxy-3-thienyl Alanine, L-dialkoxy-3-thienylalanine, L-monoalkoxymonoalkyl-3-thienylalanine, L-monoalkoxymonoamino-3-thienylalanine, L-monoalkoxymonohalogeno-3-thienylalanine, L- Monoalkoxymonohydroxy-3-thienylalanine, L-monoalkoxymononitro-3-thienylalanine, L-monoalkylmonoamino-3-thienylalanine, L-monoalkylmonohalogeno-3-thienylalanine, L-monoalkyl Monohydroxy-3-thio Nylalanine, L-monoalkylmononitro-3-thienylalanine, L-monoaminomonohalogeno-3-thienylalanine, L-monoaminomonohydroxy-3-thienylalanine, L-monoaminomononitro-3-thienylalanine, L-monohalogenomonohydroxy-3-thienylalanine, L-monohalogenomononitro-3-thienylalanine, L-monohydroxymononitro-3-thienylalanine, L-2-furylalanine, L-monohalogeno-2-furyl Alanine, L-dihalogeno-2-furylalanine, L-mononitro-2-furylalanine, L-dinitro-2-furylalanine, L-monohydroxy-2-furylalanine, L-dihydroxy-2-furylalanine, L- Monoamino-2-furylalanine, L-diamino- 2-furylalanine, L-monoalkyl-2-furylalanine, L-dialkyl-2-furylalanine, L-monoalkoxy-2-furylalanine, L-dialkoxy-2-furylalanine, L-monoalkoxymonoalkyl -2-furylalanine, L-monoalkoxymonoamino-2-furylalanine, L-monoalkoxymonohalogeno-2-furylalanine, L-monoalkoxymonohydroxy-2-furylalanine, L-monoalkoxymononitro-2 -Furylalanine, L-monoalkylmonoamino-2-furylalanine, L-monoalkylmonohalogeno-2-furylalanine, L-monoalkylmonohydroxy-2-furylalanine, L-monoalkylmononitro-2-furyl Alanine, L-monoaminomonohalogeno-2-furyl Lanine, L-monoaminomonohydroxy-2-furylalanine, L-monoaminomononitro-2-furylalanine, L-monohalogenomonohydroxy-2-furylalanine, L-monohalogenomononitro-2-furylalanine, L-monohydroxymononitro-2-furylalanine, L-3-furylalanine, L-monohalogeno-3-furylalanine, L-dihalogeno-3-furylalanine, L-mononitro-3-furylalanine, L-dinitro- 3-furylalanine, L-monohydroxy-3-furylalanine, L-dihydroxy-3-furylalanine, L-monoamino-3-furylalanine, L-diamino-3-furylalanine, L-monoalkyl-3-furyl Alanine, L-dialkyl-3-furylalanine, L-monoalkoxy- -Furylalanine, L-dialkoxy-3-furylalanine, L-monoalkoxymonoalkyl-3-furylalanine, L-monoalkoxymonoamino-3-furylalanine, L-monoalkoxymonohalogeno-3-furylalanine, L-monoalkoxymonohydroxy-3-furylalanine, L-monoalkoxymononitro-3-furylalanine, L-monoalkylmonoamino-3-furylalanine, L-monoalkylmonohalogeno-3-furylalanine, L- Monoalkylmonohydroxy-3-furylalanine, L-monoalkylmononitro-3-furylalanine, L-monoaminomonohalogeno-3-furylalanine, L-monoaminomonohydroxy-3-furylalanine, L-monoamino Mononitro-3-furylalanine, L-monoha Logenomonohydroxy-3-furylalanine, L-monohalogenomononitro-3-furylalanine, L-monohydroxymononitro-3-furylalanine and the like.

  More specifically, L-phenylalanine analogs include L-2-fluorophenylalanine, L-3-fluorophenylalanine, L-4-fluorophenylalanine, L-2, 4-difluorophenylalanine, L-2-chlorophenylalanine. , L-3-chlorophenylalanine, L-4-chlorophenylalanine, L-2, 4-dichlorophenylalanine, L-2-bromophenylalanine, L-3-bromophenylalanine, L-4-bromophenylalanine, L-2, 4 -Dibromophenylalanine, L-2-nitrophenylalanine, L-3-nitrophenylalanine, L-4-nitrophenylalanine, L-2-hydroxyphenylalanine, L-3-hydroxyphenylalanine, L-4-hydroxyphenylalanine, L 2-methylphenylalanine, L-3-methylphenylalanine, L-4-methylphenylalanine, L-2-ethylphenylalanine, L-3-ethylphenylalanine, L-4-ethylphenylalanine, L-2-propylphenylalanine, L-3 -Propylphenylalanine, L-4-propylphenylalanine, L-2-methoxyphenylalanine, L-3-methoxyphenylalanine, L-4-methoxyphenylalanine, L-2-ethoxyphenylalanine, L-3-ethoxyphenylalanine, L-4- Ethoxyphenylalanine, L-2-pyridylalanine, L-3-pyridylalanine, L-4-pyridylalanine, L-2-thienylalanine, L-3-thienylalanine, L-2-furylalanine, L-3-furyla Nin, and the like.

The S-form selective phenylalanine aminomutase of this embodiment synthesizes the corresponding (S) -β-phenylalanine analog from the L-phenylalanine analog by catalyzing the reaction shown below.
A reaction for synthesizing (S) -β-monohalogenophenylalanine from L-monohalogenophenylalanine.
A reaction for synthesizing (S) -β-dihalogenophenylalanine from L-dihalogenophenylalanine.
A reaction for synthesizing (S) -β-trihalogenophenylalanine from L-trihalogenophenylalanine.
A reaction for synthesizing (S) -β-mononitrophenylalanine from L-mononitrophenylalanine.
A reaction for synthesizing (S) -β-dinitrophenylalanine from L-dinitrophenylalanine.
A reaction for synthesizing (S) -β-trinitrophenylalanine from L-trinitrophenylalanine.
A reaction for synthesizing (S) -β-monohydroxyphenylalanine from L-monohydroxyphenylalanine.
A reaction for synthesizing (S) -β-dihydroxyphenylalanine from L-dihydroxyphenylalanine.
A reaction for synthesizing (S) -β-trihydroxyphenylalanine from L-trihydroxyphenylalanine.
A reaction for synthesizing (S) -β-monoaminophenylalanine from L-monoaminophenylalanine.
A reaction for synthesizing (S) -β-diaminophenylalanine from L-diaminophenylalanine.
A reaction for synthesizing (S) -β-triaminophenylalanine from L-triaminophenylalanine.
A reaction for synthesizing (S) -β-monoalkylphenylalanine from L-monoalkylphenylalanine.
A reaction for synthesizing (S) -β-dialkylphenylalanine from L-dialkylphenylalanine.
A reaction for synthesizing (S) -β-trialkylphenylalanine from L-trialkylphenylalanine.
A reaction for synthesizing (S) -β-monoalkoxyphenylalanine from L-monoalkoxyphenylalanine.
A reaction for synthesizing (S) -β-dialkoxyphenylalanine from L-dialkoxyphenylalanine.
A reaction for synthesizing (S) -β-trialkoxyphenylalanine from L-trialkoxyphenylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkylphenylalanine from L-monoalkoxymonoalkylphenylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoaminophenylalanine from L-monoalkoxymonoaminophenylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogenophenylalanine from L-monoalkoxymonohalogenophenylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxyphenylalanine from L-monoalkoxymonohydroxyphenylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitrophenylalanine from L-monoalkoxymononitrophenylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoaminophenylalanine from L-monoalkylmonoaminophenylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogenophenylalanine from L-monoalkylmonohalogenophenylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxyphenylalanine from L-monoalkylmonohydroxyphenylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitrophenylalanine from L-monoalkylmononitrophenylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogenophenylalanine from L-monoaminomonohalogenophenylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxyphenylalanine from L-monoaminomonohydroxyphenylalanine.
A reaction for synthesizing (S) -β-monoaminomononitrophenylalanine from L-monoaminomononitrophenylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxyphenylalanine from L-monohalogenomonohydroxyphenylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitrophenylalanine from L-monohalogenomononitrophenylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitrophenylalanine from L-monohydroxymononitrophenylalanine.
A reaction for synthesizing (S) -β-2-pyridylalanine from L-2-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogeno-2-pyridylalanine from L-monohalogeno-2-pyridylalanine.
A reaction for synthesizing (S) -β-dihalogeno-2-pyridylalanine from L-dihalogeno-2-pyridylalanine.
A reaction for synthesizing (S) -β-mononitro-2-pyridylalanine from L-mononitro-2-pyridylalanine.
A reaction for synthesizing (S) -β-dinitro-2-pyridylalanine from L-dinitro-2-pyridylalanine.
A reaction for synthesizing (S) -β-monohydroxy-2-pyridylalanine from L-monohydroxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-dihydroxy-2-pyridylalanine from L-dihydroxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoamino-2-pyridylalanine from L-monoamino-2-pyridylalanine.
A reaction for synthesizing (S) -β-diamino-2-pyridylalanine from L-diamino-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkyl-2-pyridylalanine from L-monoalkyl-2-pyridylalanine.
A reaction for synthesizing (S) -β-dialkyl-2-pyridylalanine from L-dialkyl-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxy-2-pyridylalanine from L-monoalkoxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-dialkoxy-2-pyridylalanine from L-dialkoxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkyl-2-pyridylalanine from L-monoalkoxymonoalkyl-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoamino-2-pyridylalanine from L-monoalkoxymonoamino-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogeno-2-pyridylalanine from L-monoalkoxymonohalogen-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxy-2-pyridylalanine from L-monoalkoxymonohydroxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitro-2-pyridylalanine from L-monoalkoxymononitro-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoamino-2-pyridylalanine from L-monoalkylmonoamino-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogeno-2-pyridylalanine from L-monoalkylmonohalogen-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxy-2-pyridylalanine from L-monoalkylmonohydroxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitro-2-pyridylalanine from L-monoalkylmononitro-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogeno-2-pyridylalanine from L-monoaminomonohalogen-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxy-2-pyridylalanine from L-monoaminomonohydroxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomononitro-2-pyridylalanine from L-monoaminomononitro-2-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxy-2-pyridylalanine from L-monohalogenomonohydroxy-2-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitro-2-pyridylalanine from L-monohalogenomononitro-2-pyridylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitro-2-pyridylalanine from L-monohydroxymononitro-2-pyridylalanine.
A reaction for synthesizing (S) -β-3-pyridylalanine from L-3-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogeno-3-pyridylalanine from L-monohalogeno-3-pyridylalanine.
A reaction for synthesizing (S) -β-dihalogeno-3-pyridylalanine from L-dihalogeno-3-pyridylalanine.
A reaction for synthesizing (S) -β-mononitro-3-pyridylalanine from L-mononitro-3-pyridylalanine.
A reaction for synthesizing (S) -β-dinitro-3-pyridylalanine from L-dinitro-3-pyridylalanine.
A reaction for synthesizing (S) -β-monohydroxy-3-pyridylalanine from L-monohydroxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-dihydroxy-3-pyridylalanine from L-dihydroxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoamino-3-pyridylalanine from L-monoamino-3-pyridylalanine.
A reaction for synthesizing (S) -β-diamino-3-pyridylalanine from L-diamino-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkyl-3-pyridylalanine from L-monoalkyl-3-pyridylalanine.
A reaction for synthesizing (S) -β-dialkyl-3-pyridylalanine from L-dialkyl-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxy-3-pyridylalanine from L-monoalkoxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-dialkoxy-3-pyridylalanine from L-dialkoxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkyl-3-pyridylalanine from L-monoalkoxymonoalkyl-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoamino-3-pyridylalanine from L-monoalkoxymonoamino-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogeno-3-pyridylalanine from L-monoalkoxymonohalogeno-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxy-3-pyridylalanine from L-monoalkoxymonohydroxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitro-3-pyridylalanine from L-monoalkoxymononitro-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoamino-3-pyridylalanine from L-monoalkylmonoamino-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogeno-3-pyridylalanine from L-monoalkylmonohalogeno-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxy-3-pyridylalanine from L-monoalkylmonohydroxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitro-3-pyridylalanine from L-monoalkylmononitro-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogeno-3-pyridylalanine from L-monoaminomonohalogeno-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxy-3-pyridylalanine from L-monoaminomonohydroxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomononitro-3-pyridylalanine from L-monoaminomononitro-3-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxy-3-pyridylalanine from L-monohalogenomonohydroxy-3-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitro-3-pyridylalanine from L-monohalogenomononitro-3-pyridylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitro-3-pyridylalanine from L-monohydroxymononitro-3-pyridylalanine.
A reaction for synthesizing (S) -β-4-pyridylalanine from L-4-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogeno-4-pyridylalanine from L-monohalogeno-4-pyridylalanine.
A reaction for synthesizing (S) -β-dihalogeno-4-pyridylalanine from L-dihalogeno-4-pyridylalanine.
A reaction for synthesizing (S) -β-mononitro-4-pyridylalanine from L-mononitro-4-pyridylalanine.
A reaction for synthesizing (S) -β-dinitro-4-pyridylalanine from L-dinitro-4-pyridylalanine.
A reaction for synthesizing (S) -β-monohydroxy-4-pyridylalanine from L-monohydroxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-dihydroxy-4-pyridylalanine from L-dihydroxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoamino-4-pyridylalanine from L-monoamino-4-pyridylalanine.
A reaction for synthesizing (S) -β-diamino-4-pyridylalanine from L-diamino-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkyl-4-pyridylalanine from L-monoalkyl-4-pyridylalanine.
A reaction for synthesizing (S) -β-dialkyl-4-pyridylalanine from L-dialkyl-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxy-4-pyridylalanine from L-monoalkoxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-dialkoxy-4-pyridylalanine from L-dialkoxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkyl-4-pyridylalanine from L-monoalkoxymonoalkyl-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoamino-4-pyridylalanine from L-monoalkoxymonoamino-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogeno-4-pyridylalanine from L-monoalkoxymonohalogeno-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxy-4-pyridylalanine from L-monoalkoxymonohydroxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitro-4-pyridylalanine from L-monoalkoxymononitro-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoamino-4-pyridylalanine from L-monoalkylmonoamino-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogeno-4-pyridylalanine from L-monoalkylmonohalogeno-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxy-4-pyridylalanine from L-monoalkylmonohydroxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitro-4-pyridylalanine from L-monoalkylmononitro-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogeno-4-pyridylalanine from L-monoaminomonohalogeno-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxy-4-pyridylalanine from L-monoaminomonohydroxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-monoaminomononitro-4-pyridylalanine from L-monoaminomononitro-4-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxy-4-pyridylalanine from L-monohalogenomonohydroxy-4-pyridylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitro-4-pyridylalanine from L-monohalogenomononitro-4-pyridylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitro-4-pyridylalanine from L-monohydroxymononitro-4-pyridylalanine.
A reaction for synthesizing (S) -β-2-thienylalanine from L-2-thienylalanine.
A reaction for synthesizing (S) -β-monohalogeno-2-thienylalanine from L-monohalogeno-2-thienylalanine.
A reaction for synthesizing (S) -β-dihalogeno-2-thienylalanine from L-dihalogeno-2-thienylalanine.
A reaction for synthesizing (S) -β-mononitro-2-thienylalanine from L-mononitro-2-thienylalanine.
A reaction for synthesizing (S) -β-dinitro-2-thienylalanine from L-dinitro-2-thienylalanine.
A reaction for synthesizing (S) -β-monohydroxy-2-thienylalanine from L-monohydroxy-2-thienylalanine.
A reaction for synthesizing (S) -β-dihydroxy-2-thienylalanine from L-dihydroxy-2-thienylalanine.
A reaction for synthesizing (S) -β-monoamino-2-thienylalanine from L-monoamino-2-thienylalanine.
A reaction for synthesizing (S) -β-diamino-2-thienylalanine from L-diamino-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkyl-2-thienylalanine from L-monoalkyl-2-thienylalanine.
A reaction for synthesizing (S) -β-dialkyl-2-thienylalanine from L-dialkyl-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxy-2-thienylalanine from L-monoalkoxy-2-thienylalanine.
A reaction for synthesizing (S) -β-dialkoxy-2-thienylalanine from L-dialkoxy-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkyl-2-thienylalanine from L-monoalkoxymonoalkyl-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoamino-2-thienylalanine from L-monoalkoxymonoamino-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogeno-2-thienylalanine from L-monoalkoxymonohalogeno-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxy-2-thienylalanine from L-monoalkoxymonohydroxy-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitro-2-thienylalanine from L-monoalkoxymononitro-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoamino-2-thienylalanine from L-monoalkylmonoamino-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogeno-2-thienylalanine from L-monoalkylmonohalogeno-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxy-2-thienylalanine from L-monoalkylmonohydroxy-2-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitro-2-thienylalanine from L-monoalkylmononitro-2-thienylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogeno-2-thienylalanine from L-monoaminomonohalogeno-2-thienylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxy-2-thienylalanine from L-monoaminomonohydroxy-2-thienylalanine.
A reaction for synthesizing (S) -β-monoaminomononitro-2-thienylalanine from L-monoaminomononitro-2-thienylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxy-2-thienylalanine from L-monohalogenomonohydroxy-2-thienylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitro-2-thienylalanine from L-monohalogenomononitro-2-thienylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitro-2-thienylalanine from L-monohydroxymononitro-2-thienylalanine.
A reaction for synthesizing (S) -β-3-thienylalanine from L-3-thienylalanine.
A reaction for synthesizing (S) -β-monohalogeno-3-thienylalanine from L-monohalogeno-3-thienylalanine.
A reaction for synthesizing (S) -β-dihalogeno-3-thienylalanine from L-dihalogeno-3-thienylalanine.
A reaction for synthesizing (S) -β-mononitro-3-thienylalanine from L-mononitro-3-thienylalanine.
A reaction for synthesizing (S) -β-dinitro-3-thienylalanine from L-dinitro-3-thienylalanine.
A reaction for synthesizing (S) -β-monohydroxy-3-thienylalanine from L-monohydroxy-3-thienylalanine.
A reaction for synthesizing (S) -β-dihydroxy-3-thienylalanine from L-dihydroxy-3-thienylalanine.
A reaction for synthesizing (S) -β-monoamino-3-thienylalanine from L-monoamino-3-thienylalanine.
A reaction for synthesizing (S) -β-diamino-3-thienylalanine from L-diamino-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkyl-3-thienylalanine from L-monoalkyl-3-thienylalanine.
A reaction for synthesizing (S) -β-dialkyl-3-thienylalanine from L-dialkyl-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxy-3-thienylalanine from L-monoalkoxy-3-thienylalanine.
A reaction for synthesizing (S) -β-dialkoxy-3-thienylalanine from L-dialkoxy-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkyl-3-thienylalanine from L-monoalkoxymonoalkyl-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoamino-3-thienylalanine from L-monoalkoxymonoamino-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogeno-3-thienylalanine from L-monoalkoxymonohalogeno-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxy-3-thienylalanine from L-monoalkoxymonohydroxy-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitro-3-thienylalanine from L-monoalkoxymononitro-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoamino-3-thienylalanine from L-monoalkylmonoamino-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogeno-3-thienylalanine from L-monoalkylmonohalogeno-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxy-3-thienylalanine from L-monoalkylmonohydroxy-3-thienylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitro-3-thienylalanine from L-monoalkylmononitro-3-thienylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogeno-3-thienylalanine from L-monoaminomonohalogeno-3-thienylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxy-3-thienylalanine from L-monoaminomonohydroxy-3-thienylalanine.
A reaction for synthesizing (S) -β-monoaminomononitro-3-thienylalanine from L-monoaminomononitro-3-thienylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxy-3-thienylalanine from L-monohalogenomonohydroxy-3-thienylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitro-3-thienylalanine from L-monohalogenomononitro-3-thienylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitro-3-thienylalanine from L-monohydroxymononitro-3-thienylalanine.
A reaction for synthesizing (S) -β-2-furylalanine from L-2-furylalanine.
A reaction for synthesizing (S) -β-monohalogen-2-furylalanine from L-monohalogen-2-furylalanine.
A reaction for synthesizing (S) -β-dihalogen-2-furylalanine from L-dihalogen-2-furylalanine.
A reaction for synthesizing (S) -β-mononitro-2-furylalanine from L-mononitro-2-furylalanine.
A reaction for synthesizing (S) -β-dinitro-2-furylalanine from L-dinitro-2-furylalanine.
A reaction for synthesizing (S) -β-monohydroxy-2-furylalanine from L-monohydroxy-2-furylalanine.
A reaction for synthesizing (S) -β-dihydroxy-2-furylalanine from L-dihydroxy-2-furylalanine.
A reaction for synthesizing (S) -β-monoamino-2-furylalanine from L-monoamino-2-furylalanine.
A reaction for synthesizing (S) -β-diamino-2-furylalanine from L-diamino-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkyl-2-furylalanine from L-monoalkyl-2-furylalanine.
A reaction for synthesizing (S) -β-dialkyl-2-furylalanine from L-dialkyl-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxy-2-furylalanine from L-monoalkoxy-2-furylalanine.
A reaction for synthesizing (S) -β-dialkoxy-2-furylalanine from L-dialkoxy-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkyl-2-furylalanine from L-monoalkoxymonoalkyl-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoamino-2-furylalanine from L-monoalkoxymonoamino-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogen-2-furylalanine from L-monoalkoxymonohalogen-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxy-2-furylalanine from L-monoalkoxymonohydroxy-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitro-2-furylalanine from L-monoalkoxymononitro-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoamino-2-furylalanine from L-monoalkylmonoamino-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogeno-2-furylalanine from L-monoalkylmonohalogen-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxy-2-furylalanine from L-monoalkylmonohydroxy-2-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitro-2-furylalanine from L-monoalkylmononitro-2-furylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogeno-2-furylalanine from L-monoaminomonohalogen-2-furylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxy-2-furylalanine from L-monoaminomonohydroxy-2-furylalanine.
A reaction for synthesizing (S) -β-monoaminomononitro-2-furylalanine from L-monoaminomononitro-2-furylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxy-2-furylalanine from L-monohalogenomonohydroxy-2-furylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitro-2-furylalanine from L-monohalogenomononitro-2-furylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitro-2-furylalanine from L-monohydroxymononitro-2-furylalanine.
-Reaction which synthesize | combines (S)-(beta) -3-furylalanine from L-3-furylalanine.
A reaction for synthesizing (S) -β-monohalogeno-3-furylalanine from L-monohalogeno-3-furylalanine.
A reaction for synthesizing (S) -β-dihalogeno-3-furylalanine from L-dihalogeno-3-furylalanine.
A reaction for synthesizing (S) -β-mononitro-3-furylalanine from L-mononitro-3-furylalanine.
A reaction for synthesizing (S) -β-dinitro-3-furylalanine from L-dinitro-3-furylalanine.
A reaction for synthesizing (S) -β-monohydroxy-3-furylalanine from L-monohydroxy-3-furylalanine.
A reaction for synthesizing (S) -β-dihydroxy-3-furylalanine from L-dihydroxy-3-furylalanine.
A reaction for synthesizing (S) -β-monoamino-3-furylalanine from L-monoamino-3-furylalanine.
A reaction for synthesizing (S) -β-diamino-3-furylalanine from L-diamino-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkyl-3-furylalanine from L-monoalkyl-3-furylalanine.
A reaction for synthesizing (S) -β-dialkyl-3-furylalanine from L-dialkyl-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxy-3-furylalanine from L-monoalkoxy-3-furylalanine.
A reaction for synthesizing (S) -β-dialkoxy-3-furylalanine from L-dialkoxy-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoalkyl-3-furylalanine from L-monoalkoxymonoalkyl-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonoamino-3-furylalanine from L-monoalkoxymonoamino-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohalogeno-3-furylalanine from L-monoalkoxymonohalogeno-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymonohydroxy-3-furylalanine from L-monoalkoxymonohydroxy-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkoxymononitro-3-furylalanine from L-monoalkoxymononitro-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmonoamino-3-furylalanine from L-monoalkylmonoamino-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohalogeno-3-furylalanine from L-monoalkylmonohalogeno-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmonohydroxy-3-furylalanine from L-monoalkylmonohydroxy-3-furylalanine.
A reaction for synthesizing (S) -β-monoalkylmononitro-3-furylalanine from L-monoalkylmononitro-3-furylalanine.
A reaction for synthesizing (S) -β-monoaminomonohalogeno-3-furylalanine from L-monoaminomonohalogeno-3-furylalanine.
A reaction for synthesizing (S) -β-monoaminomonohydroxy-3-furylalanine from L-monoaminomonohydroxy-3-furylalanine.
A reaction for synthesizing (S) -β-monoaminomononitro-3-furylalanine from L-monoaminomononitro-3-furylalanine.
A reaction for synthesizing (S) -β-monohalogenomonohydroxy-3-furylalanine from L-monohalogenomonohydroxy-3-furylalanine.
A reaction for synthesizing (S) -β-monohalogenomononitro-3-furylalanine from L-monohalogenomononitro-3-furylalanine.
A reaction for synthesizing (S) -β-monohydroxymononitro-3-furylalanine from L-monohydroxymononitro-3-furylalanine.

Specifically, the S-form selective phenylalanine aminomutase of this embodiment catalyzes the reaction exemplified below to synthesize the corresponding (S) -β-phenylalanine analogue from the L-phenylalanine analogue.
A reaction for synthesizing (S) -β-2-fluorophenylalanine from L-2-fluorophenylalanine.
A reaction for synthesizing (S) -β-3-fluorophenylalanine from L-3-fluorophenylalanine.
A reaction for synthesizing (S) -β-4-fluorophenylalanine from L-4-fluorophenylalanine.
A reaction for synthesizing (S) -β-2,4-difluorophenylalanine from L-2,4-difluorophenylalanine. A reaction for synthesizing (S) -β-2-chlorophenylalanine from L-2-chlorophenylalanine.
A reaction for synthesizing (S) -β-3-chlorophenylalanine from L-3-chlorophenylalanine.
A reaction for synthesizing (S) -β-4-chlorophenylalanine from L-4-chlorophenylalanine.
A reaction for synthesizing (S) -β-2,4-dichlorophenylalanine from L-2,4-dichlorophenylalanine.
A reaction for synthesizing (S) -β-2-bromophenylalanine from L-2-bromophenylalanine.
A reaction for synthesizing (S) -β-3-bromophenylalanine from L-3-bromophenylalanine.
A reaction for synthesizing (S) -β-4-bromophenylalanine from L-4-bromophenylalanine.
A reaction for synthesizing (S) -β-2,4-dibromophenylalanine from L-2,4-dibromophenylalanine.
A reaction for synthesizing (S) -β-2-nitrophenylalanine from L-2-nitrophenylalanine.
A reaction for synthesizing (S) -β-3-nitrophenylalanine from L-3-nitrophenylalanine.
A reaction for synthesizing (S) -β-4-nitrophenylalanine from L-4-nitrophenylalanine.
A reaction for synthesizing (S) -β-2-hydroxyphenylalanine from L-2-hydroxyphenylalanine.
A reaction for synthesizing (S) -β-3-hydroxyphenylalanine from L-3-hydroxyphenylalanine.
A reaction for synthesizing (S) -β-4-hydroxyphenylalanine from L-4-hydroxyphenylalanine.
A reaction for synthesizing (S) -β-2-methylphenylalanine from L-2-methylphenylalanine.
A reaction for synthesizing (S) -β-3-methylphenylalanine from L-3-methylphenylalanine.
A reaction for synthesizing (S) -β-4-methylphenylalanine from L-4-methylphenylalanine.
A reaction for synthesizing (S) -β-2-ethylphenylalanine from L-2-ethylphenylalanine.
A reaction for synthesizing (S) -β-3-ethylphenylalanine from L-3-ethylphenylalanine.
A reaction for synthesizing (S) -β-4-ethylphenylalanine from L-4-ethylphenylalanine.
A reaction for synthesizing (S) -β-2-propylphenylalanine from L-2-propylphenylalanine.
A reaction for synthesizing (S) -β-3-propylphenylalanine from L-3-propylphenylalanine.
A reaction for synthesizing (S) -β-4-propylphenylalanine from L-4-propylphenylalanine.
A reaction for synthesizing (S) -β-2-methoxyphenylalanine from L-2-methoxyphenylalanine.
A reaction for synthesizing (S) -β-3-methoxyphenylalanine from L-3-methoxyphenylalanine.
A reaction for synthesizing (S) -β-4-methoxyphenylalanine from L-4-methoxyphenylalanine.
A reaction for synthesizing (S) -β-2-ethoxyphenylalanine from L-2-ethoxyphenylalanine.
-Reaction which synthesize | combines (S)-(beta) -3-ethoxyphenylalanine from L-3-ethoxyphenylalanine.
A reaction for synthesizing (S) -β-4-ethoxyphenylalanine from L-4-ethoxyphenylalanine.
A reaction for synthesizing (S) -β-2-pyridylalanine from L-2-pyridylalanine.
A reaction for synthesizing (S) -β-3-pyridylalanine from L-3-pyridylalanine.
A reaction for synthesizing (S) -β-4-pyridylalanine from L-4-pyridylalanine.
A reaction for synthesizing (S) -β-2-thienylalanine from L-2-thienylalanine.
A reaction for synthesizing (S) -β-3-thienylalanine from L-3-thienylalanine.
A reaction for synthesizing (S) -β-2-furylalanine from L-2-furylalanine.
-Reaction which synthesize | combines (S)-(beta) -3-furylalanine from L-3-furylalanine.

  In this specification, the S-form selective phenylalanine aminomutase refers to a protein having S-form selective phenylalanine aminomutase activity. The “S-form selective phenylalanine aminomutase activity” means that β-phenylalanine can be synthesized in an S-form selective manner. “S-form selective” means that the optical purity of (S) -β-phenylalanine contained in β-phenylalanine is 50% ee or more, preferably 80% ee or more, Desirably, it is 90% ee or more, and it is most desirable that it is 99% ee or more.

The S-isomer-selective phenylalanine aminomutase according to the present embodiment has the following physicochemical properties.
(1) Action: Synthesizes (S) -β-phenylalanine from L-phenylalanine or (S) -β-phenylalanine from cinnamic acid and ammonia.

(2) Optimal pH: pH 8.0 to 9.5
When the enzyme activity in Tris-HCl buffer (pH 9.0) is 100%, the relative activity of the enzyme is 80% or more when the pH is about 8.0 to 9.5, and the pH is about 8.2 to 8.2. When 9.2, the relative activity of the enzyme is 90% or more.

  (3) Molecular weight: 57,000 ± 5,000 (SDS-polyacrylamide gel electrophoresis)

  The S-selective phenylalanine aminomutase according to this embodiment has the amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence represented by SEQ ID NO: 1 may have an amino acid sequence in which one to a plurality of amino acids are substituted, deleted, inserted or added.

  In this specification, the term “plurality” varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein, but specifically 2 to 30, preferably 2 to 15, more preferably 2 to 2. 10 pieces.

  In addition, the S-isomer-selective phenylalanine aminomutase according to this embodiment has a homology of 70% or more, desirably 80% or more, more desirably 85% or more, and most desirably 90% or more with the amino acid sequence represented by SEQ ID NO: 1. It may be a protein having an amino acid sequence or a partial fragment thereof.

  Protein homology search is based on, for example, databases related to amino acid sequences of proteins such as SWISS-PROT and PIR, databases related to DNA nucleotide sequences such as DNA Data Bank of JAPAN (DDBJ), EMBL and GenBank, and DNA base sequences. For example, it can be performed on the Internet using a FASTA program, a BLAST program, or the like for a database related to the predicted amino acid sequence.

The S-form selective phenylalanine aminomutase according to this embodiment can be obtained from an S-form selective phenylalanine aminomutase-producing organism. Examples of S-selective phenylalanine aminomutase-producing organisms include Pantoea agglomerans strain Eh335 (Non-patent Document 3).
Alternatively, a host cell may be transformed with DNA encoding S-selective phenylalanine aminomutase to express and isolate S-selective phenylalanine aminomutase. According to this method, S-form selective phenylalanine aminomutase can be easily and efficiently obtained.

(Second Embodiment)
The second embodiment of the present invention is DNA encoding S-selective phenylalanine aminomutase. This DNA is any of the following DNAs (a) to (d).
(A) DNA encoding the S-isomer-selective phenylalanine aminomutase according to the first embodiment,
(B) DNA having a base sequence represented by SEQ ID NO: 3 and encoding a protein having S-selective phenylalanine aminomutase activity,
(C) a protein having a base sequence in which one to a plurality of bases are substituted, deleted, inserted or added in the base sequence represented by SEQ ID NO: 3 and having S-selective phenylalanine aminomutase activity DNA to encode,
(D) A DNA that hybridizes with a DNA having the base sequence shown in SEQ ID NO: 3 under stringent conditions and encodes a protein having S-selective phenylalanine aminomutase activity.

The DNA according to this embodiment is a protein that does not show a significant difference in enzyme activity even if there is a slight difference in amino acid sequence from the S-selective phenylalanine aminomutase according to the first embodiment. It may contain DNA that encodes.
Specifically, a DNA encoding a protein having an enzyme activity substantially equivalent to that of the S-isomer-selective phenylalanine aminomutase according to the first embodiment, although there are slight differences in amino acid sequences due to DNA polymorphisms and mutations. Is mentioned.

  In addition to those described above, the DNA encoding S-selective phenylalanine aminomutase may be DNA having the base sequence shown in SEQ ID NO: 2. Further, it hybridizes under stringent conditions with a DNA having a base sequence represented by base numbers 1 to 1626 in SEQ ID NO: 2 and a base sequence represented by base numbers 24 to 1649 in SEQ ID NO: 3 or a complementary sequence thereof, at least 20 bases. It may be a DNA encoding a protein that is soybean and has S-selective phenylalanine aminomutase activity. Any DNA may be used as long as the DNA has the above base sequence.

  The DNA having the base sequence shown in SEQ ID NO: 2 is the AdmH gene (Genbank accession number: AY192157) derived from Pantoea agglomerans strain Eh335 (Genbank accession number: AY192157). The base sequences shown in base numbers 24-1649 in SEQ ID NO: 3 The base sequence of the AdmH gene was changed to a codon suitable for Escherichia coli for expressing the base sequence so that the expression efficiency in Escherichia coli host cells was improved. For changing to a codon suitable for the host cell, for example, DNA Designer (DNA2.0, Inc.) software: Gene Designer can be used.

  DNA encoding S-selective phenylalanine aminomutase may be synthesized, for example, chemically and / or genetically. The synthesis can be outsourced to a general DNA synthesis contractor. It can also be extracted from Pantoea agglomerans strain Eh335 (Pantoea agglomerans strain Eh335) (Non-patent Document 3). Furthermore, it is also possible to obtain DNA encoding S-selective phenylalanine aminomutase from an organism producing S-selective phenylalanine aminomutase by using a normal PCR method or hybridization method.

Further, by introducing a mutation including substitution, deletion, insertion or addition into DNA having the nucleotide sequence shown in nucleotide numbers 1-1626 in SEQ ID NO: 2 or the nucleotide sequence shown in nucleotide numbers 24-1649 in SEQ ID NO: 3. A DNA encoding a protein having an amino acid sequence including substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 and having S-selective phenylalanine aminomutase activity It is also possible to obtain
As a method for introducing a mutation including substitution, deletion, insertion or addition into DNA, a site-specific mutagenesis method or the like can be used. In addition, the number of bases for introducing a mutation including substitution, deletion, insertion or addition into DNA is within a range that can hybridize under stringent conditions, and there is a large difference in the enzyme activity of the encoded protein. It suffices if it is within the range not allowed.

  “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNA having high homology, that is, complementary strands of DNA having 50% or more, preferably 70% or more, more desirably 80% or more, and most desirably 90% or more, hybridizes and is more homologous. A condition in which a complementary strand of a nucleic acid having a low value does not hybridize.

More specifically, the hybridization under the stringent conditions can be exemplified by conditions commonly used by those skilled in the art to detect a specific hybridization signal. For example, it can be performed by the method described in Molecular Cloning: Cold Spring Harbor Laboratory Press, Current Protocols in Molecular Biology; Wiley Interscience, and a commercially available system is a Gene Image system (Amarsham). Specifically, hybridization can be performed by the following operation. The membrane to which the DNA or RNA to be tested is transferred is hybridized with a labeled probe in a hybridization buffer specified in the protocol according to the product protocol. The composition of the hybridization buffer consists of 0.1 wt% SDS, 5 wt% dextran sulfate, 1/20 of the total volume of blocking reagent attached to the kit, and 5 x SSC. The blocking reagent is, for example, 100 × Denhardt's solution (2% (weight / volume) Bovine serum albumin, 2% (weight / volume) Ficoll ^™ 400, 2% (weight / volume) polyvinylpyrrolidone). / 20 can be used after diluting. The hybridization temperature is in the range of 40 ° C. or higher and 80 ° C. or lower, more preferably 50 ° C. or higher and 70 ° C. or lower. After incubation for several hours to one day, the plate is washed with a washing buffer. The washing temperature is preferably room temperature, more preferably the temperature during hybridization. The composition of the washing buffer is 6 × SSC + 0.1 wt% SDS solution, more preferably 4 × SSC + 0.1 wt% SDS solution, and further preferably 1 × SSC + 0.1 wt% SDS solution. The membrane can be washed with such a washing buffer, and the DNA or RNA hybridized with the probe can be identified using the label used for the probe.

  In order to determine the homology between two amino acid sequences or base sequences, the sequences are preprocessed to the optimal state for comparison. For example, by making a gap in one sequence, the alignment with the other sequence is optimized. Thereafter, the amino acid residues or bases at each site are compared. When the same amino acid residue or base as the site corresponding to the second sequence is present at a site in the first sequence, the sequences are identical at that site. Homology between the two sequences is shown as a percentage of the total number of sites (total amino acids or total bases) of the number of sites that are identical between the sequences.

(Third embodiment)
The third embodiment of the present invention is a recombinant DNA having DNA encoding S-selective phenylalanine aminomutase. This recombinant DNA is obtained by incorporating the DNA according to the second embodiment into a vector.

The recombinant DNA of this embodiment can be obtained by incorporating DNA encoding S-selective phenylalanine aminomutase into a vector.
As a vector for cloning, a vector constructed for gene recombination from a phage or plasmid capable of autonomously growing in a host cell is suitable. Examples of the phage include Lambda gt10 and Lambda gt11 when Escherichia coli is used as a host cell. As the plasmid, for example, when Eschenchia coli is used as a host cell, pBTrp2, pBTac1, pBTac2 (Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen X-1), pGEM-1 Promega), pQE-8, pQE-30 (QIAGEN), pBluescript II SK (+), pBluescript II SK (-) (Stratagene), pET-3 (Novagen), pUC18, pSTV28, pST Examples thereof include pUC118 (Takara Shuzo).

  Any promoter can be used as long as it can be expressed in the host cell. For example, promoters derived from Escherichia coli or phages such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PSE promoter and the like can be mentioned. In addition, artificially modified promoters such as tac promoter and lacT7 promoter can also be used. Further, an Np promoter (Japanese Patent Publication No. 8-24586) can be used for expression in bacteria belonging to the genus Bacillus.

Any ribosome binding sequence can be used as long as it can be expressed in the host cell, but the distance between the Shine-Dalgarno sequence and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases). It is preferable to use the prepared plasmid.
In order to efficiently perform transcription / translation, a protein in which the N-terminal or part of the protein having S-selective phenylalanine aminomutase activity is deleted and the N-terminal part of the protein encoded by the expression vector are fused. It may be expressed.
A transcription termination sequence is not necessarily required for the expression of the target protein, but it is desirable to place the transcription termination sequence directly under the structural gene.

  At the time of cloning, a vector DNA fragment can be obtained by cleaving the above-mentioned vector with the restriction enzyme used for cleaving the DNA encoding the S-isomer-selective phenylalanine aminomutase described above. It is not necessary to use the same restriction enzyme as that used in the above. The DNA fragment and the vector DNA fragment may be combined by any method using a known DNA ligase. For example, after annealing the sticky end of the DNA fragment and the sticky end of the vector DNA fragment, an appropriate DNA ligase is used. To produce a recombinant DNA of the DNA fragment and the vector DNA fragment. If necessary, after annealing, it can be transferred to a host such as a microorganism and recombinant DNA can be produced using in vivo DNA ligase.

(Fourth embodiment)
The fourth embodiment of the present invention is a transformant.
This transformant is a transformant having the DNA according to the second embodiment or the recombinant DNA according to the third embodiment.

  Any host organism can be used as a transformant as long as the recombinant DNA can be stably and autonomously propagated and can express a trait of the foreign DNA. Examples of host organisms include Escherichia coli, but are not limited to Escherichia coli, and bacteria such as Escherichia bacteria, Bacillus subtilis bacteria, Pseudomonas bacteria, Saccharomyces Yeasts such as Genus, Pichia and Candida, and filamentous fungi such as Aspergillus can be used.

  As a method for transferring the recombinant DNA into the host organism, for example, when the host cell is Escherichia coli, a competent cell method using calcium treatment, an electroporation method, or the like can be used. By culturing the transformant thus obtained, a large amount of S-selective phenylalanine aminomutase can be stably produced.

  Recombinant DNA carrying DNA encoding S-selective phenylalanine aminomutase can be removed from the transformant and can be transferred to other microorganisms. In addition, a recombinant DNA having a DNA encoding S-selective phenylalanine aminomutase is amplified with a DNA fragment encoding S-selective phenylalanine aminomutase using PCR, treated with a restriction enzyme, etc. And can be easily transferred to a host organism.

  As the medium, any of a synthetic medium or a natural medium can be used as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance and other nutrients. Culturing can be performed in a liquid medium containing the above-described culture components by using a normal culture method such as shaking culture, aeration-agitation culture, continuous culture, or fed-batch culture.

The culture conditions may be appropriately selected depending on the type of culture medium and the culture method, and are not particularly limited as long as the strain can grow and produce S-selective phenylalanine aminomutase.
For example, when the culture is performed under aerobic conditions and the pH and temperature are appropriately controlled within the range of 6 to 8 ° C. and the temperature within the range of 25 ° C. to 40 ° C., the time required for the cultivation is 48 hours or less. .

  The S-form-selective phenylalanine aminomutase produced by the transformant can be used by collecting a culture solution containing the transformant in the culture as it is. In addition, when S-selective phenylalanine aminomutase is present in the culture solution, the S-selective phenylalanine aminomutase-containing solution and the transformant can be separated by filtration, centrifugation, or the like. When S-selective phenylalanine aminomutase is present in the transformant, the transformant can be collected from the obtained culture by means of filtration or centrifugation and used. In addition, the collected transformant is destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and / or a surfactant is added to the S-form selective phenylalanine. Aminomutase can be solubilized and separated and collected as a solution.

  The S-selective phenylalanine aminomutase-containing solution thus obtained is concentrated under reduced pressure, membrane concentration, salting out such as ammonium sulfate or sodium sulfate, or fractionation with a hydrophilic organic solvent such as methanol, ethanol or acetone. It can be precipitated by a precipitation method. Heat treatment and isoelectric point treatment are also effective purification means. Thereafter, purified S-isomer-selective phenylalanine aminomutase can be obtained by performing gel filtration with an adsorbent or gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography.

(Fifth embodiment)
The fifth embodiment of the present invention is a method for producing (S) -β-phenylalanine. (S) -β-phenylalanine is produced from L-phenylalanine in the presence of the S-form-selective phenylalanine aminomutase according to the first embodiment, the transformant according to the fourth embodiment or a processed product thereof.

  The processed product of the transformant is for the purpose of cell destruction, mechanically disrupting the transformant, ultrasonic treatment, freeze-thaw treatment, drying treatment, pressurization or decompression treatment, osmotic treatment, autolysis, surfactant It can be obtained as an immobilized product of a fraction or a transformant containing an S-selective phenylalanine aminomutase obtained by treatment, enzyme treatment, and S treatment.

  The reaction conditions are not particularly limited, but the reaction is preferably carried out while appropriately controlling the pH and temperature. For example, the pH is 6 or more and 10 or less, preferably 7 or more and 10 or less. The temperature is preferably in the range of 4 ° C. or more and 60 ° C. or less, and the reaction is preferably performed under shaking or stirring conditions.

  The amount of the transformant or the processed product of the transformant is not particularly limited as long as the reaction with L-phenylalanine proceeds sufficiently, but it is usually at least 1 unit, preferably 10 units or more with respect to 10 g of L-phenylalanine. It is desirable to add an amount that provides S-selective phenylalanine aminomutase activity. As a method for adding the transformant or the processed product of the transformant, it may be added all at once at the start of the reaction, or may be divided or continuously added during the reaction.

The ability of S-selective phenylalanine aminomutase activity to synthesize 1 μmol of (S) -β-phenylalanine per minute is defined as 1 unit.
The S-isomer-selective phenylalanine aminomutase activity was obtained by adding an enzyme solution to 0.1 mol / l Tris-HCl buffer (pH 8.0) containing 25 mmol / l L-phenylalanine and keeping the temperature at 25 ° C. (S) − The amount of β-phenylalanine produced is measured and calculated.

  A part of L-phenylalanine in the reaction solution only needs to be dissolved in the reaction medium, and is not necessarily completely dissolved in the reaction solution medium. Regarding the method of adding L-phenylalanine, it may be added all at once at the start of the reaction, or may be added separately or continuously as the reaction proceeds.

  As a medium for the reaction liquid, water, an aqueous medium, an organic solvent, or a mixed liquid of water or an aqueous medium and an organic solvent is used. Examples of the aqueous medium include a buffer solution such as a phosphate buffer solution, a CHES [N-cyclohexyl-2-aminoethanesulfonic acid] buffer solution, and a tris [tris (hydroxymethyl) aminomethane] hydrochloric acid buffer solution. Any organic solvent may be used as long as it does not inhibit the reaction.

  Separation and purification of (S) -β-phenylalanine from the reaction solution is performed by a method used in ordinary organic synthetic chemistry, for example, extraction with an organic solvent, crystallization, thin layer chromatography, high performance liquid chromatography, etc. Can do.

(Sixth embodiment)
The sixth embodiment of the present invention is also a method for producing (S) -β-phenylalanine. (S) -β-phenylalanine is produced from cinnamic acid and ammonia in the presence of the S-form-selective phenylalanine aminomutase according to the first embodiment, the transformant according to the fourth embodiment or a processed product thereof.

  In the fifth embodiment, L-phenylalanine is used as a raw material that becomes a substrate of the S-selective phenylalanine aminomutase, but in this embodiment, cinnamic acid and ammonia are reacted instead of L-phenylalanine. In the present embodiment, only differences from the contents described in the fifth embodiment will be described, and description of contents to which the fifth embodiment can be applied will be omitted.

The production of (S) -β-phenylalanine is preferably carried out under shaking or stirring conditions at a pH of 7 to 11, desirably 8 to 10, and a temperature of 4 ° C. to 60 ° C.
The amount of the transformant or the treated product of the transformant is not particularly limited as long as the reaction between cinnamic acid and ammonia proceeds sufficiently. Usually, it is at least 1 unit, preferably 10 units or more with respect to 10 g of cinnamic acid. It is desirable to add an amount of S-selective phenylalanine aminomutase activity.

  Examples of the ammonia source to be added to the reaction solution include gaseous ammonia, ammonia aqueous solution or alcohol solution, ammonium acetate, ammonium carbonate, ammonium chloride, ammonium sulfate, or ammonium hydroxide. A part of cinnamic acid or ammonia in the reaction solution may be dissolved in the reaction medium, and does not necessarily have to be completely dissolved in the reaction solution medium. Regarding the method for adding cinnamic acid or ammonia, it may be added all at once at the start of the reaction, or may be divided or continuously added as the reaction proceeds.

  As the medium for the reaction liquid, the same medium as described in the fifth embodiment can be used.

(Seventh embodiment)
The seventh embodiment of the present invention is a method for producing an (S) -β-phenylalanine analog. According to the method of the present embodiment, the corresponding (S) -β-phenylalanine analog is produced from the L-phenylalanine analog in the presence of the S-form selective phenylalanine aminomutase according to the first embodiment. The (S) -β-phenylalanine analog can also be produced from the L-phenylalanine analog in the presence of the transformant according to the fourth embodiment or a processed product thereof.

  Specifically, the present embodiment is a method for producing an (S) -β-phenylalanine analog represented by the following general formulas (1) to (3).

  In formula (1), Q represents an optionally substituted phenyl group, pyridyl group, thienyl group or furyl group. X, Y, and Z each independently represent a substituent substituted with Q, and may be substituted with a hydrogen atom, a fluoro group, a chloro group, a bromo group, a nitro group, a hydroxyl group, or an alkyl group having 1 to 3 carbon atoms. A good amino group, an alkyl group having 1 to 3 carbon atoms, or an alkoxy group having 1 to 3 carbon atoms is represented.

  In formula (2), Q represents an optionally substituted phenyl group, pyridyl group, thienyl group or furyl group. X and Y each independently represent a substituent substituted with Q, and each represents a hydrogen atom, a fluoro group, a chloro group, a bromo group, a nitro group, a hydroxyl group, an amino group, a methyl group, or a methoxy group.

  In the formula (3), Q is a phenyl group, 2-fluorophenyl group, 3-fluorophenyl group, 4-fluorophenyl group, 2-chlorophenyl group, 3-chlorophenyl group, 4-chlorophenyl group, 2,4-dichlorophenyl group. 4-bromophenyl group, 4-nitrophenyl group, 4-hydroxyphenyl group, 2-methylphenyl group, 3-methylphenyl group, 4-methylphenyl group, 4-methoxyphenyl group, 3-pyridyl group, 2- Represents a thienyl group or a 2-furyl group.

Specifically, the following method is illustrated as a manufacturing method of this embodiment.
A method for producing (S) -β-2-fluorophenylalanine from L-2-fluorophenylalanine.
A method for producing (S) -β-3-fluorophenylalanine from L-3-fluorophenylalanine.
A method for producing (S) -β-4-fluorophenylalanine from L-4-fluorophenylalanine.
A method for producing (S) -β-2,4-difluorophenylalanine from L-2,4-difluorophenylalanine.
A method for producing (S) -β-2-chlorophenylalanine from L-2-chlorophenylalanine.
A method for producing (S) -β-3-chlorophenylalanine from L-3-chlorophenylalanine.
A method for producing (S) -β-4-chlorophenylalanine from L-4-chlorophenylalanine.
A method for producing (S) -β-2,4-dichlorophenylalanine from L-2,4-dichlorophenylalanine.
A method for producing (S) -β-2-bromophenylalanine from L-2-bromophenylalanine.
A method for producing (S) -β-3-bromophenylalanine from L-3-bromophenylalanine.
A method for producing (S) -β-4-bromophenylalanine from L-4-bromophenylalanine.
A method for producing (S) -β-2,4-dibromophenylalanine from L-2,4-dibromophenylalanine.
A method for producing (S) -β-2-nitrophenylalanine from L-2-nitrophenylalanine.
A method for producing (S) -β-3-nitrophenylalanine from L-3-nitrophenylalanine.
A method for producing (S) -β-4-nitrophenylalanine from L-4-nitrophenylalanine.
A method for producing (S) -β-2-hydroxyphenylalanine from L-2-hydroxyphenylalanine.
A method for producing (S) -β-3-hydroxyphenylalanine from L-3-hydroxyphenylalanine.
A method for producing (S) -β-4-hydroxyphenylalanine from L-4-hydroxyphenylalanine.
A method for producing (S) -β-2-methylphenylalanine from L-2-methylphenylalanine.
A method for producing (S) -β-3-methylphenylalanine from L-3-methylphenylalanine.
A method for producing (S) -β-4-methylphenylalanine from L-4-methylphenylalanine.
A method for producing (S) -β-2-ethylphenylalanine from L-2-ethylphenylalanine.
A method for producing (S) -β-3-ethylphenylalanine from L-3-ethylphenylalanine.
A method for producing (S) -β-4-ethylphenylalanine from L-4-ethylphenylalanine.
A method for producing (S) -β-2-propylphenylalanine from L-2-propylphenylalanine.
A method for producing (S) -β-3-propylphenylalanine from L-3-propylphenylalanine.
A method for producing (S) -β-4-propylphenylalanine from L-4-propylphenylalanine.
A method for producing (S) -β-2-methoxyphenylalanine from L-2-methoxyphenylalanine.
A method for producing (S) -β-3-methoxyphenylalanine from L-3-methoxyphenylalanine.
A method for producing (S) -β-4-methoxyphenylalanine from L-4-methoxyphenylalanine.
A method for producing (S) -β-2-ethoxyphenylalanine from L-2-ethoxyphenylalanine.
A method for producing (S) -β-3-ethoxyphenylalanine from L-3-ethoxyphenylalanine.
A method for producing (S) -β-4-ethoxyphenylalanine from L-4-ethoxyphenylalanine.
A method for producing (S) -β-2-pyridylalanine from L-2-pyridylalanine.
A method for producing (S) -β-3-pyridylalanine from L-3-pyridylalanine.
A method for producing (S) -β-4-pyridylalanine from L-4-pyridylalanine.
A method for producing (S) -β-2-thienylalanine from L-2-thienylalanine.
A method for producing (S) -β-3-thienylalanine from L-3-thienylalanine.
A method for producing (S) -β-2-furylalanine from L-2-furylalanine.
A method for producing (S) -β-3-furylalanine from L-3-furylalanine.

  In 5th Embodiment, although L-phenylalanine was used as a raw material used as the substrate of S body selective phenylalanine aminomutase, it replaces with L-phenylalanine and makes the L-phenylalanine analog react in this embodiment. In the present embodiment, only differences from the contents described in the fifth embodiment will be described, and description of contents to which the fifth embodiment can be applied will be omitted.

  The (S) -β-phenylalanine analog is preferably produced at a pH of 6 to 10, preferably 7 to 10 and a temperature of 4 ° C. to 60 ° C. under shaking or stirring conditions. The amount of the transformant or the processed product of the transformant is not particularly limited as long as the S-form-selective phenylalanine aminomutase sufficiently acts on the L-phenylalanine analog. Usually, the amount used is 10 g of the L-phenylalanine analog. On the other hand, it is desirable to add at least 1 unit, preferably 10 units or more of an amount that provides S-selective phenylalanine aminomutase activity.

  The L-phenylalanine analog in the reaction solution only needs to be partially dissolved in the reaction medium, and does not necessarily have to be completely dissolved in the reaction solution medium. Regarding the method for adding the L-phenylalanine analog, it may be added all at once at the start of the reaction, or may be divided or continuously added as the reaction proceeds.

  As the medium for the reaction liquid, the same medium as described in the fifth embodiment can be used.

(Analysis conditions)
(S) -β-phenylalanine, (S) -β-phenylalanine analog, (R) -β-phenylalanine, (R) -β-phenylalanine analog, L-phenylalanine, L-phenylalanine analog and cinnamic acid are fast Quantified by liquid chromatography. These analysis conditions are as follows.

(1) Analysis conditions for (S) -β-phenylalanine, (S) -β-phenylalanine analog, (R) -β-phenylalanine and (R) -β-phenylalanine analog (1) The purpose is to measure the optical purity of β-phenylalanine and β-phenylalanine analogues.
Column: CHIRALPAK WH 4.6 × 250 (Daicel Chemical Industries, Ltd.)
Column temperature: 50 ° C
Pump flow rate; 1.5 ml / min
Eluent: 2 mmol / l copper sulfate detection; UV 254 nm

(2) Analysis conditions for β-phenylalanine, β-phenylalanine analog, L-phenylalanine, L-phenylalanine analog, and cinnamic acid (2) are mainly for the purpose of measuring enzyme activity or reaction yield.
Column; Develosil TMS-UG-5 4.6 × 250 (Nomura Chemical Co., Ltd.)
Column temperature: 40 ° C
Pump flow rate; 1.0 ml / min
Eluent: 5 mmol / l citrate buffer (pH 6.0): methanol = 8: 2 (v / v)
Detection; UV254nm

(Evaluation method)
The optical purities of (S) -β-phenylalanine and (S) -β-phenylalanine analogs were calculated from the peak area values of chromatograms obtained under the analytical conditions shown in (1) above.
The reaction yields of (S) -β-phenylalanine and (S) -β-phenylalanine analogs were calculated from the peak area values of chromatograms obtained under the analytical conditions shown in (2) above.

Example 1
<Synthesis of DNA encoding S-selective phenylalanine aminomutase>
DNA having the base sequence shown in SEQ ID NO: 3 was synthesized by consigning to DNA2.0. The synthetic DNA had EcoRI and HindIII restriction enzyme recognition sequences near the 5 ′ end and the 3 ′ end, respectively.

(Example 2)
<Production of recombinant DNA>
A recombinant plasmid was prepared as recombinant DNA.
The DNA synthesized in Example 1 and the plasmid pUC18 were digested with EcoRI and HindIII and ligated using Ligation High (manufactured by Toyobo Co., Ltd.), and then Escherichia coli DH5α (Toyobo Co., Ltd.) was used using the resulting recombinant plasmid. The product was transformed. The transformant was cultured in an LB agar medium containing 100 μg / ml of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), and was Am-resistant and white colony. The resulting transformant was obtained. A plasmid was extracted from the transformant thus obtained.
In accordance with the usual method for determining the base sequence, it was confirmed that the base sequence of the DNA fragment introduced into the plasmid was the base sequence shown in SEQ ID NO: 3.
The obtained plasmid having DNA encoding S-selective phenylalanine aminomutase was named pSPAM.

(Example 3)
<Production and expression of transformant>
Escherichia coli DH5α was transformed with pSPAM by a conventional method, and the resulting transformant was designated as MT-11046.
The transformant was inoculated into 100 ml of LB medium containing 100 μg / ml of Am in a 500 ml baffled Erlenmeyer flask, shake-cultured at 30 ° C. until OD (660 nm) became 0.5, and then IPTG (isopropyl-β -Thiogalactopyranoside) was added to 1 mmol / l, and the mixture was further cultured with shaking for 16 hours. The culture solution was centrifuged at 8000 rpm for 20 minutes, and the resulting cells were suspended in 0.1 mol / l Tris-HCl buffer (pH 8.0) and stored frozen at -20 ° C.

Example 4
<Confirmation of expression by SDS-polyacrylamide gel electrophoresis>
1.0 ml of the suspension of the transformant prepared in Example 3 was crushed in ice water for 5 minutes using a bioraptor (Olympus). The processed product of the obtained transformant was centrifuged, and the obtained supernatant was used as a soluble fraction. Moreover, what added 1.0 ml of 0.1 mol / l Tris hydrochloric acid buffer (pH 8.0) to the deposit was made into the insoluble fraction. The soluble fraction and the insoluble fraction were each analyzed by SDS-polyacrylamide gel electrophoresis. In SDS-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel is NuPAGE 4-12% Bis-Tris gel (manufactured by Invitrogen), NuPAGE MOPS SDS running buffer (manufactured by Invitrogen) is used as the running buffer, and SeeBlue Plus2 is used as the molecular weight marker. Invitrogen) was used. The band of S-selective phenylalanine aminomutase was confirmed at a position between the molecular weight markers of 51,000 and 64,000 in the lane of the soluble fraction, and was estimated to have a molecular weight of about 55,000. Therefore, it was shown that the S-form selective phenylalanine aminomutase produced by the prepared transformant is in the range of molecular weight 57,000 ± 5000. On the other hand, this band was not confirmed in the lane of the insoluble fraction.

(Example 5)
<Purification of S-isomer-selective phenylalanine aminomutase>
The cells obtained in the same manner as in Example 3 were suspended in 20 mmol / l Tris-HCl buffer (pH 8.0) and crushed at 0 ° C. for 3 minutes and 30 seconds using a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.). . The disrupted solution was centrifuged, and the resulting supernatant was treated with Benzonase Nuclease (Merck) to prepare a crude enzyme solution. The crude enzyme solution was adsorbed on an anion exchange resin (HiTrap Q XL, manufactured by Amersham), and 20 mmol / l Tris-HCl buffer (pH 8) containing 1 mol / l sodium chloride from 20 mmol / l Tris-HCl buffer (pH 8.0). Elution with a linear gradient to. The active fractions were collected, and the buffer solution was exchanged with a 50 mmol / l phosphate buffer solution (pH 8.0) containing 1 mol / l ammonium sulfate, and then adsorbed onto a hydrophobic chromatographic resin (HiTrap Butyl FF, manufactured by Amersham). Elution was performed with a linear gradient from 50 mmol / l phosphate buffer (pH 8.0) containing 1 ammonium sulfate to 50 mmol / l phosphate buffer (pH 8.0). The active fractions were collected, the buffer was exchanged with 20 mmol / l Tris-HCl buffer (pH 8.0), and then adsorbed on an anion exchange resin (HiTrap DEAE FF, Amersham), and 20 mmol / l Tris-HCl buffer. Elution was performed with a linear gradient from (pH 8.0) to 20 mmol / l Tris-HCl buffer (pH 8.0) containing 1 mol / l sodium chloride. The active fraction was fractionated with a 50 mmol / l phosphate buffer (pH 7.0) containing 0.2 mol / l sodium chloride using a gel filtration resin (TSKgel G3000SWXL, manufactured by Tosoh Corporation). The active fraction was collected, and the buffer solution was exchanged with 0.1 mol / l Tris-HCl buffer (pH 8.0) to obtain a purified enzyme preparation. In addition, the above operation was implemented at about 4 degreeC.

(Example 6)
<Optimum pH>
Enzyme activity at each pH was measured. The reaction solution (1.0 ml) contains 25 mmol / l L-phenylalanine, 0.1 mol / l of various buffer solutions and the purified enzyme preparation (appropriate amount) prepared in Example 5 and reacted at 25 ° C. for 30 minutes. It was. The result is as shown in FIG. 1, and the optimum pH was 8.0 to 9.5. Various buffer solutions used for the measurement are shown below. FIG. 1 shows pH on the horizontal axis and relative activity (%) on the vertical axis. The relative activity was determined by calculating the percentage of the enzyme activity in the Tris-HCl buffer (pH 9.0) with the enzyme activity in the Tris-HCl buffer (pH 9.0) being 100%.
Phosphate buffer solution: pH 6.0 to 8.0
Tris-HCl buffer: pH 7.5-9.0
CHES buffer: pH 9.0-10.0

(Example 7)
<Synthesis of (S) -β-phenylalanine using L-phenylalanine as a substrate>
The reaction solution (1.0 ml) was 10 mmol / l L-phenylalanine, 0.1 mol / l Tris-HCl buffer (pH 8.0) and MT-11046 supernatant prepared in the same manner as in Example 3 (appropriate The reaction was carried out at 30 ° C. for 24 hours. The reaction yield of (S) -β-phenylalanine was 36%, and the optical purity was 100% ee.

(Example 8)
<Synthesis of (S) -β-phenylalanine using cinnamic acid and ammonia as substrates>
The reaction solution (1.0 ml) contains 5 mmol / l cinnamic acid, 10% ammonium carbonate (pH 9.2) and MT-11046 supernatant solution (appropriate amount) prepared in the same manner as in Example 3. The reaction was allowed to proceed at 72 ° C. for 72 hours. The reaction yield of (S) -β-phenylalanine with respect to cinnamic acid was 5%, and the optical purity was 100% ee.

Example 9
<Synthesis of the corresponding (S) -β-phenylalanine analog from L-phenylalanine analog>
The reaction solution (1.0 ml) was 10 mmol / l L-phenylalanine analog (manufactured by Peptech) shown in Table 1, 0.1 mol / l Tris-HCl buffer (pH 8.0), and the same method as in Example 3. The supernatant (appropriate amount) of MT-11046 prepared in the above was included and reacted at 30 ° C. for 24 hours. The reaction yield of the corresponding (S) -β-phenylalanine analog was as shown in Table 1, and the optical purity was 100% ee. The reaction yield of the (S) -β-phenylalanine analog was obtained under the analytical conditions shown in (2) of (Analytical Conditions), and the optical purity was obtained under the analytical conditions shown in (1) of (Analytical Conditions). It calculated from the peak area value of the chromatogram. A reagent manufactured by Peptech was used for the preparation of the (S) -β-phenylalanine analog.

  As described above, according to the present invention, (S) -β-phenylalanine can be stereoselectively produced in one step. Therefore, (S) -β-phenylalanine can be produced efficiently at low cost.

  As mentioned above, although the structure of this invention was demonstrated, this invention is not restricted to this, Various aspects are included.

Claims (29)

  An S-selective phenylalanine aminomutase having an enzymatic activity for synthesizing (S) -β-phenylalanine from L-phenylalanine or an enzymatic activity for synthesizing (S) -β-phenylalanine from cinnamic acid and ammonia.   The S-selective phenylalanine aminomutase according to claim 1, which has an enzyme activity for synthesizing (S) -β-phenylalanine from L-phenylalanine.   The S-selective phenylalanine aminomutase according to claim 1, which has an enzyme activity for synthesizing (S) -β-phenylalanine from cinnamic acid and ammonia. S-selective phenylalanine aminomutase having the following physicochemical properties:
(1) Action: Synthesizes (S) -β-phenylalanine from L-phenylalanine or (S) -β-phenylalanine from cinnamic acid and ammonia.
(2) Optimal pH: pH 8.0 to 9.5
(3) Molecular weight: 57,000 ± 5,000 (SDS-polyacrylamide gel electrophoresis)   S-selective phenylalanine aminomutase having the amino acid sequence represented by SEQ ID NO: 1.   A S-selective phenylalanine aminomutase having an amino acid sequence in which one to a plurality of amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1.   DNA encoding the S-isomer-selective phenylalanine aminomutase according to any one of claims 1 to 6.   DNA which has the base sequence shown by sequence number 3, and codes the protein which has S body selection phenylalanine aminomutase activity.   DNA encoding a protein having a base sequence in which one to a plurality of bases are substituted, deleted, inserted, or added in the base sequence represented by SEQ ID NO: 3 and having S-selective phenylalanine aminomutase activity .   A DNA that hybridizes with a DNA having the base sequence represented by SEQ ID NO: 3 under stringent conditions and encodes a protein having S-selective phenylalanine aminomutase activity.   A recombinant DNA obtained by incorporating the DNA according to claim 7 into a vector.   Recombinant DNA obtained by incorporating the DNA according to claim 8 into a vector.   A transformant comprising the DNA according to claim 7.   A transformant comprising the DNA according to claim 8 or the recombinant DNA according to claim 12.   A transformant comprising the recombinant DNA according to claim 11.   The transformant according to claim 13, wherein the host organism is Escherichia coli.   The transformant according to claim 14, wherein the host organism is Escherichia coli.   The transformant according to claim 15, wherein the host organism is Escherichia coli.   A method for producing (S) -β-phenylalanine, comprising producing (S) -β-phenylalanine from L-phenylalanine in the presence of the S-isomer-selective phenylalanine aminomutase according to any one of claims 1 to 6. .   (S)-(beta) -phenylalanine is manufactured from L-phenylalanine in presence of the transformant of Claim 13, or its processed material, The manufacturing method of (S)-(beta) -phenylalanine characterized by the above-mentioned.   (S)-(beta) -phenylalanine is manufactured from L-phenylalanine in presence of the transformant of Claim 14, or its processed material, The manufacturing method of (S)-(beta) -phenylalanine characterized by the above-mentioned.   (S)-(beta) -phenylalanine is manufactured from L-phenylalanine in presence of the transformant of Claim 15, or its processed material, The manufacturing method of (S)-(beta) -phenylalanine characterized by the above-mentioned.   (S) -β-phenylalanine is produced from cinnamic acid and ammonia in the presence of the S-selective phenylalanine aminomutase according to any one of claims 1 to 6. Production method.   A method for producing (S) -β-phenylalanine, comprising producing (S) -β-phenylalanine from cinnamic acid and ammonia in the presence of the transformant according to claim 13 or a processed product thereof.   (S)-(beta) -phenylalanine is manufactured from a cinnamic acid and ammonia in presence of the transformant of Claim 14, or its processed material, The manufacturing method of (S)-(beta) -phenylalanine characterized by the above-mentioned.   A method for producing (S) -β-phenylalanine, comprising producing (S) -β-phenylalanine from cinnamic acid and ammonia in the presence of the transformant according to claim 15 or a processed product thereof. In the presence of the S-isomer selective phenylalanine aminomutase according to any one of claims 1 to 6, the general formula (1)

(In the formula (1), Q represents an optionally substituted phenyl group, pyridyl group, thienyl group or furyl group. X, Y, and Z each independently represent a substituent substituted with Q, and a hydrogen atom. , A fluoro group, a chloro group, a bromo group, a nitro group, a hydroxyl group, an amino group optionally substituted by an alkyl group having 1 to 3 carbon atoms, an alkyl group having 1 to 3 carbon atoms, or an alkoxy group having 1 to 3 carbon atoms A corresponding (S) -β-phenylalanine analog is produced from the L-phenylalanine analog represented by the formula (1). In the presence of the S-isomer-selective phenylalanine aminomutase according to any one of claims 1 to 6, the general formula (2)

(In the formula (2), Q represents an optionally substituted phenyl group, pyridyl group, thienyl group or furyl group. X and Y each independently represent a substituent substituted with Q, a hydrogen atom, fluoro A corresponding (S) -β-phenylalanine analog from the L-phenylalanine analog represented by the group, chloro group, bromo group, nitro group, hydroxyl group, amino group, methyl group or methoxy group. A process for producing an (S) -β-phenylalanine analog characterized by In the presence of the S-isomer-selective phenylalanine aminomutase according to any one of claims 1 to 6, the general formula (3)

(In the formula (3), Q is a phenyl group, 2-fluorophenyl group, 3-fluorophenyl group, 4-fluorophenyl group, 2-chlorophenyl group, 3-chlorophenyl group, 4-chlorophenyl group, 2,4-dichlorophenyl. Group, 4-bromophenyl group, 4-nitrophenyl group, 4-hydroxyphenyl group, 2-methylphenyl group, 3-methylphenyl group, 4-methylphenyl group, 4-methoxyphenyl group, 3-pyridyl group, 2 (S) -β-phenylalanine analog, wherein the corresponding (S) -β-phenylalanine analog is produced from the L-phenylalanine analog represented by -thienyl group or 2-furyl group. Manufacturing method.

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