KR101223665B1 - Method for preparing a carboxylic acid using nitrilase RMN2 - Google Patents

Abstract

The present invention relates to a novel nitrile and its use, and more specifically, the present invention is a nitrile represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 It relates to a method for producing a carboxylic acid comprising the step of reacting with a nitrile substrate, and a biocatalyst composition for preparing a carboxylic acid comprising the nitrile.

Description

Method for preparing carboxylic acid using nitrified RMN2 {Method for preparing a carboxylic acid using nitrilase RMN2}

The present invention relates to novel nitriles and their use. More specifically, the present invention provides a carboxylic acid comprising reacting a nitrile represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 with a nitrile substrate. It relates to a method for producing and a biocatalyst composition for producing carboxylic acid containing the nitrile.

In recent years, the use of optically active carboxylic acids and derivatives thereof in the pharmaceutical and pharmaceutical fields has increased significantly. In particular, the optically active carboxylic acid has been attracting more attention in recent years as it is known to have high usefulness as a raw material for the manufacture of medicines such as hyperlipidemia treatment, antiviral agent (AIDS treatment), anticancer agent, anticoagulant, heart disease treatment and Alzheimer's treatment. There is a need to establish a highly efficient biological manufacturing method.

The chemical process for the production of carboxylic acids has the disadvantage of producing racemic mixtures at various stages of reaction and at high temperatures and pressures, whereas the enzymatic reaction process using nitrilase is a one-step process. The production of optically active pharmaceutical intermediates (chiral drugs) from phototoxic toxic substances (nitrile compounds) has shown considerable potential both economically and economically. Thus, methods have been attempted to biochemically produce optically active carboxylic acids from nitriles using microorganisms and enzymes thereof (Banerjee et al., (2006) Appl. Microbiol. Biotechnol. 72: 77-87; Robertson et al., (2004) Appl. Environ.Microbiol. 70: 2429-2436; Rustler et al., (2008) Appl. Microbiol. Biotechnol. 80: 87-97). Most nitriles have three catalytic residues that are conserved, which are called catalytic traid motifs. Catalytic trivalent elements consist of glutamate (E), lysine (K) and cysteine (C) residues and are always conserved residues.

The present inventors have made efforts to develop novel nitriles that can produce carboxylic acids with high efficiency from various nitrile substrates. As a result, we can isolate and purify proteins that preserve catalytic trivalent elements from marine microorganisms. there was. Furthermore, the present inventors have completed the present invention by identifying that the proteins have nitrile activity capable of producing carboxylic acids from various nitrile substrates.

One object of the present invention is a carboxylic acid comprising the step of reacting a nitrile represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 with a nitrile substrate It is to provide a method of manufacturing.

Another object of the present invention to provide a biocatalyst composition for preparing a carboxylic acid comprising a nitrile represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 will be.

In one embodiment, the present invention provides a method for reacting a nitrile represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 or a biocatalyst composition comprising the same with a nitrile substrate It relates to a method of preparing a carboxylic acid comprising the step.

More specifically, the present invention comprises the steps of preparing a nitrile represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or a biocatalyst composition comprising the nitrile ; And reacting the nitrile or biocatalyst composition with a nitrile substrate.

As another aspect, the present invention relates to a biocatalyst composition for preparing a carboxylic acid comprising a nitrile represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 .

Hereinafter, the present invention will be described in more detail.

As used herein, the term "nitrilase" refers to an enzyme that catalyzes a reaction for converting a nitrile compound to a carboxylic acid, and in the form of a partially purified enzyme or purified enzyme as long as it has nitrile activity. Can be.

As used herein, the term "biocatalyst composition comprising nitrile" is a concept including all types of compositions including nitrile enzyme and exhibit nitrile activity, preferably complete with nitrile activity. The microbial cell or the nitrile may be a transformed microbial cell. It may also be a cell culture or cell disruption comprising the microbial cells.

In a preferred embodiment, the nitriles used in the present invention are nitrile RMN1 proteins represented by the amino acid sequence of SEQ ID NO: 1.

As another preferred embodiment, the nitrile used in the present invention is a nitrile RMN2 protein represented by the amino acid sequence of SEQ ID NO: 3.

As another preferred embodiment, the nitrile used in the present invention is a nitrile ORN protein represented by the amino acid sequence of SEQ ID NO: 5.

As another preferred embodiment, the nitrile used in the present invention is a nitrile VMN1 protein represented by the amino acid sequence of SEQ ID NO.

In addition, the nitrile protein of the present invention exhibits a substantial identity to the amino acid sequence as well as the protein having its normal type amino acid sequence, and at the same time maintains nitrile activity. Amino acid sequence of a. By substantial identity is meant functional equivalents in which one or more amino acid residues of a normal amino acid sequence have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, but exhibit biological activity equivalent to that of a native protein. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).

In addition, the nitrified protein of the present invention may be a variant or modified form that modifies the properties of its protein while maintaining nitrile activity. Preferably, the protein may be a protein having increased structural stability or increased protein activity against heat, pH, etc. of the protein by variation and modification on the amino acid sequence. For example, the nitrile protein of the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, or farnesylation in some cases. .

Nitrile protein of the present invention can be obtained by extraction and purification in nature by methods well known in the art. For example, the RMN1 and RMN2 proteins of the present invention are Reinekea sp. MED297 strain can be extracted and purified, ORN protein is Oceanobacter sp. RED65 strains can be extracted and purified, VMN1 protein is Vibrio sp. It can be extracted and purified from the MED222 strain.

In addition, the nitrile protein of the present invention may be synthesized chemically (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or genes based on amino acid sequence information of SEQ ID NOs: 1, 3, 5, and 7. Obtained using recombinant technology. When prepared by chemical synthesis, it can be obtained using a polypeptide synthesis method well known in the art. When using recombinant technology, the nucleic acid encoding the nitrile protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured to express the nitrile protein, and then the nitrile is removed from the host cell. Can be obtained by recovering the protein.

In the present invention, the nucleotide sequence encoding the nitrile protein is a nucleotide sequence encoding the nitrile protein represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: Nucleic acids with one nucleotide sequence may be single or double stranded and may be DNA molecules (genomic, cDNA) or RNA molecules.

As used herein, the term "nucleic acid molecule" or "nucleic acid" is a concept that encompasses DNA and RNA molecules inclusively. Nucleotide, which is a constituent unit of a nucleic acid molecule, is substituted with one or more bases as well as natural nucleotides. Analogs modified by the combination of (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

In a preferred embodiment, the nucleotide sequence encoding the nitrified RMN1 protein is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 2 can be illustrated as a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

In another preferred embodiment, the nucleotide sequence encoding the nitrified RMN2 protein is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 4 can be illustrated as a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3.

In another preferred embodiment, the nucleotide sequence encoding the nitrified ORN protein is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5. The nucleotide sequence of SEQ ID NO: 6 can be illustrated as a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5.

In another preferred embodiment, the nucleotide sequence encoding the nitrified VMN1 protein is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7. The nucleotide sequence of SEQ ID NO: 8 can be illustrated as a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7.

Nucleic acid molecules of the invention can be isolated from nature or prepared using chemical synthesis. When chemically synthesizing a nucleotide sequence encoding a nitrile protein, synthesis methods well known in the art, for example, Engels and Uhlmann, Angew Chem Int Ed Engl. 37: 73-127, 1988 The methods described can be used, including tryster, phosphate, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on solid supports, and the like.

In a more preferred embodiment, the nucleic acid having the nucleotide sequence encoding the nitrile protein of the present invention is inserted into and expressed in a recombinant vector.

As used herein, the term "vector" means a means for introducing a nucleotide sequence encoding a protein of interest into a host cell. Vectors of the present invention include plasmid vectors, cosmid vectors, viral vectors and the like. Suitable expression vectors include signal or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers and can be prepared in various ways depending on the purpose. The initiation codon and the termination codon are generally considered to be part of the nucleotide sequence encoding the protein of interest and must be functional in the individual when the gene construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible. The expression vector also includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host genomic DNA.

Preferably, the gene inserted into the vector and delivered is irreversibly fused into the genome of the host cell so that gene expression in the cell can be stably maintained for a long time.

As a host cell capable of continuously cloning and expressing the vector of the present invention, any host cell known in the art may be used, and a host having high DNA introduction efficiency and a high expression efficiency of introduced DNA may be commonly used. have. Specifically bacteria, for example, known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera fruitgifer (SF9), CHO, COS1, COS7 Animal cells such as BSC1, BSC40, and BMT10 may be used, but are not limited thereto. Preferably E. coli. In a specific embodiment of the present invention, DH5α and BL21-CodonPlus (DE3) -RIL cells (Stratagene, LaJolla, CA) were used as strains for plasmid growth and gene expression, respectively.

Introduction of vectors into host cells is well known, for example, the calcium chloride method (Journal of Molecular Biology, Vol. 53, p. 159, 1970), the Method of Enzymology (Vol. 68, p. 253, 1979), Electro Current Protocols in Molecular Biology (Vol. 1, p. 184, 1994), and Invitro Packaging (Current Protocols in Molecular Biology, Vol. 1, p. 571, 1994) may be used.

Nitrilas are expressed in selected host cells and then subjected to conventional biochemical separation techniques such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration). , And can be separated and purified using a variety of chromatography such as adsorption chromatography, ion exchange chromatography, affinity chromatography, etc., usually used in combination to separate proteins of high purity.

In a specific embodiment of the present invention, after culturing the strain transformed by a recombinant vector comprising a nucleic acid molecule encoding the nitrile of the present invention, the culture medium is recovered by centrifuging the culture medium and then crushed cells The precipitated solution was removed by centrifugation of the crushed solution to separate a recombinant nitrile coenzyme solution, and the crude enzyme solution thus obtained was purified by a known column chromatography method to obtain a novel nitrile enzyme of the present invention. Example 5).

The nitrile protein obtained as described above can be used to prepare carboxylic acid by reaction with nitrile substrate.

For use in the preparation of carboxylic acid, in the present invention, a nitrile protein represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 as long as it has nitrile activity Or nucleic acid molecules encoding the nitrile proteins, or nucleic acid constructs or vectors to which the nucleic acid molecules and regulatory signal sequences are linked may be used. Alternatively, it may be used in the form of a complete microbial cell having nitrile activity or a microbial cell transformed with the nitrile, a cell culture comprising the cell or a lysate thereof.

As a preferred embodiment, 1) transforming a vector comprising a nucleic acid molecule encoding a nitrile protein of the present invention to prepare a transformant, 2) purifying the nitrile from the transformant , And 3) reacting the purified nitrile with a nitrile substrate to prepare a carboxylic acid.

Nitriles of the invention cause reactions with various nitrile substrates.

As used herein, the term "nitrile" refers to an organic compound (R-CN) in which a cyano group (-C≡N) is directly bonded to a carbon atom of a hydrocarbon group (R). If the organic compound containing a cyano group can be used as a substrate of the nitrile of the present invention without limitation, preferably, acetonitrile (ACN), acrylonitrile (ACRN), adiponitrile (adiponitrile) , ADN), benzonitrile (benzonitrile, BEN), butyronitrile (BUTN), 4-chlorobenzonitrile (4-chlorobenzonitrile, CBEN) and mandelonitrile (MAN) have.

As used herein, the term "carboxylic acid" refers to a compound having a carboxy group (-COOH).

The process according to the invention is preferably carried out under conditions of pH 4 to pH 11, preferably pH 4 to pH 9. In addition, the process according to the invention is preferably carried out at a temperature of 0 ° C to 80 ° C, preferably 10 ° C to 60 ° C, more preferably 15 ° C to 50 ° C.

Carboxylic acids prepared by the process according to the invention can be isolated from the aqueous reaction solution by extraction or crystallization. The aqueous reaction solution can be acidified with inorganic or organic acids and then extracted with an organic solvent. Extraction can be repeated several times to increase yield. The organic solvent can be used any solvent which shows a phase boundary with water after salt addition, for example, solvents such as toluene, benzene, hexane, methyl tertiary butyl ethyr or ethyl acetate.

When the organic phase comprising the product carboxylic acid is crystallized and separated, it is preferable to cool the solution to a temperature of 0 ° C to 10 ° C. Crystallization can occur directly from the organic solution. To repeat the crystallization, the crystallized product can be dissolved again in the same or different solvent to crystallize once again, with subsequent crystallization further increasing the enantiomeric purity of the product. In addition, the carboxylic acid can be acidified with acid and immediately crystallized from the aqueous reaction solution. In this case, the aqueous solution is preferably heated and concentrated to reduce the volume to 10 to 90%, preferably 20 to 80%, particularly preferably 30 to 70%, and the crystallization is cooled at a temperature of 0 ° C to 10 ° C. It is preferable to perform by.

In a specific embodiment of the present invention, to determine whether the proteins RMN1 (SEQ ID NO: 1), RMN2 (SEQ ID NO: 3), ORN (SEQ ID NO: 5), and VMN1 (SEQ ID NO: 7) actually exhibit nitrile activity, After the protein was reacted with various nitrile substrates, color change was observed using colorimetric analysis by pH sensitivity. As a result, it was found that all the enzymes used for the mandelonitrile (MAN) substrate turned yellow in 2 hours and exhibited nitrile enzyme activity.All enzymes except RMN2 had different substrates (ACN, ACRN, AND, BEN, BUTN and CBEN) were observed to be yellow at 4 hours (Fig. 6).

Therefore, the proteins of the present invention can be confirmed that the enzymes have a high nitrile activity against the mandelonitrile substrate and exhibit a wide range of substrate specificity. The enzymes of the present invention are expected to contribute to the production and production of a wide range of carboxylic acids in the future.

Since the nitrified protein of the present invention has various substrate specificities, it may be usefully used for biosynthesis of carboxylic acid with high efficiency. In addition, the carboxylic acid produced by the nitrile of the present invention can be used as a material for the synthesis of pharmaceuticals with high added value industrially.

Figure 1 shows the schematic diagram of the catalytic reaction of nitrile and the result of measuring the reaction of nitrile by colorimetric analysis using the sensitivity of pH. Pi buffer stands for phosphate buffer.
Figure 2 shows the chemical structure of the nitrile substrates used in the present invention. A is acetonitrile (ACN), B is acrylonitrile (ACRN), C is adiponitrile (ADN), D is benzonitrile (BEN), E is butyl Nitrile (butyronitrile (BUTN)), F stands for 4-chlorobenzonitrile (CBEN), and G stands for mandelonitrile (MAN).
Figure 3 shows the manufacturing process of the expression vector comprising a nucleic acid molecule encoding the nitrile protein of the present invention.
Figure 4 shows the results of comparative analysis of the amino acid sequence of the isolated and purified nitrile RMN1, RMN2, VMN1, VMN2, ORN and LBN in the present invention. The boxed moiety represents the catalytic trivalent elements glutamate (E), lysine (K) and cysteine (C).
5 is a photograph showing the isolated and purified nitrile RMN1, RMN2, VMN1, VMN2, ORN and LBN by SDS-PAGE in the present invention. (A) shows the expression pattern of an enzyme, respectively. M is the molecular weight standard marker, BL is E. coli BL-21 cell extract. Lanes 1, 3, 5, 7, 9 and 11 are E. coli BL-21 cell extracts that do not induce overexpression by adding isopropyl-β-D-thiogalactoside (IPTG), lanes 2, 4, 6, 8, 10 and 12 is an E. coli BL-21 cell extract induced by overexpression with isotpropyl-β-D-thiogalactoside (IPTG). (B) shows the purification pattern of an enzyme, respectively.
Figure 6 shows the results of measuring the nitrile activity through the colorimetric analysis for the isolated and purified nitrile RMN1, RMN2, VMN1, VMN2, ORN and LBN in the present invention. EB (enzyme blank) represents the enzyme untreated group, SB (substrate blank) the substrate untreated group, and DB (dye blank) represents the non-dye group.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Materials and reagents

The basal medium used for strain cultivation was a product of Diffco Corporation (USA). Acetonitrile (ACN), acrylonitrile (ACRN), adiponitrile (ADN), benzonitrile (BEN) and butyl as the various nitrile substrates used in this study Nitrile (butyronitrile, BUTN), 4-chlorobenzonitrile (CBEN) and mandelonitrile (MAN) were purchased from Aldrich (USA). Figure 2 shows the chemical structure of the nitrile substrates used in the present invention. All products used in this study were purchased from Aldrich as HPLC Grade.

Example  2. Microbial Strains and Culture Conditions

In this studyLeeuwenhoekiella blandensis MED217,Oceanobacter sp. RED65,Reinekea sp. MED297 andVibrio sp. MED222 strain was used. The four strains were distributed by Dr. Jarone Pinhassi of the University of Kalmar, Sweden.Leeuwenhoekiella blandensis The genomic sequence of the MED217 strain is gene bank registration number NZ_AANC00000000,Oceanobacter sp. The genomic sequence of the RED65 strain is GenBank Accession No. NZ_AAQH00000000,Reinekea sp. The genomic sequence of the MED297 strain is GenBank Accession No. NZ_AAOE00000000, andVibrio sp. The genomic sequence of the MED222 strain is disclosed in GenBank Accession No. NZ_AAND00000000.

In the present invention, the strains were inoculated in the marine microbial culture medium (marine broth), and then cultured for 3-4 days at a stirring speed of 180 rpm using a shaking incubator at a temperature of 30 ° C.

DH5α and BL21-CodonPlus (DE3) -RIL cells (Stratagene, LaJolla, Calif., USA) were used as strains for plasmid propagation and gene expression, respectively, and cultured at 37 ° C. in LB (Luria-Bertani) medium with the appropriate antibiotics. It was.

Example  3. Derived from marine microorganisms Night Relay  Gene ORF  Sequencing

Leeuwenhoekiella blandensis MED217, Oceanobacter sp. RED65, Reinekea sp. MED297 and Vibrio sp. To identify nitrile genes from MED222 strains, a catalytic traid corresponding to an open reading frame (ORF) of each genomic DNA sequence of the strains analyzed by the Moore foundation (www.moore.org). motifs) were analyzed using Ensoltek's ProteinFinder (www.ensoltek.com) and the BLAST program. Candidate nitrile amino acid sequences were also analyzed by the CLUSTAL W program (Thompson, et al., (1994) Nucleic. Acids. 22: 4673-4680).

On the other hand, it was determined by a conventional method whether there is an intrinsic nitrile activity-site for the strains. To this end, a sequence containing catalytic traid motifs was selected and analyzed.

Example  4. Night Relay  gene Cloning

Leeuwenhoekiella blandensis MED217, Oceanobacter sp. RED65, Reinekea sp. MED297 and Vibrio sp. To clone the nitrile gene in the ORF sequence of the MED222 strains, the genomic DNA of the strain was first amplified by PCR using forward and reverse primers. At this time, restriction enzymes NdeI and XhoI were attached to both ends of the primer, respectively , and the forward and reverse primer sequences and PCR conditions of the lbn , orn , rmn1 , rmn2 , vmn1 and vmn2 genes are shown in Table 1, respectively.

gene Type of primer order SEQ ID NO: Annealing (℃)
lbn
Forward ( lbn F) 5'-CGACCCGG CATATG AAAGAAGTAGATCACATAGAT-3 ' 13 63, 45 Reverse ( lbn RH) 5'-CTCCACAT CTCGAG TTTTTTCTTCCACTTAAGCTGAAA-3 ' 14 63, 49 Reverse ( lbn RX) 5'-CTCCACAT CTCGAG CTATTTTTTCTTCCACTTAAGCTG-3 ' 15 64, 51
orn
Forward direction ( orn F) 5'-CGACCCGG CATATG ACGCAAACTGTGATTACCGT-3 ' 16 57, 50 Reverse ( orn RH) 5'-CTCCACAT CTCGAG TAATCGTTTATGCTTCAACAATGG-3 ' 17 64, 51 Reverse ( orn RX) 5'-CTCCACAT CTCGAG TCATAATCGTTTATGCTTCAACAA-3 ' 18 63, 49
rmn1
Forward direction ( rmn1 F) 5'-CGACCCGG CATATG AGAGAAGTTACCGTAGCGG-3 ' 19 68, 51 Reverse direction ( rmn1 RH) 5'-CTCCACAT CTCGAG AGCGCGTCCGTCTTTGGTCATT-3 ' 20 69, 57 Reverse direction ( rmn1 RX) 5'-CTCCACAT CTCGAG CTAAGCGCGTCCGTCTTTGGTC-3 ' 21 70, 59
rmn2
Forward direction ( rmn2 F) 5'-CGACCCGG CATATG AAGTTAGCATTGGCCCAGC-3 ' 22 68, 51 Reverse direction ( rmn2 RH) 5'-CTCCACAT CTCGAG GACCCCCGGCGCCCAGCCAT-3 ' 23 74, 64 Reverse ( rmn2 RX) 5'-CTCCACAT CTCGAG TCAGACCCCCGGCGCCCAGC-3 ' 24 74, 64
vmn1
Forward direction ( vmn1 F) 5'-CGACCCGG CATATG CTTCAACCATTGGCGCTCG-3 ' 25 69, 57 Reverse ( vmn1 RH) 5'-CTCCACAT CTCGAG ACGATTCAACGCAGACGCTATC-3 ' 26 68, 55 Reverse ( vmn1 RX) 5'-CTCCACAT CTCGAG TTAACGATTCAACGCAGACGCTA-3 ' 27 67, 53
vmn2
Forward direction ( vmn2 F) 5'-CGACCCGG CCATGGCT AAGAAAGACATCAAAGTAGCG-3 ' 28 69, 49 Reverse ( vmn2 RH) 5'-CTCCACAT CTCGAG TTTCTCAGCAAAACGCGCTTGG-3 ' 29 68, 55 Reverse ( vmn2 RX) 5'-CTCCACAT CTCGAG TTATTTCTCAGCAAAACGCGCTT-3 ' 30 66, 52

The underlined portions of the forward and reverse primers in Table 1 indicate the NdeI and XhoI sites , respectively , and for the expression of the histidine-tagged lbn , orn , rmn1 , rmn2 , vmn1 and vmn2 genes, lbn RX, described in Table 1, orn RX, rmn1 RX, rmn2 RX, vmn1 RX and vmn2 RX reverse primers were further prepared. PCR reaction conditions were as follows: (1) initial denaturation at 95 ° C for 1 minute, (2) denaturation at 94 ° C for 20 seconds, annealing at 45 to 74 ° C (typically 50 to 55 ° C) for 30 seconds, and 1 at 72 ° C. Elongation for 30 minutes (30 cycles), and (3) at 72 ° C. for 10 minutes at the final elongation conditions. The exact annealing temperature in each gene amplification is shown in Table 1. After the PCR reaction, the amplified fragment having the NdeI and XhoI restriction enzyme sites was linked to the pET-24a (+) vector having the NdeI / XhoI site, and then the recombinant expression vector was transformed into DH5α. It was introduced into BL21-CodonPlus (DE3) -RP (Novagen) strain to confirm the expression.

Example  5. Night Relay  Gene Expression and Purification

In order to confirm that the nitrile gene expression vector prepared in Example 4 is substantially expressed in cells, the transformant was cultured at 37 ° C., and then the OD value was 0.4 to 0.6 at 600 nm. IPTG (isopropyl-β-D-thiogalactoside) was added to induce expression. After incubation for 3 hours, the cells were obtained by centrifugation at 5,000 g for 20 minutes, and the obtained cells were suspended in a solution containing 50 mM phosphate (pH 7.0), 0.5M KCl and 10% glycerol and homogenized by a grinder. Cells were removed by centrifugation at 15,000 g for 30 minutes using His.Bind Purification Kit (Novagen).

The soluble fragment was loaded on a Ni-nitrilotriacetic (Ni-NTA) column equilibrated with a binding solution containing 500 mM NaCl, 20 mM phosphate (pH 7.0) and 5 mM imidazole, followed by 500 mM NaCl, 20 mM It was washed with a washing solution containing phosphate (pH 7.0) and 60 mM imidazole. Next, the bound enzyme was eluted with an eluent containing 500 mM NaCl, 20 mM phosphate (pH 7.0) and 1 M imidazole, and dialyzed with 50 mM phosphorylated solution (pH 7.0). Protein purification was isolated by SDS-PAGE by the method described in Laemmli (1970). Protein concentration was measured by Bradford method using the Bio-Rad protein assay kit with the standard protein BSA (Bradford (1976) Anal. Biochem 72: 248-254).

Example  6. Night Relay  Active measurement

In order to confirm the nitrile activity of the protein obtained in Example 5, the inventors used a colorimetric assay based on pH sensitivity (Banerjee et al., (2003) J. Biomol. Screen. 8: 559-565). In a 96-well microplate, add 201 μl of phosphate solution [10 mM phosphate (pH 7.2)] to 230 μl of the total reaction amount, add bromothymol blue to 0.01%, and 5.75 μl of nitrile substrates (500 mM). stock in ethanol) was added and finally 23.25 μl of nitrile was added to react at 30 to 50 ° C. for 2 to 4 hours. Nitrile activity results in the nitrile substrates changing from blue to yellow as the pH is acidified to produce carboxylic acids (FIG. 1). As described above, various nitriles were used to measure the activity of various nitrile substrates.

Experiment result

One. Marine microorganisms Night Relay  Gene ORF  Sequence analysis result

Leeuwenhoekiella blandensis MED217, Oceanobacter sp. RED65, Reinekea sp. MED297 and Vibrio sp. The result of comparative analysis of the ORF sequence of the nitrile gene from the MED222 strain is shown in FIG. That is, Figure 4 is a comparative analysis of the amino acid sequence of the isolated and purified nitriles, the National Center for Biotechnology Information (NCBI) registration number and sequence number of the comparative protein is as follows:

RMN1 ( Reinekea sp. MED297), ZP_01112744, SEQ ID NO: 1;

RMN2 ( Reinekea sp.MED297), ZP_01116337, SEQ ID NO: 3;

ORN (Oceanobacter sp. RED65), ZP — 01308306, SEQ ID NO: 5;

VMN1 ( Vibrio sp. MED222), ZP_01063455, SEQ ID NO: 7;

VMN2 ( Vibrio sp. MED222), ZP_01063485, SEQ ID NO: 9; And

LBN ( Leeuwenhoekiella blandensis MED217), ZP_01060838, SEQ ID NO: 11.

As described above, Reinekea sp. MED297 and Vibrio sp. Two genes could be selected from the MED222 strain, respectively, and Oceanobacter sp. With RED65 One gene was selected from each of Leeuwenhoekiella blandensis MED217 strains. In addition, all of the selected nitrase enzymes had conserved catalytic triid motifs (FIG. 4).

Specifically, Reinekea sp. RMN1 derived from MED297 strain consists of 289 amino acids with ORF and catalytic trivalent elements (E43, K116, and C153). RMN2 derived from the same strain consists of 589 amino acids with ORF. There were valent elements (E41, K108, and C144). Vibrio sp. VMN1 from MED222 strain consists of 297 amino acids with ORF and catalytic trivalent elements (E77, K148 and C180). VMN2 from the same strain consists of 320 amino acids with ORF. Elements E45, K120 and C151 were present. Also, Oceanobacter sp. ORN from RED65 strain consists of 274 amino acids with ORF and catalytic trivalent elements (E45, K118, and C160), Leeuwenhoekiella LBN of the blandensis MED217 strain was composed of 523 amino acids with ORF and catalytic trivalent elements (E279, K354, and C388) (FIG. 4 and Table 2).

Strain
Length
(amino acid) Molecular Weight
(kDa) Catalytic trivalent elements SEQ ID NO:
Enzyme Name
Glutamate Lysine Cysteine Reinekea sp. MED297 289 32.1 E43 K116 C153 One RMN1 Reinekea sp. MED297 538 60.2 E41 K108 C144 3 RMN2 Oceanobacter sp. RED65 274 30.0 E45 K118 C160 5 ORN Vibrio sp. MED222 297 32.8 E77 K148 C180 7 VMN1 Vibrio sp. MED222 320 35.7 E45 K120 C151 9 VMN2 Leeuwenhoekiella blandensis MED217 523 60.5 E279 K354 C388 11 LBN

2. Night Relay  gene Cloning , Expression and purification results

Leeuwenhoekiella blandensis MED217, Oceanobacter sp. RED65, Reinekea sp. MED297 and Vibrio sp. Cloning, expression and purification of nitriles from the MED222 strain resulted in Reinekea sp. Recombinant nitro relay's of RMN1 and RMN2 molecular weight of MED297 derived was 32.1 kDa and 60.2 kDa, respectively, Vibrio sp. The molecular weights of the recombinant nitrified VMN1 and VMN2 derived from MED222 were 32.8 kDa and 35.7 kDa, respectively, and Leeuwenhoekiella blandensis. MED217 and Oceanobacter sp. The molecular weights of the recombinant nitrified LBN and ORN derived from RED65 were 60.5 kDa and 30.0 kDa, respectively (Table 2).

5 shows well the expression pattern and purification of the nitrile enzymes.

3. Night Relay  Active measurement result

Using colorimetric analysis by pH sensitivity, 201 μl of phosphate solution [10 mM phosphate (pH 7.2)] was added to 230 μl of the total reaction amount in a 96-well microplate, bromothymol blue was added to 0.01%, 5.75 μl of various nitrile substrates (FIG. 2; 500 mM stock in ethanol) were added and finally 23.25 μl of various enzymes (Table 2 and FIG. 5) were added and reacted at 30 to 50 ° C. for 2 to 4 hours.

As a result, it was found that all the enzymes used for the mandelonitrile (MAN) substrate turned yellow in 2 hours, and showed nitrile enzyme activity.All enzymes except RMN2 had different substrates (ACN, ACRN, AND). , BEN, BUTN and CBEN) can be observed to be yellow at 4 hours (FIG. 6).

Taken together, these results indicate that the enzymes isolated by the present inventors have high nitrile activity against mandelonitrile substrates and exhibit a wide range of substrate specificities.

<110> GYEONGBUK INSTITUTE FOR MARINE BIOINDUSTRY          KOREA OCEAN RESEARCH AND DEVELOPMENT INSTITUTE <120> Method for preparing a carboxylic acid using nitrilase RMN2 <130> DPP20110089KR <160> 30 <170> Kopatentin 1.71 <210> 1 <211> 289 <212> PRT <213> Reinekea sp. MED297 <220> <221> PEPTIDE (222) (1) .. (289) <223> amnino acid sequences of nitrilase RMN1 <400> 1 Met Arg Glu Val Thr Val Ala Ala Thr Gln Met Pro Cys Gly Trp Asp   1 5 10 15 Val Ser Glu Asn Leu Lys Thr Ala Glu Arg Leu Val Arg Glu Ala Ala              20 25 30 Ala Ser Gly Ala Gln Val Ile Leu Leu Gln Glu Leu Phe Glu Arg Pro          35 40 45 Tyr Phe Cys Gln His Gln Lys Glu Glu Phe Arg Arg Phe Ala Thr Ala      50 55 60 Ile Asp Asp Asn Pro Ala Ile Ala His Phe Ala Pro Ile Ala Arg Glu  65 70 75 80 Leu Gly Val Val Leu Pro Ile Ser Phe Phe Glu Gln Cys Gly Pro Val                  85 90 95 Ala Tyr Asn Ser Val Val Val Leu Asp Ala Asp Gly Glu Asn Leu Gly             100 105 110 Leu Tyr Arg Lys Thr His Ile Pro Asp Gly Pro Gly Tyr Cys Glu Lys         115 120 125 Phe Tyr Phe Thr Pro Gly Asp Thr Gly Phe Gln Val Phe Ser Thr Arg     130 135 140 Phe Gly Arg Ile Gly Val Gly Ile Cys Trp Asp Gln Trp Phe Pro Glu 145 150 155 160 Thr Ala Arg Ala Met Thr Leu Met Gly Ala Glu Leu Leu Phe Tyr Pro                 165 170 175 Thr Ala Ile Gly Ser Glu Pro Tyr Asn Pro Asp Ile Asp Ser Ser Gly             180 185 190 His Trp Gln Arg Thr Gln Gln Gly His Ala Ala Ala Asn Val Ile Pro         195 200 205 Leu Ile Ala Ser Asn Arg Ile Gly Thr Glu Val Ile Asp Asp Thr Gln     210 215 220 Ile Thr Phe Tyr Gly Ser Ser Phe Ile Ala Asp Asn Thr Gly Ala Leu 225 230 235 240 Val Thr Ser Met Asp Arg Thr Ser Thr Gly Phe Ile Gln Ala Thr Phe                 245 250 255 Asp Leu Asp Ala Leu Asn Ala Gln Arg Ser Glu Trp Gly Leu Phe Arg             260 265 270 Asp Arg Arg Pro Ser Gln Tyr Gly Thr Leu Met Thr Lys Asp Gly Arg         275 280 285 Ala     <210> 2 <211> 870 <212> DNA <213> Reinekea sp. MED297 <220> <221> gene (222) (1) .. (870) <223> Nucleotide sequences of nitrilase RMN1 <400> 2 atgagagaag ttaccgtagc ggccacacag atgccctgtg gttgggacgt cagtgaaaat 60 ctcaagacgg cggaaaggct ggtacgcgaa gccgcggcca gcggggctca ggtcattctt 120 ttgcaggagc tgtttgaacg gccttacttc tgtcagcatc agaaagaaga atttcggcgc 180 tttgccaccg ccatcgatga taacccggcc attgcgcatt ttgcccccat cgcccgggaa 240 ctgggcgtgg tcttacccat cagctttttt gagcaatgcg gtccggtggc ctacaactcc 300 gtcgttgtgc tggatgcgga tggcgaaaac ctgggtctgt atcggaaaac gcacattccg 360 gacggtcccg gctactgcga gaagttttat ttcaccccgg gagataccgg ttttcaggtg 420 ttttcgacgc gcttcgggcg catcggcgtc gggatctgct gggatcaatg gtttccggaa 480 actgcgcggg cgatgacact gatgggggcg gaactgctgt tttacccgac cgccatcggc 540 agtgaaccct acaatccgga catcgattct tccggtcatt ggcagcgtac ccaacagggt 600 cacgcggcgg ccaatgtgat accgctgatt gccagcaacc gtatagggac agaggtcatc 660 gacgacactc agatcacctt ttacggttct tcgttcatcg ccgataacac gggcgcactg 720 gtgacgtcga tggatcgcac aagtaccggg tttatccagg caacgtttga tctggatgcc 780 ctgaacgctc agcgcagtga atgggggctg ttccgtgatc gccggcccag tcagtatggc 840 acgttaatga ccaaagacgg acgcgcttag 870 <210> 3 <211> 538 <212> PRT <213> Reinekea sp. MED297 <220> <221> PEPTIDE (222) (1) .. (538) <223> amnino acid sequences of nitrilase RMN2 <400> 3 Met Lys Leu Ala Leu Ala Gln Gln Arg Phe Pro Val Gly Asp Ile Asp   1 5 10 15 Gly Asn Val Asp Lys Ile Leu Thr Leu Ser Arg Glu Ala Met Ala Gln              20 25 30 Gly Ala Asp Met Ile Val Phe Pro Glu Leu Thr Leu Thr Gly Tyr Pro          35 40 45 Pro Glu Asp Leu Leu Leu Arg Pro Ser Leu Ala Lys Arg Val Ser Gln      50 55 60 Ala Met His Arg Leu Phe Asp Ala Arg Leu Pro Ile Ala Met Val Val  65 70 75 80 Gly Tyr Pro Gln Arg Glu Asp Gly Lys Leu Tyr Asn Lys Val Met Val                  85 90 95 Ile Ser Glu Gly Gln Val Ile Ala Asp Tyr Arg Lys Gln His Leu Pro             100 105 110 Asn Tyr Gln Val Phe Asp Glu Lys Arg Tyr Phe Gln Lys Gly Asp Gln         115 120 125 Thr Cys Val Phe Asp Phe Met Gly Ala Arg Ile Gly Leu Ser Ile Cys     130 135 140 Glu Asp Ile Trp Tyr Asp Gly Pro Ala Arg Arg Ala Tyr Glu Ala Gly 145 150 155 160 Ala Gln Ile Asn Leu Asn Ile Asn Gly Ser Pro Tyr Ser Ile Asn Arg                 165 170 175 Thr Ala Glu Arg His Glu Gln Val Thr Arg Val Val Ser Gln Trp Pro             180 185 190 Met Ala Thr Val Tyr Val Asn His Val Gly Gly Gln Asp Glu Leu Val         195 200 205 Phe Asp Gly Gly Ser Phe Val Val Gly Ala Asp Ala Thr Val Gln Ala     210 215 220 Ser Leu Pro Glu Phe Gln Glu Ala Leu Gln Tyr Val Glu Leu Thr Gln 225 230 235 240 Glu Ala Asn Gly Trp Gln Val Lys Ser Gly Glu Val Thr Pro Pro Met                 245 250 255 Ser Val Glu Ala Ala Leu Tyr Glu Ala Leu Lys Thr Gly Leu Ala Asp             260 265 270 Tyr Val Asn Arg Asn Arg Phe Pro Gly Val Val Leu Gly Met Ser Gly         275 280 285 Gly Ile Asp Ser Ala Leu Ser Ala Ala Ile Ala Val Asp Ala Leu Gly     290 295 300 Pro Asp Arg Val Met Gly Val Met Met Pro Tyr His Tyr Thr Ala Lys 305 310 315 320 Ile Ser Leu Glu Asp Ala Glu Asp Glu Ala Arg Arg Leu Gly Ile Arg                 325 330 335 Tyr Glu Val Leu Pro Ile Gly Glu Ala Phe Glu Ala Ala Leu Thr Thr             340 345 350 Leu Gln Pro Gln Phe Gly Asp Arg Pro Ala Asp Val Thr Glu Gln Asn         355 360 365 Met Gln Ser Arg Met Arg Gly Leu Phe Leu Met Ala Leu Ser Asn Lys     370 375 380 Thr Gly Asn Met Val Leu Thr Thr Gly Asn Lys Ser Glu Met Ala Val 385 390 395 400 Gly Tyr Ala Thr Leu Tyr Gly Asp Met Cys Gly Gly Tyr Asn Cys Leu                 405 410 415 Lys Asp Val Pro Lys Leu Trp Val Tyr Arg Leu Ser Arg Trp Arg Asn             420 425 430 Ser Phe Gly Glu Val Ile Pro Glu Arg Val Ile Thr Arg Pro Pro Ser         435 440 445 Ala Glu Leu Ala Pro Asp Gln Leu Asp Glu Asp Ser Leu Pro Pro Tyr     450 455 460 Glu Val Leu Asp Ala Ile Ile Glu Arg Tyr Val Glu His Asp Glu Ser 465 470 475 480 Gln Gln Thr Ile Ile Glu Ser Gly Phe Asn Glu Asp Asp Val Lys Arg                 485 490 495 Val Ile Arg Leu Ile Asp Leu Asn Glu Tyr Lys Arg Arg Gln Ala Pro             500 505 510 Glu Gly Val Arg Val Thr Lys Arg Gly Phe Gly Arg Asp Arg Arg Tyr         515 520 525 Pro Ile Thr His Gly Trp Ala Pro Gly Val     530 535 <210> 4 <211> 1617 <212> DNA <213> Reinekea sp. MED297 <220> <221> gene (222) (1) .. (1617) <223> Nucleotide sequences of nitrilase RMN2 <400> 4 tcagaccccc ggcgcccagc catgtgtaat cgggtagcgc cggtcacgac caaaaccccg 60 cttggtgaca cgaacaccct caggtgcctg acggcgctta tactcattca gatcaatcaa 120 ccggatgaca cgtttgacat cgtcttcatt gaaaccggac tcgataatgg tttgctgact 180 ttcatcatgc tcgacatagc gttcaatgat ggcatccagc acttcatagg gcggcaagct 240 gtcttcgtcc agctgatccg gagccagctc ggccgatggc ggtcgggtaa tgacccgttc 300 agggattacc tctccgaagg agttgcgcca gcgagacaag cgatacaccc agagtttcgg 360 cacgtctttc aaacagttat agcccccaca catatcgccg tacaaggtgg cgtatccgac 420 cgccatctca ctcttattgc cggtggtcag caccatattg ccggttttgt tcgacaacgc 480 catcaaaaac aacccgcgca tacgcgattg catgttctgc tcggtaacat ccgccggccg 540 gtcaccaaac tgaggctgaa gcgtggttaa cgcggcttca aatgcctcac cgatgggcag 600 cacttcgtat cggataccga gacgacgagc ttcgtcttct gcatcctcca gagaaatttt 660 cgcggtgtag tgatagggca tcatcacgcc catcacccga tcaggtccca gggcatccac 720 agcaatggcg gcacttaacg ccgagtcgat tccgcctgac atacccagaa ctacgccggg 780 gaagcggttt cgattgacat agtcggccag ccccgtcttg agcgcttcat agagcgctgc 840 ctcgaccgac atcggcggtg ttacctcgcc tgatttcacc tgccagccat ttgcttcctg 900 cgtcagctca acgtactgca atgcctcctg aaactcaggc aacgaagcct gcacggtggc 960 gtcagctccg acaacaaagg agccaccatc aaaaaccagt tcgtcctgac cacctacatg 1020 gtttacgtag acggtcgcca tcggccattg agacaccacc cgagtcacct gttcgtgacg 1080 ctctgctgtt cgattaatgg agtaagggct gccgttaata ttcagattga tctgcgcacc 1140 ggcctcatag gcacgacgcg ccgggccgtc ataccagatg tcttcgcaaa tactcaatcc 1200 gatacgtgca cccatgaaat caaagacaca ggtctgatcc cccttctgaa aataccgctt 1260 ttcatcaaag acctgatagt ttggcaaatg ctgtttgcgg tagtcggcaa tcacctgccc 1320 ttcgctgatc accatgacct tgttgtaaag cttgccgtct tcacgttgag gatagccaac 1380 gaccatggca attggtaagc gcgcatcaaa caagcgatgc atggcctgac taacacgttt 1440 cgccagactc gggcgcagca aaagatcttc tggcgggtac ccggtcaagg tcagttccgg 1500 aaagacgatc atatctgcgc cctgcgccat ggcctcacgg ctcagggtga gaattttatc 1560 cacattgccg tcaatatctc cgactggaaa acgctgctgg gccaatgcta acttcat 1617 <210> 5 <211> 274 <212> PRT <213> Oceanobacter sp. RED65 <220> <221> PEPTIDE (222) (1) .. (274) <223> amnino acid sequences of nitrilase ORN <400> 5 Met Thr Gln Thr Val Ile Thr Val Gly Leu Val Gln Met Thr Ser Gly   1 5 10 15 Lys Ala Val Gln Pro Asn Leu Arg Ala Ala Glu Ala Ala Ile Lys Arg              20 25 30 Cys Val Glu Gln Gly Ala Thr Thr Val Leu Leu Pro Glu Met Phe Val          35 40 45 Cys Leu Gly Val Lys Asn Gln Val Glu Ile Ala Gln Thr Gln Cys Gln      50 55 60 Lys Gly Gly Pro Val Arg Ser Gln Leu Ser Ala Leu Ala Lys Asp Phe  65 70 75 80 Lys Val Asn Ile Ile Ala Gly Ser Met Pro Leu Met Ser Ser Val Glu                  85 90 95 Asp Lys Val Leu Ala Ala Cys Leu Val Phe Ala Ala Asp Gly Ser Glu             100 105 110 Val Cys Gln Tyr Asp Lys Val His Leu Phe Asp Val Asp Val Ser Asp         115 120 125 Asn Lys Gly Arg Tyr Arg Glu Ser Asp Thr Phe Ile Ala Gly Thr Gln     130 135 140 Ser Lys Thr Val Ser Leu Asp Gly Thr Leu Tyr Gly Leu Ser Val Cys 145 150 155 160 Tyr Asp Leu Arg Phe Pro Glu Leu Tyr Gln Gln Tyr Gln Lys Gln Ser                 165 170 175 Cys Gln Val Val Thr Val Pro Ser Ala Phe Thr Tyr Thr Thr Gly Gln             180 185 190 Lys His Trp Leu Thr Leu Leu Lys Ala Arg Ala Ile Glu Thr Gln Ser         195 200 205 Phe Val Met Ala Ala Asn Gln Val Gly Thr His Glu Asp Gly Arg Ile     210 215 220 Thr Trp Gly Gln Ser Ile Val Ile Asn Pro Asp Gly Glu Ile Val Gly 225 230 235 240 Glu Leu Asp Ser Glu Lys Ala Gly Glu Leu Val Val Glu Leu Asp Leu                 245 250 255 Glu Leu Cys Gln Lys Ile Arg Gln Ser Met Pro Leu Leu Lys His Lys             260 265 270 Arg leu         <210> 6 <211> 825 <212> DNA <213> Oceanobacter sp. RED65 <220> <221> gene (222) (1) .. (825) <223> nucleotide sequences of nitrilase ORN <400> 6 tcataatcgt ttatgcttca acaatggcat agactgacga atcttttgac agagttcaag 60 atcaagttca acaaccaact cacctgcttt ttcgctgtct aactcgccca ctatttctcc 120 gtctggattg atgacaatag attgccccca ggtgatgcgt ccatcctcgt gggtgcccac 180 ctgattcgcg gccataacaa aactttgagt ttcaatagct cttgccttta ataaggtcaa 240 ccaatgcttt tgacctgtgg tataggtaaa cgcagagggt acggtcacga cctgacatga 300 ctgcttttga tactgttgat acaattcggg aaaacgcaaa tcatagcaaa cacttaaacc 360 atacagagta ccgtcaaggc taaccgtctt actttgagtc ccagctatga aagtatcaga 420 ttcacgataa cgacccttat tatcgcttac gtctacatca aacaaatgga ctttatcgta 480 ctggcaaacc tcgcttccat ctgcagcaaa aactaaacag gcagccaaca ccttatcttc 540 aacgctggac atcaacggca tagaacctgc gataatattt accttaaaat cctttgcaag 600 agcagataat tgagagcgga caggcccccc tttctgacat tgagtttgtg cgatctccac 660 ttgattcttt actcccaagc aaacaaacat ttcaggaagt aaaacggtag tggcgccttg 720 ttctacacag cgctttatcg cagcctctgc tgctcgtaaa ttaggttgga ctgctttgcc 780 actggtcatc tgtaccaagc cgacggtaat cacagtttgc gtcat 825 <210> 7 <211> 297 <212> PRT <213> Vibrio sp. MED222 <220> <221> PEPTIDE (222) (1) .. (297) <223> amnino acid sequences of nitrilase VMN1 <400> 7 Met Leu Gln Pro Leu Ala Leu Glu Ser Ala Ser Asp Gly Gly Leu Ser   1 5 10 15 Tyr Pro Val Val Leu Ser Leu Lys Phe Glu Val Leu Pro Ser Lys Asp              20 25 30 Ser Ile Leu Asn Ser Val Ser Val Thr Leu Val Gln Leu Glu Val Glu          35 40 45 Tyr Lys Asn Lys Gln Met Asn Ile Ser Arg Val Ser Glu Leu Leu Glu      50 55 60 Ala Glu Thr Ala Val Gly Asp Ile Thr Leu Leu Pro Glu Leu Phe Ser  65 70 75 80 Thr Gly Tyr Ile Phe Asn Asp Ala Ala Glu Ile His Glu Leu Cys Glu                  85 90 95 Asp Phe Asn Asn Ser Pro Thr Ile Asp Ser Leu Thr Ala Leu Ala Thr             100 105 110 Lys His Gln Thr Leu Ile Val Ala Gly Val Ala Glu Glu Asp Asn Gly         115 120 125 Gln Tyr Tyr Asn Ser Val Val Val Val Asp Gly Ser Gly Leu Arg His     130 135 140 Lys Tyr Arg Lys Val Ser Gln Thr Lys Phe Asp Lys Glu Tyr Phe Ser 145 150 155 160 Arg Gly Asn Glu Leu Leu Thr Phe Glu Tyr Lys Gly Leu Lys Phe Gly                 165 170 175 Val Ala Ile Cys Phe Asp Ile Trp Phe Pro Glu Ile Met Arg Pro Tyr             180 185 190 Gln Ser Val Asp Val Ile Leu His Pro Ala Asn Phe Gly Gly His His         195 200 205 Ser Phe Ala Ile Ala Gln Ala Arg Ala Leu Glu Glu Gly Cys His Ile     210 215 220 Val Thr Cys Asn Arg Val Gly Gln Asp Val Val Asp Ala Phe Thr Ala 225 230 235 240 Thr Tyr Cys Gly Gly Ser Arg Thr Tyr Ser Pro Lys Gly Asp Leu Met                 245 250 255 Leu Gln Leu Ser Glu His Gln Ser Val Glu Thr Ile Asn Ile Gln Asp             260 265 270 Leu Ser Ile Ala Pro Gln Tyr Asn Gly Val Asp Val Leu Asp Glu Ile         275 280 285 Gln Gln Ile Ala Ser Ala Leu Asn Arg     290 295 <210> 8 <211> 894 <212> DNA <213> Vibrio sp. MED222 <220> <221> gene (222) (1) .. (894) <223> nucleotide sequences of nitrilase VMN1 <400> 8 ttgcttcaac cattggcgct cgagtcagca agcgatgggg gattatctta ccctgtcgtt 60 cttagtctca agtttgaagt gttaccgagt aaggattcga ttttgaatag tgtcagcgtc 120 actcttgtcc agcttgaagt tgaatacaaa aataaacaaa tgaacatctc gcgagtctct 180 gagcttttgg aagctgagac ggcagtgggt gatatcacgt tgctgcctga actgttctct 240 actggatata tctttaacga tgccgcagaa attcatgaac tgtgtgaaga cttcaataac 300 agcccgacca ttgattcatt aactgcgctt gccacgaaac atcagacgtt aatcgttgcg 360 ggtgtcgctg aggaagataa tggtcagtat tacaatagcg ttgtggtggt tgatggttca 420 ggtttgcgtc ataaatacag aaaagtcagt caaacgaaat tcgacaaaga gtacttttct 480 agaggaaatg aacttcttac ttttgaatac aaaggcttga agttcggtgt cgcgatttgc 540 tttgatatat ggttcccgga gatcatgaga ccgtatcagt cggtagacgt tattctccat 600 cctgcgaatt ttggtggtca tcatagcttt gccattgcac aagcaagagc gttagaagaa 660 gggtgtcata tcgtgacctg taatcgcgtc gggcaagatg tggttgatgc ctttaccgcg 720 acatattgtg gcggcagtag aacctattca ccgaagggag acttaatgct tcaactcagt 780 gagcatcagt ctgttgaaac gattaatatt caagacttgt ctattgcgcc tcaatacaat 840 ggtgttgatg tgttagatga aatacaacag atagcgtctg cgttgaatcg ttaa 894 <210> 9 <211> 320 <212> PRT <213> Vibrio sp. MED222 <220> <221> PEPTIDE (222) (1) .. (320) <223> amnino acid sequences of nitrilase VMN2 <400> 9 Met Lys Lys Asp Ile Lys Val Ala Ser Val Gln Phe Asn His His Ala   1 5 10 15 Gly Asp Lys Ala Tyr Asn Leu Ser Val Ile Glu Gln Tyr Val Gln Lys              20 25 30 Ala Ala Asp Ser Asp Val Gln Ile Val Ser Phe Pro Glu Met Cys Ile          35 40 45 Thr Gly Tyr Trp His Val Ser Ala Leu Ser Arg Asp Glu Ile Glu Ala      50 55 60 Leu Ala Glu Pro Val Pro Gly Gly Glu Ser Thr Gln Lys Leu Ile Ala  65 70 75 80 Leu Ala Thr Gln Phe Gly Ile Ser Val Gly Ala Gly Leu Ile Glu Gln                  85 90 95 Gly Ile Asp Gly Glu Leu Tyr Asn Thr Tyr Val Phe Ala Met Pro Asn             100 105 110 Gly Glu Val Gln Lys His Arg Lys Leu His Thr Phe Val Ser Pro His         115 120 125 Met Ser Ser Gly Asp Gln Tyr Thr Val Phe Asp Thr Pro His Gly Cys     130 135 140 Lys Val Gly Ile Leu Ile Cys Trp Asp Asn Asn Leu Val Glu Asn Val 145 150 155 160 Arg Ile Thr Val Leu Lys Gly Ala Asp Ile Leu Ile Ala Pro His Gln                 165 170 175 Thr Gly Gly Cys His Ser Arg Ser Pro Asn Ala Met Lys Arg Ile Asp             180 185 190 Pro Glu Leu Trp Phe Asn Arg Asp Glu Asn Pro Asp Ala Ile Arg Ala         195 200 205 Glu Met Gln Gly Lys Asn Gly Arg Glu Trp Leu Met Arg Trp Leu Pro     210 215 220 Ala Arg Ala His Asp Asn Gly Leu Phe Val Val Phe Ser Asn Gly Val 225 230 235 240 Gly Val Asp Met Asp Glu Val Arg Thr Gly Asn Ala Met Ile Leu Ser                 245 250 255 Pro Tyr Gly Glu Ile Ile Ala Glu Thr Asp Ser Val Asp Asn Asp Met             260 265 270 Val Ile Ala Glu Leu Lys Ala Glu Glu Leu Asp Met Cys Thr Gly Arg         275 280 285 Arg Trp Ile Arg Gly Arg Lys Pro Glu Leu Tyr His Ser Leu Thr Gln     290 295 300 Pro Leu Gly His Glu Leu Asp Pro His Gln Ala Arg Phe Ala Glu Lys 305 310 315 320 <210> 10 <211> 963 <212> DNA <213> Vibrio sp. MED222 <220> <221> gene (222) (1) .. (963) <223> nucleotide sequences of nitrilase VMN2 <400> 10 atgaagaaag acatcaaagt agcgtcggtt caatttaacc accacgcagg tgataaggcg 60 tataacttat cggttataga gcaatacgtt caaaaggctg cggacagtga cgtgcagatc 120 gtcagtttcc cggagatgtg catcactggc tactggcatg tgtctgcttt gtcgcgagat 180 gagattgaag cgttagcgga acctgtaccg ggtggtgaat cgactcaaaa gctgattgca 240 ctggcaacac agtttggaat cagtgttggc gcgggcttga tagagcaggg catcgatggc 300 gaattataca acacctacgt attcgccatg cccaatggtg aggtacaaaa gcaccgtaaa 360 ctgcacacct tcgttagccc acatatgagc agtggcgacc aatacacagt gtttgatacg 420 cctcatggtt gcaaagtcgg tatcttgatt tgttgggata acaacttggt tgagaacgtc 480 aggatcactg ttttgaaagg cgccgatata ttgattgcgc cacaccaaac gggcggttgt 540 cattcacgaa gcccgaacgc gatgaagcga attgaccctg agttatggtt taaccgagat 600 gaaaacccag atgcgattcg tgcagaaatg cagggcaaga atggccgcga gtggctaatg 660 cgttggctgc ctgcaagagc gcacgataac ggcctctttg tggtgttcag taatggtgta 720 ggtgttgata tggacgaagt gaggacaggc aacgcgatga tcttgagccc ttacggtgaa 780 atcattgctg agaccgatag tgttgataac gatatggtga ttgcagagct aaaagcagaa 840 gagttagata tgtgtacggg cagacgttgg attcgtggtc gtaagccaga gctttatcat 900 tcactcacac aacctcttgg tcatgaactt gatccgcacc aagcgcgttt tgctgagaaa 960 taa 963 <210> 11 <211> 523 <212> PRT <213> Leeuwenhoekiella blandensis MED217 <220> <221> PEPTIDE (222) (1) .. (523) <223> amnino acid sequences of nitrilase LBN <400> 11 Met Lys Glu Val Asp His Ile Asp Asn Ser Asp Ser Ala Glu Lys Arg   1 5 10 15 Glu Arg Ile Asp Thr Ile Asn Leu Glu Tyr Leu Lys Val Lys Asp Phe              20 25 30 Glu Glu Leu Lys Glu Ile Met Ile Ala Val Tyr Pro Asn Ile Pro Asp          35 40 45 Pro Tyr Trp Lys Lys Lys Glu Leu Gln Lys Leu Val Ser Ile Phe Pro      50 55 60 Glu Gly Gln Val Val Val Lys Val Asn Gly Glu Ile Ala Gly Cys Ala  65 70 75 80 Leu Ser Ile Val Val Asp Tyr Glu Lys Phe Ser Asp Ser His Thr Tyr                  85 90 95 Met Glu Ile Thr Gly Asp Glu Thr Phe Ser Thr His Thr Pro Lys Gly             100 105 110 Asn Val Leu Tyr Gly Ile Asp Ile Phe Ile Ser His Lys Tyr Arg Gly         115 120 125 Met Arg Ile Gly Arg Arg Leu Tyr Asp Tyr Arg Lys Glu Leu Cys Glu     130 135 140 Asn Leu Asn Leu Glu Arg Ile Val Phe Gly Gly Arg Leu Pro Asn Tyr 145 150 155 160 His Gln Tyr Ala Lys Glu Leu Ser Pro Lys Ala Tyr Ile Glu Lys Val                 165 170 175 Arg Ala Arg Glu Ile Asn Asp Pro Val Leu Asp Phe Gln Leu Ser Asn             180 185 190 Asp Phe His Val Lys Arg Ile Ile Thr Asn Tyr Leu Glu Gly Asp Lys         195 200 205 Gln Ser Lys Glu Tyr Ala Ala Leu Leu Glu Trp Asp Asn Ile Tyr Tyr     210 215 220 Gln Lys Pro Ala Lys Lys Pro Asn Ala Ile Lys Thr Val Val Arg Leu 225 230 235 240 Gly Leu Val Gln Trp Gln Met Arg Thr Tyr Lys Asn Phe Glu Glu Leu                 245 250 255 Phe Glu Gln Ala Glu Tyr Phe Ile Asp Thr Ile Ser Gly Tyr Arg Ser             260 265 270 Asp Phe Ala Leu Phe Pro Glu Phe Phe Asn Ala Pro Leu Met Ala Ala         275 280 285 Tyr Asn His Leu Ser Glu Pro Asp Ala Ile Arg Glu Leu Ala Lys Tyr     290 295 300 Thr Glu Arg Ile Arg Asp Arg Phe Ser Glu Leu Ser Val Ser Tyr Asn 305 310 315 320 Ile Asn Ile Ile Thr Gly Ser Met Pro Tyr Met Glu Asp Gly Thr Leu                 325 330 335 Tyr Asn Val Gly Phe Leu Cys Lys Arg Asp Gly Thr Ile Glu Lys Phe             340 345 350 Tyr Lys Leu His Val Thr Pro Asp Glu Ala Lys Ala Trp Gly Met Ser         355 360 365 Gly Gly Glu Lys Leu Lys Thr Phe Asn Thr Asp Cys Gly Lys Ile Gly     370 375 380 Ile Leu Ile Cys Tyr Asp Val Glu Phe Pro Glu Leu Gly Arg Leu Leu 385 390 395 400 Ala Asp Glu Gly Met Asp Ile Leu Phe Val Pro Phe Leu Thr Asp Thr                 405 410 415 Gln Asn Gly Tyr Ser Arg Val Arg His Cys Ala Gln Ala Arg Ala Ile             420 425 430 Glu Asn Glu Cys Tyr Val Ala Ile Ala Gly Ser Val Gly Asn Ile Pro         435 440 445 Lys Val His Asn Met Asp Met Gln Phe Ala Gln Ser Met Val Leu Thr     450 455 460 Pro Cys Asp Phe Gln Phe Pro Thr Asn Gly Val Lys Ala Glu Ala Thr 465 470 475 480 Pro Asn Ala Glu Met Ile Leu Val Ala Asp Val Asp Leu Asp Leu Leu                 485 490 495 Lys Glu Leu Asn Gln Phe Gly Ser Val His Asn Leu Val Asp Arg Arg             500 505 510 Lys Asp Leu Phe Gln Leu Lys Trp Lys Lys Lys         515 520 <210> 12 <211> 1572 <212> DNA <213> Leeuwenhoekiella blandensis MED217 <220> <221> gene (222) (1) .. (1572) <223> nucleotide sequences of nitrilase LBN <400> 12 ctattttttc ttccacttaa gctgaaataa atcttttctt cggtctacca aattatgaac 60 actaccaaat tgatttagtt ctttcaatag atcaagatct acatctgcaa cgagaatcat 120 ctcagcattt ggcgtcgctt ccgccttaac gccgtttgta ggaaactgaa agtcacaggg 180 tgttaacacc atactttgtg caaattgcat atccatatta tgtacttttg ggatgttccc 240 cacgcttccg gcgatagcca cataacattc gttctcaata gcccgtgcct gcgcacaatg 300 acgcacacgg ctgtatccat tttgcgtatc ggttaagaag ggaacgaaga gaatatccat 360 tccttcatct gctagtaaac gacccaattc tggaaactca acatcataac aaatgaggat 420 tccaatctta ccgcaatcgg tattgaaggt tttcagtttt tcgccaccgc tcatccccca 480 ggctttggct tcgtccggtg ttacgtgcag tttataaaac ttttctatcg taccgtcacg 540 tttacataag aatcccacgt tatacaacgt accgtcttcc atatacggca tactccctgt 600 aatgatattg atattatagc tcaccgaaag ctcactaaac cgatctctta tgcgctcggt 660 atattttgct aattctcgta tcgcatcagg ttctgataag tgattgtacg ctgccataag 720 cggagcatta aagaactctg gaaacaatgc aaagtctgaa cgatacccag aaatggtgtc 780 gataaaatat tcggcctgct caaaaagttc ttcaaaattt ttataggtac gcatctgcca 840 ttgcaccaag cctaaacgta caacggtttt gatcgcattt ggcttttttg ccggcttttg 900 ataatagata ttgtcccatt ctaaaagcgc cgcatactct tttgattgct tatctccttc 960 taggtaattt gtaataatcc gttttacgtg aaaatcatta ctcaactgaa agtcaagcac 1020 gggatcgtta atctcacgcg cacgcacttt ctctatgtag gcttttggag aaagttcttt 1080 ggcatactga tgatagttgg gtaatcttcc gccaaaaaca atgcgttcta aattaagatt 1140 ctcacacaat tctttacgat aatcataaag acgtcgacca atgcgcatgc cacggtactt 1200 atgagaaata aagatatcta taccgtacaa tacatttccc ttgggcgtat gcgtactaaa 1260 agtttcatcg cctgtgattt ccatataggt atgcgaatca gaaaacttct cgtaatctac 1320 cacgatagaa agggcacaac cagcgatctc cccgtttacc ttgaccacta cttgaccttc 1380 cggaaaaatg gaaaccagtt tttgcagctc ctttttcttc caataaggat ctggaatatt 1440 gggataaaca gcgatcatga tctccttgag ttcttcaaag tcttttactt ttaaatactc 1500 aagattgatg gtgtctatgc gttcgcgctt ttctgcactg tctgaattat ctatgtgatc 1560 tacttctttc at 1572 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer lbnF <400> 13 cgacccggca tatgaaagaa gtagatcaca tagat 35 <210> 14 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> reverse primer lbnRH <400> 14 ctccacatct cgagtttttt cttccactta agctgaaa 38 <210> 15 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> reverse primer lbnRX <400> 15 ctccacatct cgagctattt tttcttccac ttaagctg 38 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> forward primer ornF <400> 16 cgacccggca tatgacgcaa actgtgatta ccgt 34 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> reverse primer or nRH <400> 17 ctccacatct cgagtaatcg tttatgcttc aacaatgg 38 <210> 18 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> reverse primer ornRX <400> 18 ctccacatct cgagtcataa tcgtttatgc ttcaacaa 38 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer rmn1F <400> 19 cgacccggca tatgagagaa gttaccgtag cgg 33 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer rmn1RH <400> 20 ctccacatct cgagagcgcg tccgtctttg gtcatt 36 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer rmn1RX <400> 21 ctccacatct cgagctaagc gcgtccgtct ttggtc 36 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer rmn2F <400> 22 cgacccggca tatgaagtta gcattggccc agc 33 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> reverse primer rmn2RH <400> 23 ctccacatct cgaggacccc cggcgcccag ccat 34 <210> 24 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> reverse primer rmn2RX <400> 24 ctccacatct cgagtcagac ccccggcgcc cagc 34 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer vmn1F <400> 25 cgacccggca tatgcttcaa ccattggcgc tcg 33 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer vmn1RH <400> 26 ctccacatct cgagacgatt caacgcagac gctatc 36 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> reverse primer vmn1RX <400> 27 ctccacatct cgagttaacg attcaacgca gacgcta 37 <210> 28 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer vmn2F <400> 28 cgacccggcc atggctaaga aagacatcaa agtagcg 37 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer vmn2RH <400> 29 ctccacatct cgagtttctc agcaaaacgc gcttgg 36 <210> 30 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> reverse primer vmn2RX <400> 30 ctccacatct cgagttattt ctcagcaaaa cgcgctt 37

Claims (10)

Preparing a biocatalyst composition comprising nitrile RMN2 or the nitrile RMN2 represented by the amino acid sequence of SEQ ID NO: 3; And
Reacting the nitrile RMN2 or biocatalyst composition with a nitrile substrate.
The method of claim 1, wherein the biocatalyst composition comprising nitrile RMN2 comprises a cell comprising nitrile RMN2, a cell culture comprising the cell or a cell lysate.
The method of claim 1, wherein the nitrile substrate is mandelonitrile.
The method of claim 1, wherein the nitrified RMN2 is obtained from a transformant transformed with a vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 3. The method of claim 4, wherein the nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 3 has a nucleotide sequence of SEQ ID NO: 4. 6.
A biocatalyst composition for preparing carboxylic acid comprising nitrile RMN2 represented by the amino acid sequence of SEQ ID NO: 3.
The composition of claim 6, wherein the biocatalyst composition comprises a cell comprising nitrile RMN2, a cell culture comprising the cell or a cell lysate.
The composition of claim 6, wherein the nitrified RMN2 is obtained from a transformant transformed with a vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 3. 8.
The composition of claim 8, wherein the nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 3 has a nucleotide sequence of SEQ ID NO: 4. 10. The composition of claim 6, wherein the nitrile RMN2 has a degrading activity against mandelonitrile.

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