BR112016026869B1 - IMPROVED NITRILE HYDRATASE AND METHOD FOR PRODUCING AN AMIDE COMPOUND - Google Patents

Abstract

IMPROVED NITRILE HYDRATASE AND METHOD FOR PRODUCING AN AMIDE COMPOUND. The present invention relates to new improved nitrile hydratase with greater resistance to amide compounds under high temperatures. Specifically there is provided a nitrile hydratase having at least one amino acid selected from (a) to (d) below, in the amino acid sequence expressed in SEQ ID NO:50 (X1 to X27 represent independent arbitrarily defined amino acid residues). (a) X1 is valine or glycine, (b) X9 is valine or threonine, (c) X23 is an amino acid selected from the group consisting of isoleucine, leucine, methionine and threonine, (d) X24 is leucine.

Description

field of invention

[0001] The present invention relates to an improved (mutated) nitrile hydratase and a method for producing the improved nitrile hydratase. Furthermore, the present invention relates to DNA encoding the enzyme, a recombinant vector containing the DNA, a transformant containing the recombinant vector and a method for producing an amide compound.

Background of the invention

[0002] A nitrile hydratase is an enzyme having a nitrile hydration activity that catalyzes the hydration of a nitrile group to an amide group. Furthermore, the corresponding amide compounds can be produced from the nitrile compounds using the enzyme or a microbial cell or the like which contains the enzyme. Compared to conventional chemical synthesis methods, this method is known to have high conversion rate and high selectivity rate of a nitrile compound to a corresponding amide compound.

[0003] Examples of microorganisms that produce a nitrile hydratase include the genus Corynebacterium, genus Pseudomonas, genus Rhodococcus, genus Rhizobium, genus Klebsiella, genus Pseudonocardia and the like. Among these, the J1 strain of Rhodococcus rhodochrous has been used for the industrial production of acrylamides and its usefulness has been verified. In addition, a gene encoding a nitrile hydratase produced by the strain has been identified (see Patent Publication 1).

[0004] However, introducing a mutation into a nitrile hydratase has been attempted not only to use a nitrile hydratase isolated from a naturally occurring microorganism or its genes, but also to alter its activity, substrate specificity, Vmax, Km, stability to heat, stability against a substrate, stability against a subsequent product and the like of a nitrile hydratase. As for the nitrile hydratase in Pseudonocardia thermophila JCM 3095, from its three-dimensional structure data, presumed sites relating to substrate specificity or thermal stability are obtained, and mutant enzymes with modified substrate specificity were obtained among them (see publications by patent 2 to 4). Also, nitrile hydratase genes with increased heat resistance and resistance to amide compounds were produced by the inventors of the invention (see patent publications 5 to 9).

[0005] However, developing a nitrile hydratase which further improved heat resistance and resistance to amide compounds and which can react at high temperatures and using nitrile hydratase to produce an amide compound are very useful from the point of view of in view of production costs such as costs involved with the catalyst, and obtaining enzymes with this performance is especially desired in order to obtain a reduction in the amount of enzyme for reactions and in production costs or the like.

CITATION LIST PATENT PUBLICATION

[0006] Patent Publication 1: JP 3162091 B

[0007] Patent Publication 2: WO 2004/056990 A

[0008] Patent Publication 3: JP 2004-194588 A

[0009] Patent Publication 4: JP 2005-160403 A

[0010] Patent Publication 5: WO 2005/116206 A

[0011] Patent Publication 6: JP 2007-143409 A

[0012] Patent Publication 7: JP 2007-43910 A

[0013] Patent literature 8: JP 2008-253182 A

[0014] Patent Literature 9: JP 2010-172295 A

SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0015] The object of the invention is to have a method for producing an amide compound with greater production efficiency providing a new improved nitrile hydratase with greater resistance to amide compounds under high temperatures.

WAYS TO SOLVE THE PROBLEM

[0016] To solve the problems described above, the inventors of the invention carried out intensive studies and, as a result, found that a protein in which a specific amino acid residue in the amino acid sequence of a nitrile hydratase is replaced by another amino acid residue which has a nitrile hydratase activity and shows greater resistance to amide-type compounds under high temperatures. The invention is completed accordingly.

[0017] Namely, the invention provides the following [1] to [13].

[0018] [1] An improved nitrile hydratase having at least one amino acid sequence represented by the following SEQ ID NO: 46 to 49 in the α subunit;

[0019] (a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E

[0020] (with the proviso that, R indicates arginine, K indicates lysine, A indicates alanine, E indicates glutamic acid, X1 indicates an amino acid other than tyrosine, and X2 to X8 each independently indicates any amino acid residue);

[0021] (b) SEQ ID NO: 47: X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC

[0022] (with the proviso that, N indicates asparagine, V indicates valine, C indicates cysteine, T indicates threonine, L indicates leucine, X9 indicates an amino acid other than serine, and X10 to X22 each independently indicates any amino acid residue );

[0023] (c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V

[0024] (with the proviso that, W indicates tryptophan, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, V indicates valine, X23 indicates an amino acid other than valine, and X25 to X29 each independently indicates any amino acid residue);

[0025] (d) SEQ ID NO: 49: VX24DSX25X26EX27RX28X29V

[0026] (with the proviso that, V indicates valine, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, X24 indicates an amino acid other than tryptophan, and X25 to X29 each independently indicates any residue of amino acid);

[0027] [2] The improved nitrile hydratase described above in the above [1], wherein the improved nitrile hydratase has at least one amino acid sequence represented by the following SEQ ID NO: 46 to 49 in the α-subunit; (a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E

[0028] (with the proviso that, R indicates arginine, K indicates lysine, A indicates alanine, E indicates glutamic acid, X1 indicates an amino acid other than tyrosine, and X2 to X8 each independently indicates any amino acid residue); (b) SEQ ID NO: 47:X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC

[0029] (with the proviso that, N indicates asparagine, V indicates valine, C indicates cysteine, T indicates threonine, L indicates leucine, X9 indicates an amino acid other than serine, and X10 to X20 each independently indicates any amino acid residue , X21 indicates valine and X22 indicates threonine);

[0030] (c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V

[0031] (with the proviso that, W indicates tryptophan, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, V indicates valine, X23 indicates an amino acid other than valine, and X25 to X29 each independently indicates any amino acid residue);

[0032] (d) SEQ ID NO: 49: VX24DSX25X26EX27RX28X29V

[0033] (with the proviso that, V indicates valine, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, X24 indicates an amino acid other than tryptophan, and X25 to X29 each independently indicates any residue of amino acid);

[0034] [3] The improved nitrile hydratase described above in the above [1], wherein the improved nitrile hydratase has at least one amino acid sequence represented by the following SEQ ID NO: 46 to 49 in the α-subunit;

[0035] (a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E

[0036] (with the proviso that, R indicates arginine, K indicates lysine, A indicates alanine, E indicates glutamic acid, X1 indicates glycine or valine, and X2 to X8 each independently indicates any amino acid residue); (b) SEQ ID NO: 47: X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC

[0037] (provided that, N indicates asparagine, V indicates valine, C indicates cysteine, T indicates threonine, L indicates leucine, X9 indicates valine or threonine, X10 to X20 each independently indicates any amino acid residue, X21 indicates valine and X22 indicates threonine);

[0038] (c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V

[0039] (with the proviso that, W indicates tryptophan, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, V indicates valine, X23 is selected from the group consisting of isoleucine, leucine, methionine and threonine, and X25 to X29 each independently indicate any amino acid residue);

[0040] (d) SEQ ID NO: 49: VX24DSX25X26EX27RX28X29V

[0041] (with the proviso that, V indicates valine, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, X24 indicates leucine, and X25 to X29 each independently indicates any amino acid residue);

[0042] [4] The improved nitrile hydratase described in any of the above [1] to [3], wherein

[0043] in SEQ ID NO: 46 above, X2 indicates T (threonine), X3 indicates E (glutamic acid), X4 indicates Y (tyrosine), X5 indicates E (glutamic acid), X6 indicates A (alanine), X7 indicates T (threonine) and X8 indicates I (isoleucine),

[0044] in SEQ ID NO: 47 above, X10 indicates A (alanine), X11 indicates V (valine), X12 indicates F (phenylalanine), X13 indicates D (asparaginic acid) X14 indicates S (serine), X15 indicates Q ( glutamine), X16 indicates T (threonine), X17 indicates H (histidine), X18 indicates H (histidine), X19 indicates V (valine), and X20 indicates V (valine) and

[0045] in SEQ ID NO: 48 and 49 above, X25 indicates S (serine), X26 indicates S (serine), X27 indicates I (isoleucine), X28 indicates Y (tyrosine) and X29 indicates I (isoleucine); It is

[0046] the amino acid sequence represented by SEQ ID NO: 46 corresponds to positions 8 to 19 of the amino acid sequence of the α subunit of a nitrile hydratase, the amino acid sequence represented by SEQ ID NO: 47 corresponds to positions 8 to 105 of same sequence and the amino acid sequence represented by SEQ ID NO: 48 and 49 corresponds to positions 153 to 164 of the same sequence;

[0047] [5] The improved nitrile hydratase described in any one of [1] to [4] above, wherein the improved nitrile hydratase has at least one amino sequence represented by SEQ ID NO: 50;

[0048] [6] An improved nitrile hydratase having at least one amino acid sequence represented by SEQ ID NO: 50 in the α subunit, characterized in that at least one mutation of amino acids selected from the following (i) to (iv) is included:

[0049] (i) X1 indicates G (glycine) or V (valine),

[0050] (ii) X9 indicates V (valine) or T (threonine),

[0051] (iii) X23 is an amino acid selected from the group consisting of I (isoleucine), L (leucine), M (methionine) and T (threonine), and

[0052] (iv) X24 indicates L (leucine);

[0053] [7] The nitrile hydratase described above in [6], where X2 indicates T (threonine), X3 indicates E (glutamic acid), X4 indicates Y (tyrosine), X5 indicates E (glutamic acid), X6 indicates A (alanine), X7 indicates T (threonine), X8 indicates I (isoleucine), X10 indicates A (alanine), X11 indicates V (valine), X12 indicates F (phenylalanine), X13 indicates D (asparaginic acid) X14 indicates S (serine), X15 indicates Q (glutamine), X16 indicates T (threonine), X17 indicates H (histidine), X18 indicates H (histidine), X19 indicates V (valine), X20 indicates V (valine), X25 indicates S ( serine), X26 indicates S (serine), X27 indicates I (isoleucine), X28 indicates Y (tyrosine) and X29 indicates I (isoleucine);

[0054] [8] The improved nitrile hydratase described in any one of [1] to [7] above, in which the nitrile hydratase is derived from Rhodococcus bacterium or Nocardia bacterium;

[0055] [9] The DNA encoding the improved nitrile hydratase described in any one of [1] to [8] above, or the DNA which hybridizes under stringent conditions with DNA having a base sequence complementary to the aforementioned DNA and encodes a protein having a nitrile hydratase activity with increased resistance to amide compounds under high temperatures;

[0056] [10] A recombinant vector containing the DNA described in [9] above;

[0057] [11] A transformant containing the recombinant vector described in [10] above;

[0058] [12] A method for producing a nitrile hydratase, the method including culturing the transformant described in [11] above and collecting nitrile hydratase from the obtained culture; It is

[0059] [13] A method for producing an amide compound, the method including contacting a nitrile compound with the improved nitrile hydratase described in any one of [1] to [8] above, or with a culture which is obtained by culturing the transformant described in [11] above or a processed product of the culture.

[0060] EFFECT OF THE INVENTION

[0061] According to the invention, a new improved (mutated) nitrile hydratase with increased resistance to amide compounds under high temperatures can be provided. The improved nitrile hydratase of the invention has excellent resistance to amide compounds under high temperatures and allows for improved efficiency for producing amide-type compounds.

[0062] BRIEF DESCRIPTION OF THE FIGURES

[0063] Fig. 1 is a schematic illustration illustrating plasmid pSJ034;

[0064] Fig. 2-1 is a drawing illustrating the amino acid sequence (part of the N-terminal side) of the α-subunit of nitrile hydratase derived from various microorganisms.

[0065] Fig. 2-2 is a drawing illustrating the same amino acid sequences as Fig. 2-1, and shows sequences subsequent to the amino acid sequences of Fig. 2-1.

[0066] Fig. 3 illustrates the amino acid sequence of the α subunit of the invention which is represented by SEQ ID NO: 50.

[0067] This application claims priority benefit from Japanese Patent Application No. 2014-118041 (filed June 6, 2013), which is incorporated in full herein by reference.

MODE(S) FOR CARRYING OUT THE INVENTION 1. Nitrile Hydratase 1.1 Known Nitrile Hydratase

[0068] A “nitrile hydratase” has a high dimensional structure consisting of a group of α and β subunit domains, and contains a non-heme iron atom or non-corrin cobalt atom as a prosthetic molecule. These nitrile hydratases are identified and referred to as an iron-containing nitrile hydratase and cobalt-containing nitrile hydratase, respectively.

[0069] A representative example of iron-containing nitrile hydratase includes a hydratase derived from the N-771 strain of Rhodococcus. The three-dimensional structure of such an iron-containing nitrile hydratase has been clearly identified by X-ray crystal structural analysis. The enzyme is linked with nonheme iron through four amino acid residues in a cysteine cluster (Cys-Ser-Leu-Cys- Ser-Cys) forming the active site of the α subunit.

[0070] As for the cobalt-containing nitrile hydratase, examples are those derived from the J1 strain of Rhodococcus rhodochrous (which may be further referred to as “J1 strain”) or those derived from Pseudonocardia thermophila.

[0071] A cobalt-containing nitrile hydratase derived from the J1 strain is linked with a cobalt atom through a region identified as a cysteine cluster (Cys-Thr-Leu-Cys-Ser-Cys) that forms the active site of the α subunit . In the cobalt-containing nitrile hydratase cysteine cluster derived from Pseudonocardia thermophila, the cysteine (Cys) at position 4 from the upstream side (N-terminal side) of the cysteine cluster derived from the J1 strain is cysteine sulfinic acid (Csi) , and the cysteine (Cys) at position 6 from the far downstream side (C-terminal side) of the cysteine cluster derived from the J1 strain is sulfenic cysteic acid (Cse).

[0072] As described above, a prosthetic molecule is linked with a region identified as "C(S/T)LCSC" cysteine clusters in the α-subunit. Examples of nitrile hydratase containing a binding region with a prosthetic molecule are those that have the amino acid sequences and are encoded by gene sequences derived from the following: Rhodococcus rhodochrous J1 (FERM BP-1478), Rhodococcus rhodochrous M8 (Old Union patent Soviet No. 1731814 (SU 1731814), Rhodococcus Rhodochrous M33 (VKM AC-1515D), Rhodococcus Rhodochrous ATCC 39484 (JP 2001292772 a), Bacillus Smithii (JP 9-248188 a), Pseudonocardia Thermophila (JP 9-2 75978 a) or geobacillus thermoglucosidasius.On the other hand, the β subunit is thought to be attributed to structural stability.

[0073] The nitrile hydratase derived from the J1 strain of Rhodococcus rhodochrous (FERM BP-1478) has the GenBank accession number “P21220”. Furthermore, the GenBank accession number of the α subunit derived from Rhodococcus rhodochrous M8 (SU 1731814) is “ATT79340” and the GenBank accession number of the β subunit is “AAT 79339”. The GenBank accession number of the nitrile hydratase gene derived from Rhodococcus pyridinivorans MW3 is “AJ582605” and the GenBank accession number of the nitrile hydratase gene derived from Rhodococcus pyridinivorans S85-2 is “AJ582605”. The Rhodococcus ruber RH nitrile hydratase gene (CGMCC No. 2380) is described in Chinese Patent No. 101463358 (CN1463358). Furthermore, the GenBank accession number of the nitrile hydratase gene derived from Nocardia YS-2002 is “X86737” and the GenBank accession number of the nitrile hydratase gene derived from Nocardia sp. JBRs is “AY141130.0”.

[0074] In SEQ ID NOs: 1 to 19 of the sequence listing, the amino acid sequence and base sequence of the known nitrile hydratase are described.

[0075] SEQ ID NO: 1: base sequence of the β subunit derived from Rhodococcus rhodochrous J1

[0076] SEQ ID NO: 2: amino sequence derived from Rhodococcus rhodochrous J1 of the β subunit

[0077] SEQ ID NO: 3: base sequence derived from Rhodococcus rhodochrous J1 of the α subunit

[0078] SEQ ID NO: 4: amino sequence derived from Rhodococcus rhodochrous J1 of the α subunit

[0079] SEQ ID NO: 5: amino sequence derived from Rhodococcus rhodochrous M8 of the α subunit

[0080] SEQ ID NO: 6: amino sequence derived from Rhodococcus ruber TH of the α subunit

[0081] SEQ ID NO: 7: amino sequence derived from Rhodococcus pyridinivorans MW33 of the α subunit

[0082] SEQ ID NO: 8: amino sequence derived from Rhodococcus pyridinivorans S85-2 of the α subunit

[0083] SEQ ID NO: 9: amino sequence derived from Rhodococcus pyridinivorans MS-38 of the α subunit

[0084] SEQ ID NO: 10: amino sequence derived from Nocardia sp. α-subunit JBRs

[0085] SEQ ID NO: 11: amino sequence derived from Nocardia sp. α subunit YS-2002

[0086] SEQ ID NO: 12: amino sequence derived from non-cultured bacteria SP1 of the α subunit

[0087] SEQ ID NO: 13: amino sequence derived from non-cultured bacteria BD2 of the α subunit

[0088] SEQ ID NO: 14: amino sequence derived from Rhodococcus rhodochrous ATCC39484 of the α subunit

[0089] SEQ ID NO: 15: amino sequence of the α subunit of Sinorhizobium medicae WSM419

[0090] SEQ ID NO: 16: amino sequence of the α subunit of Sinorhizobium medicae Q6

[0091] SEQ ID NO: 17: amino sequence of the α subunit of Pseudonocardia thermophila JCM3095

[0092] SEQ ID NO: 18: amino sequence of the α subunit derived from Rhodococcus rhodochrous Cr4

[0093] SEQ ID NO: 19: amino sequence of the α subunit derived from Comamonas testosterone.

[0094] In addition, Fig. 2-1 and Fig. 2-2 show the amino acid sequence alignments (in one-letter code) in the α-subunits of known nitrile hydratase derived from various microorganisms. In each of Fig. 2-1 and Fig. 2-2, each amino acid sequence corresponds to SEQ ID NO: 4, 5 to 19 in order from above.

[0095] The nitrile hydratase according to the invention is not limited to one with the above sequence, but includes a protein having an amino acid sequence that is homologous or identical to the amino acid sequence described in any one of SEQ ID NO: 1 to 19 about 60% or greater, preferably about 70% or greater, more preferably about 80% or greater, even more preferably about 90% or greater, particularly preferably about 95% or greater and more preferably at about 98% or greater, while also having nitrile hydratase activity.

[0096] Furthermore, regarding the nitrile hydratase of the invention, a protein having the amino acid sequence described in any one of SEQ ID NOs: 1 to 19 in which 1 to various amino acids, specifically, 1 to 20, preferably 1 to 10, more preferably from 1 to 5 and even more preferably from 1 to 2 amino acids are deleted, substituted or added, and also having nitrile hydratase activity is also included in the nitrile hydratase of the invention.

1.2 Improved Nitrile Hydratase

[0097] The improved nitrile hydratase of the invention is a new improved nitrile hydratase with increased resistance to amide compounds under high temperatures.

[0098] The improved nitrile hydratase of the invention is not limited to being derived from any specific type. For example, those registered as nitrile hydratase in the GenBank database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=prote in) provided by the National Center for Biotechnology Information ( NCBI) of the United States, or those which are described as nitrile hydratase in publications, may be referred to for use.

[0099] Specific examples include nitrile hydratases which are described in WO 2005/116206 A, JP 2007-143409 A, JP 2007-43910 A, JP 2008-253182 A or JP 2010-172295 A (incorporated herein by reference) . These nitrile hydratases have heat resistance or acrylamide strength. By further adding an amino acid substitution according to the invention, a property to improve resistance to starch compounds under high temperatures can be obtained.

[00100] Examples of the improved nitrile hydratase of the invention include a nitrile hydratase of which the α subunit has the amino acid sequence (SEQ ID NO: 50) shown in Fig. 3. Among the amino acid sequences shown in Fig. 3, has the amino acid sequence represented by SEQ ID NO: 46 at the 8th to 19th amino acids when counted from the N-terminus, the amino acid sequence represented by SEQ ID NO: 47 at the 88th to 105th amino acids, the sequence of amino acids represented by SEQ ID NO: 48 or SEQ ID NO: 49 at the 153rd to 165th amino acids.

[00101] About the amino acid sequence represented by SEQ ID NO: 50, one having at least one mutation of amino acids selected from (a) to (d) can be referred to as an embodiment of the invention (X1 to X29 represent a residue independent arbitrary amino acid).

[00102] (a) X1 is glycine or valine

[00103] (b) X9 is valine or threonine,

[00104] (c) X23 is an amino acid selected from the group consisting of isoleucine, leucine, methionine and threonine

[00105] (d) X24 is leucine.

[00106] With another embodiment, an improved nitrile hydratase having the amino acid sequence represented by SEQ ID NO: 50, where X2 is T (threonine), X3 is E (glutamic acid), X4 is Y (tyrosine), X5 is E (glutamic acid), X6 is A (alanine), X7 is T (threonine), X8 is I (isoleucine), X10 is A (alanine), X11 is V (valine), X12 is F (phenylalanine), X13 is D (asparaginic acid) X14 is S (serine), X15 is Q (glutamine), X16 is T (threonine), X17 is H (histidine), X18 is H (histidine), X19 is V (valine), X20 is V (valine), X25 is S (serine), X26 is S (serine), X27 is I (isoleucine), X28 is Y (tyrosine), X29 is I (isoleucine) and also has at least one feature selected from (a) (d) above may be mentioned.

[00107] However, also included in the improved nitrile hydratase of the invention is a nitrile hydratase that is homologous or identical, at a position other than said substitution positions, to the amino acid sequence described in SEQ ID NO: 50 in about 70 % or greater, preferably about 80% or greater, more preferably about 90% or greater, even more preferably about 95% or greater, particularly preferably about 98% or greater, while also having heat resistant and/or resistance to amide compounds.

[00108] Also included in the improved nitrile hydratase of the invention is a nitrile hydratase having the amino acid sequence described in SEQ ID NO: 50 in which 1 to 10, preferably 1 to 5 and more preferably 1 to 2 amino acids are excluded , substituted or added to a position other than the mentioned substitution positions and has the same heat resistance and/or resistance to amide compounds.

[00109] As another example of an improved nitrile hydratase of the invention, with respect to the amino acid sequence represented by SEQ ID NO: 4 of a known nitrile hydratase, one having at least one characteristic selected from (e) to (h) can be mentioned.

[00110] (e) the 8th amino acid residue (tyrosine) of the α subunit is replaced with glycine or valine

[00111] (f) the 88th amino acid residue (serine) of the α subunit is replaced with valine or threonine

[00112] (g) the 153rd amino acid residue (valine) of the α subunit is replaced with an amino acid selected from isoleucine, leucine, methionine and threonine

[00113] (h) the 154th amino acid residue (tryptophan) of the α subunit is replaced with leucine

[00114] Meanwhile, also included in the improved nitrile hydratase of the invention is a nitrile hydratase that is homologous or identical to the amino acid sequence described in SEQ ID NO: 4 by about 70% or greater, preferably about 80% or greater , more preferably about 90% or greater, even more preferably about 95% or greater, particularly preferably about 98% or greater, at a position other than the aforementioned substitution positions, and also has the same heat resistance and/or resistance to amide compounds.

[00115] Furthermore, with respect to the amino acid sequence identified by SEQ ID NO: 4, a nitrile hydratase having, in a substitution position other than that described above, an amino acid sequence in which 1 to 10, preferably about 1 to 5, and even more preferred 1 to 2 amino acid residues are deleted, substituted or added and having the same heat resistance and/or resistance to amide compounds is also included in the improved nitrile hydratase of the invention.

[00116] The above amino acid substitutions from (e) to (h) are described as “Yα8G, Sα88V, Vα153I, Wα154L”. Standard amino acids are identified by a single letter alphabetic code. The letter to the left of the numeral showing the replaced position (i.e., number of amino acid residues for the substituted site) represents the amino acid in a one-letter code before the substitution, and the letter to the right represents the amino acid in a one-letter code after replacement.

[00117] In particular, with regard to the amino acid sequence of the α subunit as shown in SEQ ID NO: 4, if there is a description of “Yα8G”, it means a modality having amino acid substitution in the improved nitrile hydratase in which tyrosine ( Y) at position 8 counted from the N-terminal amino acid residues (including the N-terminal amino acid residue itself) of the α-subunit amino acid sequence (SEQ ID NO: 4) is replaced with glycine (G).

[00118] Modes of amino acid substitutions in more preferred embodiments of the improved nitrile hydratase according to the invention are shown as the following 1 to 8:

[00119] 1. Yα8G

[00120] 2. Yα8V

[00121] 3. Sα88V

[00122] 4. Sα88T

[00123] 5. Vα153I

[00124] 6. Vα153L

[00125] 7. Vα153M

[00126] 8. Vα153T

[00127] 9. Wα154L

[00128] Preferred embodiments of base substitutions to bring about the above amino acid substitutions are shown below. Table 1

[00129] With regard to the improved nitrile hydratase activity of the invention, resistance to amide compounds under high temperatures is improved over wild-type nitrile hydratase activity while naturally derived characteristics are maintained.

[00130] Here, “nitrile hydratase activity” means an enzymatic activity to catalyze hydration for the conversion of a nitrile compound to a corresponding amide compound (RCN+H2O RCONH2). Activity determination is conducted by contacting a nitrile compound as substrate with a nitrile hydratase for conversion to a corresponding amide compound and quantifying the resulting amide compound. Any nitrile compound can be used as a substrate as long as the nitrile hydratase reacts with a compound, but acrylonitrile is preferred.

[00131] The reaction conditions include a substrate concentration of 2.5%, reaction temperature of 10°C to 30°C and reaction time of 10 to 30 minutes. Enzymatic reactions are terminated by addition of phosphoric acid. Then, using HPLC (high performance liquid chromatography) or gas chromatography, the acrylamide produced is analyzed to measure the amount of the amide compound.

[00132] The expression “resistance to amide compounds under high temperatures” means that, even in the presence of amide-type compounds, the activity of nitrile hydratase is maintained under high temperatures. The expression "high temperatures" specifically denotes 40°C to 60°C and more preferably 45°C to 55°C.

[00133] The “resistance to amide compounds under high temperatures” can be assessed by analyzing a culture of transformants containing an improved nitrile hydratase, or an improved nitrile hydratase isolated from the transformant in the presence of an amide compound such as acrylamide (at a high concentration 30 to 50%, for example) under high temperatures based on the amount of consumption or rate of consumption of a nitrile compound such as acrylonitrile as a substrate. For example, when the improved nitrile hydratase is brought into contact with an amide compound in the range of 40°C to 60°C and the nitrile hydratase shows consumption amount or consumption rate of 1.1 times or more, preferably 1 .15 times or more and more preferably 1.2 times or more than the comparative example (i.e. nitrile hydratase with no mutation), it can be evaluated to be resistant to amide-type compounds under high temperatures.

[00134] As for the "amide compounds", an amide compound represented by the general formula (1) below, for example, can be cited.

[00135] R-CONH2 (1)

[00136] (in the formula, R is an optionally substituted linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, an optionally substituted cycloalkyl or aryl group having 3 to 18 carbon atoms, or a saturated heterocyclic group or optionally substituted unsaturated). Particularly preferred is an acrylamide where "R" in the formula is "CH 2 = CH-".

[00137] The above improved nitrile hydratase is obtained by substituting amino acids in a known nitrile hydratase. For example, such an improved nitrile hydratase is obtained by introducing said mutation in the amino acid sequence (SEQ ID NO: 4) of a nitrile hydratase derived from the J1 strain of Rhodococcus rhodochrous and by screening for a nitrile hydratase with an improved resistance to the amide compound at high temperatures.

[00138] Even for a nitrile hydratase derived from these other than the J1 strain, resistance to amide-type compounds under high temperatures can be enhanced by introducing the same mutation to a corresponding site for modification. Examples of bacteria for producing a nitrile hydratase include Rhodococcus rhodochrous M8 (SEQ ID NO: 5), Rhodococcus ruber TH (SEQ ID NO: 6), Rhodococcus rhodochrous M33 (VKM Ac-1515D), Rhodococcus pyridinivorans MW3 (SEQ ID NO: 7 ), Rhodococcus pyridinivorans S85-2 (SEQ ID NO: 8), Nocardia sp. JBRs (SEQ ID NO: 10) and Nocardia YS-2002 (SEQ ID NO: 11). However, Rhodococcus rhodochrous M33 (MDV Ac-1515D) was selected because it is capable of constitutive expression of a nitrile hydratase based on a natural mutation of the M8 bacterium above and the amino acid or gene sequence of the nitrile hydratase itself is not mutant (US 5,827,699).

[00139] The improved nitrile hydratase of the invention can be obtained by introducing a mutation, either randomly or site-specifically, to a gene encoding a known nitrile hydratase according to a known method and selecting the enzyme with the desired function, i.e. resistance to amide compounds under high temperatures.

[00140] Examples of a method for introducing a mutation include a random mutation introduction method like error-prone PCR and site-directed mutagenesis like Kunkel's method or Gapped Duplex method.

error-prone PCR

[00141] As a method for studying functions and characteristics of proteins through a mutant, random mutagenesis is known. Random mutagenesis is a method of introducing a random mutation into the gene encoding a specific protein so that a mutant is produced. In random mutagenesis by PCR, stringent conditions are set low for the period of DNA amplification so that a mutated base can be introduced (error-prone PCR).

[00142] In such an error-prone PCR method, a mutation is randomly introduced at any position of the entire DNA site to be amplified. Then, examining the function of the obtained mutant, in which the mutation is introduced in a random site, the amino acid information or the important domain for a specific function of a protein is obtained. As the nitrile hydratase used for the error-prone PCR model, the nitrile hydratase gene derived from a wild-type strain or DNA obtained as an amplified product by the error-prone PCR can be used.

[00143] As reaction conditions for error-prone PCR, for example, a composition ratio of any one, two or three among dNTP (dGTP, dCTP, dATP or dTTP) in the reaction mixture is reduced relative to the other dNTP. Therefore, during DNA synthesis, in a position that requires a dNTP whose ratio is reduced, another dNTP is more likely to be used by mistake and this can lead to mutation. Furthermore, other preferred reaction conditions are a composition in which the amount of MgCl2 and/or MnCl2 in the reaction mixture is increased.

Site-directed mutagenesis (site-specific introduction of mutation)

[00144] As for the method of introducing a site-specific mutation, a general method is as follows: the DNA strand containing a target gene is dissociated into a single strand and tempered to an oligonucleotide chain containing a target gene, which is then prepared as a double strand by elongating the single strand using a DNA polymerase, the double strand is combined in E. coli for replication and, after fusion replication in E. coli, a clone including the desired mutation is selected (see Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) or the like). In addition to the Kunkel method, various methods such as the Gapped Duplex method are known, and the method can be conveniently performed using a commercially available mutagenesis kit such as the QuickChange™ XL site-directed mutagenesis kit (manufactured by Stratagene), mutagenesis system GeneTailor™ site-directed mutagenesis system (manufactured by Invitrogen Corporation), TaKaRa site-directed mutagenesis system (Mutan-K, Mutan-Super Express Km and the like, manufactured by Takara Bio Inc), or the like.

[00145] In addition to the method including introducing a mutation to a known nitrile hydratase gene as described above, the improved nitrile hydratase of the invention can also be obtained by metagenome screening from environmental DNA.

1.3 DNA encoding the improved nitrile hydratase

[00146] The invention also provides the DNA encoding the improved nitrile hydratase of the invention.

[00147] The "DNA encoding the improved nitrile hydratase" of the invention also includes DNA that has hybridized under stringent conditions to a DNA having a base sequence complementary to the base sequence of the DNA encoding the improved nitrile hydratase of the invention, and also encodes a protein with nitrile hydratase activity that is resistant to amide compounds under high temperatures.

[00148] “Strict conditions” are those for washing after hybridization; a salt concentration of 300 to 2000 mM and a temperature of 40 to 75°C, preferably a salt concentration of 600 to 900 mM and a temperature of 65°C. For example, 2XSSC conditions at 50°C can be employed. In addition to such buffer salt concentration, temperature and the like, a person skilled in the art can define the conditions for obtaining DNA encoding a nitrile hydratase of the invention by adding various conditions such as probe concentration, probe length, time of reaction and the like.

[00149] For detailed information on termination of hybridization, Molecular Cloning, A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press (1989)) or the like may be referred to. DNA to be hybridized includes DNA or partial fragment thereof, containing a base sequence that has 40% or greater, preferably 60% or greater and more preferably 90% or greater homology to the DNA of the gene of the invention.

1.4 Recombinant vector, transformant

[00150] It is necessary that the DNA encoding the improved nitrile hydratase gene be implanted in a vector so that the nitrile hydratase is expressed in the host organism to be transformed. Examples of such vectors to be used include plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA and the like.

[00151] In addition to a nitrile hydratase gene, a vector can be coupled with a promoter, terminator, enhancer, splice signal, poly A plus signal, selection marker, ribosome binding sequence (SD sequence) or the like. Examples of selectable markers include kanamycin resistance gene, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, and the like.

[00152] A host to be used in the invention is not limited to any specific type as long as it can express the target nitrile hydratase after the recombinant vector is introduced into the host. Examples include bacteria such as Escherichia coli and Bacillus subtilis, yeast, cells of animal origin, insect cells, plant cells and the like.

[00153] When E. coli is used as a host, an expression vector with high expression efficiency, such as pkk 233-2 expression vector with a trc promoter (manufactured by Amersham Biosciences Corp), pTrc 99A (manufactured by Amersham Biosciences Corp) or the like is preferable.

[00154] When a bacterium is used as a host, Escherichia coli can be used, for example, and a strain of Rhodococcus such as Rhodococcus rhodochrous ATCC 12674, Rhodococcus rhodochrous ATCC 17895 and Rhodococcus rhodochrous ATCC 19140 can also be used. ATCC strains can be obtained from the American Type Culture Collection. Method for introducing a recombinant vector into a bacterium is not limited to any specific method as long as the DNA is introduced into the bacterium. For example, a method utilizing calcium ions, electroporation or the like can be employed.

[00155] When yeast is used as a host, examples are Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like. As a method for introducing a recombinant vector into yeast, it is not specifically limited as long as the DNA can be introduced into yeast. For example, an electroporation method, spheroplast method, lithium acetate method, or the like can be employed.

[00156] When animal cells are used as a host, COS-7 monkey cells, Vero cells, CHO cells, mouse L cells, mouse GH3 cells, human FL cells or the like can be employed. As a method for introducing a recombinant vector into cells of animal origin, for example, electroporation method, calcium phosphate method, lipofection method or the like can be used.

[00157] When insect cells are used as a host, Sf9 cells, Sf21 cells or the like can be used. A method for introducing a recombinant vector into insect cells, for example, electroporation method, calcium phosphate method, lipofection method or the like can be used.

[00158] When plant cells are used as a host, BY-2 tobacco cells or the like can be used, but not limited to them. A method for introducing a recombinant vector into plant cells, for example, Agrobacterium method, particle gun method, PEG method, electroporation method or the like can be used.

[00159] When E. coli is used as a host, since most of the expressed nitrile hydratases are formed by an inclusion body and are insoluble, a transformant with low catalytic activity is obtained. On the other hand, if a Rhodococcus strain is used as a host, nitrile hydratase is present in the soluble fraction and, therefore, a transformant with high activity is obtained. Host can be selected based on purposes. However, when an improved enzyme is selected under stringent conditions, a high activity transformant derived from a Rhodococcus strain is preferred.

1.5 Method for producing improved nitrile hydratase

[00160] Improved nitrile hydratase can be produced by culturing the above transformant and collecting a protein with nitrile hydratase activity from the culture obtained. The invention also provides a method of producing an improved nitrile hydratase.

[00161] In the invention, "culture" means any culture supernatant, cultured cells, cultured bacterial cells and cell homogenates or bacterial cell homogenates.

[00162] Culturing a transformant is carried out according to a method that is generally used for culturing a host. As for a culture medium of a transformant of the invention, a natural or synthetic culture medium is used as long as it contains a carbon source, a nitrogen source, inorganic salts or the like for the host bacterium to assimilate and culture of a transformant is performed. efficiently. Examples of a carbon source include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch; organic acids such as acetic acid and propionic acid; alcohols such as ethanol and propanol; and the like. Examples of a nitrogen source include inorganic acids such as ammonium, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate and ammonium salts of organic acids and other nitrogen-containing compounds.

[00163] In addition, peptone and yeast extract, meat extract, with corn steep liquor, various amino acids or the like can also be used. Examples of an inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium carbonate and the like. Also, if necessary, a defoaming agent can be used to prevent foaming during the culturing process. In addition, cobalt ions or iron ions as a nitrile hydratase prosthetic molecule, or nitriles and amides as an enzyme inducer, can also be added to the culture.

[00164] Culture can be performed by adding selective pressure to prevent the vector and target gene from being eliminated. That is, if a selectable marker is a drug-resistant gene, a corresponding drug can be added; or if a selectable marker is a complementary gene, corresponding nutritional factors can be removed.

[00165] Also, if a selectable marker is an assimilation addition gene, an equivalent assimilation factor can be added as a single factor if necessary. For example, when E. coli transformed by a vector that contains an ampicillin resistant gene is cultured, ampicillin can be added as needed during the culture process.

[00166] When culturing a transformant transformed by a recombinant vector containing, as a promoter, an inducible promoter, such an inducer can be added to the medium, if necessary. For example, when culturing a transformant transformed by an expression vector with an isopropyl-β-D-thiogalactopyranoside (IPTG) inducible promoter, IPTG or the like can be added to the medium. Likewise, when culturing a transformant transformed by an expression vector with an indoleacetic acid (IAA) inducible trp promoter, IAA or the like can be added to the medium.

[00167] The culture conditions of a transformant are not specifically limited as the productivity of the improved nitrile hydratase and the growth of the host are not prohibited. Generally, conditions are preferred to be 10°C to 40°C, more preferably 20°C to 37°C, for 5 to 100 hours. The pH value is adjusted using inorganic or organic acid, alkaline solution or the like. If it is Rhodococcus, the pH is adjusted to be 6 to 9.

[00168] As for culture methods, solid state culture, static culture, shake culture, aeration-shake culture and the like can be used. When a Rhodococcus transformant is cultured, in particular, it is preferable to use shake culture or aeration-shake culture (flask fermentation) under aerobic conditions.

[00169] When grown under the above culture conditions, the improved nitrila hydratase of the invention is accumulated in a high yield in the above culture product, i.e. at least in any of the culture supernatant, cultured cell, cultured bacterial cell, cell homogenates or bacterial cell homogenates.

[00170] After culturing, when an improved nitrile hydratase is produced in a cell or bacterial cell, the target nitrile hydratase can be collected by homogenizing the cells or bacterial cells. Bacterial cells or cells are homogenized by high-pressure treatment using a French press or homogenizer, supersonic treatment, grinding treatment with glass beads or the like, enzymatic treatment with lysozyme, cellulase, pectinase and the like, freeze-thaw treatment, with hypotonic solution, phage bacteriolysis induction treatment, and so on.

[00171] After homogenization, residues of cell homogenates or bacterial cell homogenates (including insoluble fractions of cell extract) are removed, if necessary. To remove residues, filtration or centrifuge methods are used if necessary. To increase the efficiency of residue removal, a coagulant or filter aid can be used. The supernatant obtained after waste removal is soluble fractions of the cell extract, which can be used as a crudely purified improved nitrile hydratase solution.

[00172] Also, when an improved nitrile hydratase is produced in a bacterial cell or in cells, it is also possible that the bacterial cells or the cells themselves are collected by a centrifuge or membrane filtration to be used without homogenizing them.

[00173] When an improved nitrile hydratase is produced outside the bacterial cells or cells, the culture can be used as is, or the bacterial cells or cells are removed using a filter or centrifuge method. Next, the enhanced nitrile hydratase is collected from the culture by extracting it through ammonium sulfate precipitation, if necessary. In addition, dialysis or various chromatography techniques (gel filtration, ion exchange chromatography and affinity chromatography, etc.) can be used to isolate and purify nitrile hydratase.

[00174] The efficiency to produce a nitrile hydratase, which is obtained by culturing a transformant, can be confirmed in one unit per culture solution, wet weight or dry weight of bacterial cells, crude enzyme solution protein or the like by SDS-PAGE (polyacrylamide gel electrophoresis), measurements of nitrile hydratase activity, or the like, but not particularly limited thereto. SDS-PAGE can be performed by a method known to a person skilled in the art. Also, as for the nitrile hydratase activity, the activity described above can be used.

[00175] Other than the methods described above, an improved nitrile hydratase can be produced through a cell-free protein synthesis system. In a cell-free protein synthesis system, a protein is synthesized in an artificial vessel such as a test tube using a cell extract. A cell-free protein synthesis system used in the present invention includes a cell-free transcription system that synthesizes RNA using DNA as a template.

[00176] In such a case, an organism corresponding to the above host corresponds to the organism from which the cell extract is derived. Here, for the cell extract, extracts of eukaryotic or prokaryotic origin such as wheat germ extract, E. coli and the like can be used. Such cell extracts may or may not be concentrated.

[00177] The cell extract can be obtained by ultrafiltration, dialysis, polyethylene glycol (PEG), precipitation or the like, for example. In the invention, a commercially available kit can also be used for cell-free protein synthesis. Examples of such kits include a PROTEIOS™ reagent kit (TOYOBO CO., LTD), TNT™ system (Promega Corporation), a PG-Mate™ synthesizer (TOYOBO CO., LTD), RTS (Roche Diagnostics K.K.) and the like.

[00178] An improved nitrile hydratase obtained by cell-free protein synthesis as described above can also be purified by correctly selecting a chromatography type.

2. Method for producing amide compound

[00179] The improved nitrile hydratase of the invention can be used as an enzyme catalyst for material production. For example, an amide compound is produced by bringing a nitrile compound into contact with the improved nitrile hydratase. Then, the amide compound produced upon contact is collected. Therefore, an amide compound is produced.

[00180] As an enzyme catalyst, in addition to nitrile hydratase isolated and purified as described above, a culture after culturing the transformants of the invention or a processed product of the culture can also be used. Examples of processed product include post-culture cells (i.e., transformants) immobilized with acrylamide gel or the like, those processed by glutaraldehyde, those supported by inorganic carriers such as alumina, silica, zeolite, diatomaceous earth, and the like.

[00181] Here, “contact” means that an improved nitrile hydratase and a nitrile compound are present in the same reaction system or culture system: for example, an isolated and purified improved nitrile hydratase and a nitrile compound are mixed; a nitrile compound is added into a one-cell culture vessel (transformant) to express an improved nitrile hydratase; the cells are cultured in the presence of a nitrile compound; an extract of the cells is mixed with a nitrile compound; and so on.

[00182] A nitrile compound to be used as a substrate is selected by considering the substrate specificity of the enzyme, the stability of the enzyme to the substrate, and the like. As for the nitrile compound, acrylonitrile is preferred. The reaction method and the method of collecting an amide compound after completion of the reactions are properly selected depending on the characteristics of the substrate and enzyme catalyst.

[00183] The enzyme catalyst is preferred to be recycled as long as its activity is not lost. From the point of view of preventing loss of activity and ease of recycling, enzyme catalyst is preferred for use as a processed product.

EXAMPLE

[00184] Further on in this document, the invention is more specifically explained in view of the examples. However, the invention is not limited to them. However, “%” described here indicates % by mass.

Example 1 Preparation of plasmid to express the improved nitrile hydratase

[00185] A plasmid to be a model for introducing the amino acid substitution of the invention was prepared as follows.

[00186] As a model having the nitrile hydratase gene from the J1 strain, pSJ034 was used (Fig. 1). pSJ034 is a plasmid capable of expressing nitrile hydratase in a strain of Rhodococcus. Plasmid pJD034 was produced from pSJ023 by the method disclosed in JP 10-337185 A. That is, according to partial cleavage at the XbaI site and ligation with the SSE8387I linker, plasmid pSJ033 was prepared such that an XbaI site of the plasmid pSJ023 was replaced with Sse8387I. Then, plasmid pSJ033 was partially cleaved at the Sse8387I site and a Klenow fragment was used to break the ends to cause self-ligation. Therefore, plasmid pSJ034 was obtained.

[00187] Here, the J-1 strain of Rhodococcus rhodochrous was registered under the accession number “FERM BP-1478” in the depositary of the international patent agency, Instituto Nacional de Ciência e Tecnologia Industrial Avançada (Central 6, 1 -1- 1 Higashi, Tsukuba Ibaraki, (currently NITE Patent Microorganism Depositary Center: 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Room No. 120)), (original filing date September 18, 1987).

[00188] Furthermore, pSJ023 is a transformant of “R. rhodochrous ATCC 12674/pSJ023”, and is internationally registered under accession number FERM BP-6232 at the depositary of the international patent office, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, (currently NITE Patent Microorganism Depositary Center: 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Room No. 120)), (original filing date March 4, 1997).

Example 2 Production of the improved nitrile hydratase

[00189] Using the plasmid pSJ034 prepared in Example 1, the amino acid substitution was performed. PCR was performed using the composition of a reaction solution, reaction condition, and primers described below. PCR Reaction Solution Composition Sterile Water 20 μl pSJ034 (1 ng/ml) 1 μl Forward Primer (10 mM) 2 μl Reverse Primer (10 mM) 2 μl PrimeSTAR MAX (2X) 25 μl Total 50 μl Reaction conditions for PCR

[00190] (98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 90 seconds) X 30 cycles Table 2 <Starter>

[00191] Upon completion of the PCR, 5 μL of the reaction mixture was provided for electrophoresis on a 0.7% agarose gel, an amplified fragment of 11 kb was confirmed and 1 μL DpnI (supplied with the kit) was added to the PCR reaction mix, which was then reacted at 37°C for one hour. Therefore, the model plasmid was removed. Thereafter, the reaction mixture was purified with the Wizard SV Gel and PCR Clean-Up system (Promega Corporation), and the transformation was introduced into JM109 using the purified PCR reaction product. From the obtained culture product, plasmid DNA was extracted using the QIAprep Spin miniprep kit (Qiagen), and the base sequence of the nitrile hydratase was confirmed using CEQ 8000 automated sequencer (manufactured by Beckman Coulter, Inc.). The plasmids obtained were named as shown in Table 3. Table 3

Example 3 Preparation of Rhodococcus transformant

[00192] The cells of Rhodococcus rhodochrous strain ATCC 12674 in a logarithmic growth phase were collected using a centrifuge, washed three times with ice-cold sterile water and suspended in the sterile water. Then, 1 µl of plasmid prepared in Example 2 and 10 µl of bacterial cell suspension were mixed and cooled on ice. The DNA and bacterial cell suspension were provided in a cuvette and the electrical pulse treatment was performed using an electroporation device, gene pulser (Bio-Rad Laboratories), under conditions of 2.0 kV and 200. by electrical pulse it was allowed to stand in an ice-cold condition for ten minutes, and subjected to heat shock at 37 °C for 10 minutes. After 500 μL of MYK a culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4a, 0.2% KH2PO4) was added and allowed to stand at 30°C for 5 hours, the strain was applied to an MYK agar culture medium containing 50 μg/mL of kanamycin and grown at 30°C for 3 days. The colony obtained after cell culture at 30°C for 3 days was used as a transformant.

[00193] Each transformant obtained above process was inoculated in a MYK culture medium (50 μg/mL of kanamycin), and subjected to culture agitation at 30°C for 2 days. Then, the 1% culture was inoculated into a GGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HPO4, 0.05% KH2PO4 .05%, 0.05% Mg2O4.7H2O, 1% CoCl2, 0.1% urea, 50 μg/mL kanamycin, pH 7.2) and subjected to culture agitation at 30°C for 3 days . Bacterial cells were collected using a centrifuge and washed with 100 mM phosphate buffer (pH 7.0) to prepare a bacterial cell suspension.

Example 4 Evaluation of resistance to amide compounds under high temperatures

[00194] Resistance to the amide compounds of the improved nitrile hydratase obtained in Example 3 was measured according to the following method.

[00195] 0.2 ml of the bacterial cell mixture and 4.8 ml of 50 mM phosphate buffer (pH 7.0) were mixed so that 5 ml of a 50 mM phosphate buffer solution (pH 7.0) containing acrylonitrile to 5.0% (w/v) was added further. Then, the mixture was reacted while being mixed at 10°C for ten minutes. Then, the bacterial cells were filtered and the amount of acrylamide produced was quantified using gas chromatography.

Analysis conditions

[00196] Analysis instrument: gas chromatography GC2014 (manufactured by Shimadzu Corporation)

[00197] Detector: FID (detection at 200°C)

[00198] Column: 1 m glass column filled with PoraPak PS (filling column manufactured by Waters Corporation)

[00199] Column temperature: 190°C

[00200] The activity of nitrile hydratase was determined by converting the amount of acrylamide. Here, regarding nitrile hydratase activity, the amount of enzyme to produce 1 µmol of acrylamide per 1 minute is defined as 1 U.

[00201] Then the test was performed with the following composition for reaction solution and reaction conditions. However, each cell suspension used for the reaction was conveniently diluted with 100 mM phosphate buffer solution (pH 7.0) so that it had the same amount of enzyme activity as the previously measured enzyme activity. As a comparative control, the ATCC12674/pSJ034 comparative strain was used.

Composition of the reaction solution

[00202] 50% acrylamide solution 94 g

[00203] Acrylonitrile 3 g

[00204] 1M Phosphate buffer solution 1 g

[00205] Cell solution (with the same amount of enzyme activity unit (U)) 2 g

reaction conditions

[00206] Reaction temperature 45°C

[00207] Reaction time 3 hours

[00208] 1 ml of each reaction solution was sampled either before the start of the reaction (0 hour) or 3 hours after the reaction. After filtering using a 0.45 μm filter, the obtained filter solution was subjected to gas chromatography. Result of the analysis of the remaining acrylonitrile ratio (%) is shown in Table 4. Table 4

[00209] From the above results, it was found that the acrylonitrile consumption rate of each improved nitrile hydratase is 110% or more than pSJ034 as a comparative example. In a reaction for the synthesis of amide-type compounds with a nitrile hydratase, a loss of activity due to exposure to high temperature and high product concentration and an inhibition of the reaction caused by the amide compounds as a reaction product are the problems. In this regard, as the improved nitrile hydratase of the invention maintains the activity of the nitrile hydratase even at high temperature and even in the presence of acrylamide in high concentration, it is believed to have improved resistance to acrylamide under high temperature.

Example 5 Improved Nitrile Hydratase Production

[00210] The amino acid substitution was performed in the same way as Example 2 using the nitrile hydratase described in WO 2012/164933 A (pSJ306A). The prepared plasmids are shown in Table 5. Table 5

[00211] Using the plasmids described in Table 5, the transformant ATCC12674 of Rhodococcus rhodochrous was obtained in the same way as in Example 3, and cultivated in an MYK medium. Using the cell culture obtained, the evaluation of resistance to amide compounds under high temperatures was carried out according to the following conditions.

Composition of the reaction solution

[00212] 50% acrylamide solution 94 g

[00213] Acrylonitrile 3 g

[00214] 1M Phosphate buffer solution 1 g

[00215] Cell solution (with the same amount of enzyme activity unit (U)) 1 g

[00216] <Reaction conditions>

[00217] Reaction temperature 45°C

[00218] Reaction time 5 hours Table 6

[00219] From the above results, it was found that the acrylonitrile consumption rate of each improved nitrile hydratase is 110% or more than pSJ306A as a comparative example. Consequently, as the improved nitrile hydratase of the invention maintains the activity of the nitrile hydratase even at high temperature and even in the presence of high concentration of acrylamide, it is believed to have improved resistance to acrylamide under high temperature.

Example 6 Production of Enhanced Nitrile Hydratase (JBRs)

[00220] A plasmid to express the nitrile hydratase gene from Nocardia sp. JBRs (GenBank accession number: AY141130) were produced according to the following method.

[00221] By performing the PCR in which pSJ034 is used as a template, the vector fragment was prepared with the Wizard SV Gel and PCR Clean-Up system (Promega Corporation). PCR Reaction Solution Composition Sterile Water 20 μl pSJ034 (1 ng/ml) 1 μl Forward Primer (10 mM) 2 μl Reverse Primer (10 mM) 2 μl PrimeSTAR MAX (2X) 25 μl Total 50 μl

PCR reaction conditions

[00222] (98°C for 10 seconds, 55°C for 5 seconds and 72°C for 90 seconds) X 30 cycles

[00223] NH-F: GAAGTGATCG TATGAGTGAA GACACACTCA CTG (SEQ ID NO: 51)

[00224] NH-R: GTGGATACCA TCCATTTCCT CATTCCTTTC ATC (SEQ ID NO: 52)

[00225] The vector fragment produced above and the artificially synthesized nitrile hydratase gene from Nocardia sp. JBRs (SEQ ID NO: 44) were cloned using the In-Fusion Cloning Kit (Takara Bio Inc.) and transformed into E. coil HST08 (Takara Bio Inc.). From the colonies obtained, the plasmid was collected and the DNA sequence was confirmed. Therefore, the plasmid for expressing the nitrile hydratase derived from Nocardia sp. JBRs were obtained (pSJ-JBRs).

[00226] Furthermore, using the pSJ-JBRs as a template, the amino acid substitution was performed in the same way as in Example 2. The prepared plasmids are shown in Table 7. Table 7

[00227] Using the plasmids described in Table 7, the transformant ATCC12674 of Rhodococcus rhodochrous was obtained in the same way as in Example 3, and cultured in an MYK medium. Using the cell culture obtained, the evaluation of resistance to amide compounds under high temperatures was carried out according to the conditions of Example 4. The results are shown in Table 8. Table 8

[00228] From the above results, it was found that the acrylonitrile consumption rate of each improved nitrile hydratase is 108% or more than pSJ-JBRs as a comparative example. In this regard, as the improved nitrile hydratase of the invention maintains the activity of the nitrile hydratase even at high temperature and even in the presence of high concentration of acrylamide, it is believed to have improved resistance to acrylamide under high temperature.

Example 7 Production of the improved nitrile hydratase (S85-2)

[00229] The plasmid to express the nitrile hydratase gene from Rhodococcus pyridinivorans S85-2 (GenBank accession number: AJ582605) was produced in the same way as Example 6 using an artificially synthesized nitrile hydratase gene (SEQ ID NO: 45 ). The plasmid obtained was named pSJ-S85-2.

[00230] Furthermore, using pSJ-S85-2 as a template, amino acid substitution was performed in the same way as Example 2. The prepared plasmids are shown in Table 9. Table 9

[00231] Using the plasmids described in Table 9, the transformant ATCC12674 of Rhodococcus rhodochrous was obtained in the same way as Example 3, and cultured in an MYK medium. Using the obtained cell culture, the evaluation of resistance to amide compounds under high temperatures was carried out according to the conditions of Example 4. The results are shown in Table 10. [Table 10]

[00232] From the above results, it was found that the acrylonitrile consumption rate of each improved nitrile hydratase is 130% or more than pSJ-S85-2 as a comparative example. In this regard, as the improved nitrile hydratase of the invention maintains the activity of the nitrile hydratase even at high temperature and even in the presence of high concentration of acrylamide, it is believed to have improved resistance to acrylamide under high temperature.

Example 8 Production of the improved nitrile hydratase (M8)

[00233] For the plasmid to express the nitrile hydratase gene from Rhodococcus rhodochrous M8 (GenBank accession number: AAT79340, AAT79339), the plasmid pSJ-NO1A described in JP 2011-200132 A was used and the amino acid substitution was performed in the same manner as Example 2. The plasmids prepared are shown in Table 11. Table 11

[00234] Using the plasmids described in Table 11, the transformant ATCC12674 of Rhodococcus rhodochrous was obtained in the same way as Example 3, and cultured in an MYK medium. Using the cell culture obtained, the evaluation of resistance to amide compounds under high temperatures was carried out according to the conditions of Example 4. The results are shown in Table 12. Table 12

[00235] From the above results, it was found that the acrylonitrile consumption rate of each improved nitrile hydratase is 110% or more than pSJ-NO1A as a comparative example. In this regard, as the improved nitrile hydratase of the invention maintains the activity of the nitrile hydratase even at high temperatures and even in the presence of high concentration of acrylamide, it is believed to have improved resistance to acrylamide under high temperatures.

Example 9 Rhodococcus rhodochrous (Mitsui bacterium)

[00236] For the plasmid expressing the nitrile hydratase gene from Pseudonocardia thermophila JCM 3095 (GenBank accession number: DD028560, DD028561), the plasmid pSJ-NO2A described in JP 2011-200132 A was used and the amino acid substitution was performed in the same manner as Example 2. The plasmids prepared are shown in Table 10. Table 13

[00237] Using the plasmids described in Table 13, the transformant ATCC12674 of Rhodococcus rhodochrous was obtained in the same way as Example 3, and cultured in an MYK medium. Using the cell culture obtained, the evaluation of resistance to amide compounds under high temperatures was carried out according to the conditions of Example 4. The results are shown in Table 14. Table 14

[00238] From the above results, it was found that the acrylonitrile consumption rate of each improved nitrile hydratase is 240% than pSJ-NO2A as a comparative example. In this regard, as the improved nitrile hydratase of the invention maintains the activity of the nitrile hydratase even at high temperature and even in the presence of high concentration acrylamide, it is believed to have improved resistance to acrylamide under high temperature.

INDUSTRIAL APPLICABILITY

[00239] Because the improved nitrile hydratase of the invention has greater resistance to acrylamide under high temperatures, a corresponding amide compound can be produced from a nitrile compound, and thus the nitrile hydratase is useful for the industrial production of amide compounds .

[00240] All publications, patents and patent applications cited in the invention are incorporated into the present application by reference. DEPOSITION NUMBER Rhodococcus rhodochrous J1 CEPA: FERM BP-1478 R. rhodochrous ATCC12674/pSJ023: FERM BP-6232 SEQUENCE LISTING FREE TEXT SEQ ID NO: 20: α8G-F primer SEQ ID NO: 21: α8G-R primer SEQ ID NO: 22: α8V-F primer SEQ ID NO: 23: α8V-R primer SEQ ID NO: 24: α88V-F primer SEQ ID NO: 25: α88V-R primer SEQ ID NO: 26: α153I-F primer SEQ ID NO: 27: α153I-R primer SEQ ID NO: 28: α153L-F primer SEQ ID NO: 29: α153L-R primer SEQ ID NO: 30: α153M-F primer SEQ ID NO: 31: α153M-R primer SEQ ID NO: 32: α153T-F primer SEQ ID NO: 33: α153T-R primer SEQ ID NO: 34: α154L-F primer SEQ ID NO: 35: α154L-R primer SEQ ID NO: 36: α153I-α154L- primer F SEQ ID NO: 37: α153I-α154L-R primer SEQ ID NO: 38: α153L-α154L-F primer SEQ ID NO: 39: α 153L-α154L-R primer SEQ ID NO: 40: α153M^α154L-F primer SEQ ID NO: 41: α153M^α154L-R primer SEQ ID NO: 42: α153T^α154L-F primer SEQ ID NO: 43: α153T^α154L-R primer SEQ ID NO: 46: Specific amino acids according to the invention SEQ ID NO: 47: Specific amino acids according to the invention SEQ ID NO: 48: Specific amino acids according to the invention SEQ ID NO: 49: Specific amino acids according to the invention SEQ ID NO: 50: α-subunit amino acid according to with the invention SEQ ID NO: 51: NH-F primer SEQ ID NO: 52: NH-R primer SEQ ID NO: 53: α88T-F primer SEQ ID NO: 54: α88T-R primer

Claims (5)

1. Improved nitrile hydratase having greater resistance to amide compounds under high temperatures compared to nitrile hydratase with the sequence of SEQ ID NO: 4 derived from the J1 strain of Rhodococcus rhodocrous, where the expression "high temperatures" indicates 40°C to 60 °C, characterized in that the improved nitrile hydratase has at least one amino acid sequence represented by the following SEQ ID NO: 46 to 49 in the α subunit; (a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E (with the proviso that, R indicates arginine, K indicates lysine, A indicates alanine, E indicates glutamic acid, X1 indicates an amino acid other than tyrosine), wherein in SEQ ID NO: 46 above, X2 indicates T (threonine), X3 indicates E (glutamic acid), X4 indicates Y (tyrosine), X5 indicates E (glutamic acid), X6 indicates A (alanine), X7 indicates T (threonine), and X8 indicates I (isoleucine); (b) SEQ ID NO: 47: X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC (with the proviso that, N indicates asparagine, V indicates valine, C indicates cysteine, T indicates threonine, L indicates leucine, X9 indicates an amino acid other than serine, and X21 to X22 each a independently indicates any amino acid residue), where in SEQ ID NO: 47 above, X10 indicates A (alanine), X11 indicates V (valine), X12 indicates F (phenylalanine), X13 indicates D (asparaginic acid), X14 indicates S (serine), X15 indicates Q (glutamine), X16 indicates T (threonine), X17 indicates H (histidine), X18 indicates H (histidine), X19 indicates V (valine) and X20 indicates V (valine); (c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V (with the proviso that, W indicates tryptophan, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, V indicates valine, X23 indicates an amino acid other than valine ), wherein in SEQ ID NO: 48 above, X25 indicates S (serine), X26 indicates S (serine), X27 indicates I (isoleucine), X28 indicates Y (tyrosine) and X29 indicates I (isoleucine); (d) SEQ ID NO: 49: VX24DSX25SEX27RX28X29V (with the proviso that, V indicates valine, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, X24 indicates an amino acid other than tryptophan), wherein in SEQ ID NO: 49 above, X25 indicates S (serine), X26 indicates S (serine), X27 indicates I (isoleucine), X28 indicates Y (tyrosine) and X29 indicates I (isoleucine), and wherein the nitrile hydratase is derived from Rhodococcus bacterium or Nocardia bacterium. 2. Improved nitrile hydratase according to claim 1, characterized in that the improved nitrile hydratase has at least one amino acid sequence represented by the following SEQ ID NO: 46 to 49 in the α subunit; (a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E (with the proviso that, R indicates arginine, K indicates lysine, A indicates alanine, E indicates glutamic acid, X1 indicates an amino acid other than tyrosine); (b) SEQ ID NO: 47:X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC (with the proviso that, N indicates asparagine, V indicates valine, C indicates cysteine, T indicates threonine, L indicates leucine, X9 indicates an amino acid other than serine, X21 indicates valine, and X22 indicates threonine); (c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V (with the proviso that, W indicates tryptophan, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, V indicates valine, X23 indicates an amino acid other than valine ); (d) SEQ ID NO: 49: VX24DSX25SEX27RX28X29V (with the proviso that, V indicates valine, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, X24 indicates an amino acid other than tryptophan); 3. Improved nitrile hydratase according to claim 1, characterized in that the improved nitrile hydratase has at least one amino acid sequence represented by the following SEQ ID NO: 46 to 49 in the α subunit; (a) SEQ ID NO: 46: X1X2X3X4X5X6RX7KAX8E (with the proviso that, R indicates arginine, K indicates lysine, A indicates alanine, E indicates glutamic acid, X1 indicates glycine or valine); (b) SEQ ID NO: 47: X9X10X11X12NX13X14X15X16X17X18X19X20X21CX22LC (with the proviso that, N indicates asparagine, V indicates valine, C indicates cysteine, T indicates threonine, L indicates leucine, X9 indicates valine or threonine, X21 indicates valine and X22 indicates threonine ); (c) SEQ ID NO: 48: X23WDSX25X26EX27RX28X29V (with the proviso that, W indicates tryptophan, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, V indicates valine, X23 is selected from the group consisting into isoleucine, leucine, methionine and threonine); (d) SEQ ID NO: 49: VX24DSX25SEX27RX28X29V (with the proviso that, V indicates valine, D indicates asparaginic acid, S indicates serine, E indicates glutamic acid, R indicates arginine, X24 indicates leucine); 4. Improved nitrile hydratase according to any one of claims 1 to 3, characterized in that the improved nitrile hydratase has a) an amino acid sequence represented by SEQ ID NO: 50, or an amino acid sequence represented by SEQ ID NO : 50 in which 1 to 10 amino acids are deleted, substituted or added at a position other than X1 to X29 and having the same resistance to heat and/or resistance to amide compounds. 5. Method for producing an amide compound, the method characterized by putting a nitrile compound in contact with the improved nitrile hydratase as defined in any one of claims 1 to 4.