Improved nitrile hydratase

Abstract

The present invention relates to a novel improved nitrile hydratase having improved resistance to amide compounds at high temperatures. Specifically, it relates to a nitrile hydratase (X1 to X2) having at least one amino acid mutation selected from the group consisting of (a) to (d) in the amino acid sequence shown in SEQ ID NO: 50. 27 represents an independent arbitrary amino acid residue). (a) X1 is valine or glycine; (b) X9 is valine or threonine; (c) X 23 is an amino acid selected from the group consisting of isoleucine, leucine, methionine and threonine; (d) X 24 For leucine.

Description

[0001] This application is a divisional application of Chinese patent application 201580030220.1. The application date of the original application 201580030220.1 is May 12, 2015, and its name is “Improved nitrile hydratase”. Technical Field

[0002] The present invention relates to an improved (mutated) nitrile hydratase and a method for producing the same, and further relates to a 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 Art

[0003] Nitrile hydratase is an enzyme having nitrile hydration activity that hydrates a nitrile group and converts it into an amide group. The amide compound corresponding to the nitrile compound is produced using the enzyme or a microbial cell containing the enzyme, and it is known that the method has a high conversion rate and selectivity from the nitrile compound to the corresponding amide compound compared to the existing chemical synthesis method.

[0004] Examples of microorganisms that produce nitrile hydratase include microorganisms belonging to the genera Corynebacterium, Pseudomonas, Rhodococcus, Rhizobium, Klebsiella, and Pseudonocardia. Among them, the Rhodococcus rhodochrous J1 strain is used in the industrial production of acrylamide, and its usefulness has been confirmed. In addition, the gene encoding the nitrile hydratase produced by this strain has also been identified (see Patent Document 1).

[0005] On the other hand, in addition to utilizing nitrile hydratase and its gene isolated from microorganisms existing in nature, attempts have been made to introduce mutations into nitrile hydratase for the purpose of changing the activity, substrate specificity, Vmax, Km, thermal stability, stability to substrate, stability to product, etc. of nitrile hydratase; in the nitrile hydratase of Pseudonocardia thermophila JCM3095, regions related to substrate specificity and thermal stability were predicted from stereostructure information, and mutant enzymes with altered substrate specificity were obtained therefrom (see Patent Documents 2 to 4). In addition, the present inventors have obtained nitrile hydratase genes with improved heat resistance or amide compound resistance (see Patent Documents 5 to 9).

[0006] However, from the viewpoint of production costs such as catalyst costs, it is very useful to further improve heat resistance and amide compound resistance, or to develop a nitrile hydratase that can react at high temperatures and use it in the production of amide compounds. From the viewpoint of reducing the amount of enzyme during the reaction and reducing costs, it is desired to obtain an enzyme having such performance.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent No. 3162091

[0010] Patent Document 2: International Publication No. 2004 / 056990

[0011] Patent Document 3: Japanese Patent Application Publication No. 2004-194588

[0012] Patent Document 4: Japanese Patent Application Publication No. 2005-16403

[0013] Patent Document 5: International Publication No. 2005 / 116206 Pamphlet

[0014] Patent Document 6: Japanese Patent Application Publication No. 2007-143409

[0015] Patent Document 7: Japanese Patent Application Publication No. 2007-43910

[0016] Patent Document 8: Japanese Patent Application Publication No. 2008-253182

[0017] Patent Document 9: Japanese Patent Application Publication No. 2010-172295 Summary of the invention

[0018] Problem that the invention aims to solve

[0019] An object of the present invention is to provide a novel improved nitrile hydratase having improved tolerance to amide compounds at high temperatures, thereby enabling a method for producing amide compounds with higher production efficiency.

[0020] Solutions for solving problems

[0021] The present inventors conducted intensive studies to solve the above problems and found that a protein obtained by replacing specific amino acid residues in the amino acid sequence of nitrile hydratase with other amino acid residues not only has nitrile hydratase activity but also has improved amide compound resistance at high temperatures, thereby completing the present invention.

[0022] That is, the present invention provides the following [1] to

[13] .

[0023] [1] An improved nitrile hydratase comprising at least one of the amino acid sequences shown in SEQ ID NOs: 46 to 49 in its α subunit,

[0024] (a) Serial number 46: X1X2X3X4X5X6RX7KAX8E

[0025] (wherein, R is arginine, K is lysine, A is alanine, E is glutamic acid, X1 is an amino acid other than tyrosine, and X2 to X8 independently represent any amino acid residue);

[0026] (b) Serial number 47: X9X 10 X 11 X 12 NX 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 CX 22 LC

[0027] (wherein, N is asparagine, V is valine, C is cysteine, T is threonine, L is leucine, X9 is an amino acid other than serine, 10 ~X 22 Independently of each other, represent any amino acid residue);

[0028] (c) Sequence number 48: X 23 WDSX 25 X 26 EX 27 RX 28 X 29 V

[0029] (W represents tryptophan, D represents aspartic acid, S represents serine, E represents glutamic acid, R represents arginine, V represents valine, X represents 23 represents amino acids other than valine, X 25 ~X 29 Independently of each other, represent any amino acid residue);

[0030] (d) Serial No. 49: VX 24 DSX 25 X 26 EX 27 RX 28 X 29 V

[0031] (Wherein, V represents valine, D represents aspartic acid, S represents serine, E represents glutamic acid, R represents arginine, X represents 24 represents amino acids other than tryptophan, X 25 ~X 29 represent any amino acid residues independently of each other)

[0032] [2] The improved nitrile hydratase according to [1] above, wherein the α subunit comprises at least one of the amino acid sequences shown in SEQ ID NOs: 46 to 49 below,

[0033] (a) Serial number 46: X1X2X3X4X5X6RX7KAX8E

[0034] (wherein, R is arginine, K is lysine, A is alanine, E is glutamic acid, X1 is an amino acid other than tyrosine, and X2 to X8 independently represent any amino acid residue);

[0035] (b) Serial number 47: X9X 10 X 11 X 12 NX 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 CX 22 LC

[0036] (wherein, N is asparagine, V is valine, C is cysteine, T is threonine, L is leucine, X9 is an amino acid other than serine, 10 ~X 20 Independently of each other, X represents any amino acid residue. 21 represents valine, X 22 represents threonine);

[0037] (c) Sequence number 48: X 23 WDSX 25 X 26 EX 27 RX 28 X 29 V

[0038] (W represents tryptophan, D represents aspartic acid, S represents serine, E represents glutamic acid, R represents arginine, V represents valine, X represents 23 represents amino acids other than valine, X 25 ~X 29 Independently of each other, represent any amino acid residue);

[0039] (d) Serial No. 49: VX 24 DSX 25 X 26 EX 27 RX 28 X 29 V

[0040] (Wherein, V represents valine, D represents aspartic acid, S represents serine, E represents glutamic acid, R represents arginine, X represents 24 represents amino acids other than tryptophan, X 25 ~X 29 represent any amino acid residues independently of each other)

[0041] [3] The improved nitrile hydratase according to [1] above, wherein the α subunit comprises at least one of the amino acid sequences shown in SEQ ID NOs: 46 to 49 below,

[0042] (a) Serial number 46: X1X2X3X4X5X6RX7KAX8E

[0043] (wherein, R is arginine, K is lysine, A is alanine, E is glutamic acid, X1 is glycine or valine, and X2 to X8 independently represent any amino acid residue);

[0044] (b) Serial number 47: X9X 10 X 11 X 12 NX 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 CX 22 LC

[0045] (wherein N is asparagine, V is valine, C is cysteine, T is threonine, L is leucine, X9 is valine or threonine, X 10 ~X 20 Independently of each other, X represents any amino acid residue. 21 represents valine, X 22 represents threonine);

[0046] (c) Sequence number 48: X 23 WDSX 25 X 26 EX 27 RX 28 X 29 V

[0047] (W represents tryptophan, D represents aspartic acid, S represents serine, E represents glutamic acid, R represents arginine, V represents valine, X represents 23 is selected from the group consisting of isoleucine, leucine, methionine and threonine, X 25 ~X 29Independently of each other, represent any amino acid residue);

[0048] (d) Serial No. 49: VX 24 DSX 25 X 26 EX 27 RX 28 X 29 V

[0049] (Wherein, V represents valine, D represents aspartic acid, S represents serine, E represents glutamic acid, R represents arginine, X represents 24 represents leucine, X 25 ~X 29 represent any amino acid residues independently of each other)

[0050] [4] The improved nitrile hydratase according to any one of [1] to [3] above, wherein in SEQ ID NO: 46, X2 is T (threonine), X3 is E (glutamate), X4 is Y (tyrosine), X5 is E (glutamate), X6 is A (alanine), X7 is T (threonine), and X8 is I (isoleucine),

[0051] In the aforementioned sequence number 47, X 10 A (alanine), X 11 V (valine), X 12 F (phenylalanine), X 13 D (aspartic acid), X 14 is S (serine), X 15 Q (glutamine), X 16 is T (threonine), X 17 H (histidine), X 18 H (histidine), X 19 V (valine), X 20 is V (valine),

[0052] In the aforementioned sequence numbers 48 and 49, X 25 is S (serine), X 26 is S (serine), X 27 I (isoleucine), X 28 Y (tyrosine), X 29 is I (isoleucine).

[0053] It should be noted that the amino acid sequence shown in sequence number 46 corresponds to positions 8 to 19 of the amino acid sequence of the α subunit of nitrile hydratase, the amino acid sequence shown in sequence number 47 corresponds to positions 88 to 105 of the sequence, and the amino acid sequences shown in sequence numbers 48 and 49 correspond to positions 153 to 164 of the sequence.

[0054] [5] The improved nitrile hydratase according to any one of [1] to [4] above, which has the amino acid sequence shown in SEQ ID NO: 50.

[0055] [6] An improved nitrile hydratase comprising the amino acid sequence shown in SEQ ID NO: 50 in its α subunit, and having at least one amino acid mutation selected from the following (i) to (iv).

[0056] (i) X1 is G (glycine) or V (valine)

[0057] (ii) X9 is V (valine) or T (threonine)

[0058] (iii)X 23 An amino acid selected from the group consisting of I (isoleucine), L (leucine), M (methionine) and T (threonine)

[0059] (iv)X 24 For L (Leucine)

[0060] [7] The improved nitrile hydratase according to [6] above, wherein X2 is T (threonine), X3 is E (glutamate), X4 is Y (tyrosine), X5 is E (glutamate), X6 is A (alanine), X7 is T (threonine), X8 is I (isoleucine), X9 is 10 A (alanine), X 11 V (valine), X 12 F (phenylalanine), X 13 D (aspartic acid), X 14 is S (serine), X 15 Q (glutamine), X 16 is T (threonine), X 17 H (histidine), X 18 H (histidine), X 19 V (valine), X 20 V (valine), X 25 is S (serine), X 26 is S (serine), X 27 I (isoleucine), X 28 Y (tyrosine), X 29 is I (isoleucine).

[0061] [8] The improved nitrile hydratase according to any one of [1] to [7] above, wherein the nitrile hydratase is derived from a bacterium of the genus Rhodococcus or a bacterium of the genus Nocardia.

[0062] [9] A DNA encoding the improved nitrile hydratase described in any one of [1] to [8] above; or a DNA that hybridizes with a DNA having a base sequence complementary to the aforementioned DNA under stringent conditions and encodes a protein having nitrile hydratase activity that is resistant to amide compounds at high temperatures.

[0063]

[10] A recombinant vector comprising the DNA described in [9] above.

[0064]

[11] A transformant comprising the recombinant vector described in

[10] above.

[0065]

[12] A method for producing nitrile hydratase, the method comprising culturing the transformant according to

[11] above and extracting nitrile hydratase from the obtained culture. And,

[0066]

[13] A method for producing an amide compound, characterized in that the improved nitrile hydratase according to any one of [1] to [8] above or a culture obtained by culturing the transformant according to

[11] or a processed product of the culture is contacted with a nitrile compound.

[0067] Effects of the Invention

[0068] According to the present invention, a novel improved (mutated) nitrile hydratase having improved resistance to amide compounds at high temperatures can be provided. The improved nitrile hydratase of the present invention has excellent resistance to amide compounds at high temperatures and can improve the production efficiency of amide compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The structure diagram of plasmid pSJ034.

[0070] Figure 2-1 It is a diagram showing the amino acid sequence (part of the N-terminal side) of the α-subunit of nitrile hydratase derived from various microorganisms.

[0071] Figure 2-2 To show the Figure 2-1 The same amino acid sequence diagram, which shows the Figure 2-1 The amino acid sequence of .

[0072] Figure 3 This is the amino acid sequence of the α subunit of the present invention as shown in SEQ ID NO: 50.

[0073] This application claims priority based on Japanese Patent Application No. 2014-118041 (filed on June 6, 2014 ), the contents of which are incorporated herein by reference. DETAILED DESCRIPTION

[0074] 1. Nitrile Hydratase

[0075] 1.1 Known nitrile hydratases

[0076] "Nitrile hydratase" has a higher-order structure formed by the association of domains of α subunits and β subunits, and has a non-heme iron atom or a non-corrin core cobalt atom as a cofactor. These nitrile hydratases are distinguished by the names of iron-type nitrile hydratase and cobalt-type nitrile hydratase.

[0077] As an iron-type nitrile hydratase, a nitrile hydratase derived from Rhodococcus N-771 strain can be cited as a representative example. The three-dimensional structure of the iron-type nitrile hydratase becomes clear by performing X-ray crystallographic structure analysis. As a result, the enzyme binds to non-heme iron via four amino acid residues in the cysteine ​​cluster (Cys-Ser-Leu-Cys-Ser-Cys) of the α subunit forming the active center.

[0078] Representative examples of the cobalt-type nitrile hydratase include the cobalt-type nitrile hydratase derived from Rhodococcus rhodochrous J1 strain (hereinafter sometimes referred to as “J1 bacteria”) and the cobalt-type nitrile hydratase derived from Pseudonocardiathermophila .

[0079] The cobalt-type nitrile hydratase derived from J1 bacteria is bound to the cobalt atom via the region shown by the cysteine ​​cluster (Cys-Thr-Leu-Cys-Ser-Cys) of the α subunit forming the active center. It should be noted that in the cysteine ​​cluster of the cobalt-type nitrile hydratase derived from Pseudonocardia thermophila, the cysteine ​​(Cys) at the 4th position from the upstream side (N-terminal side) in the cysteine ​​cluster derived from J1 bacteria is cysteine ​​sulfinic acid (Csi), and the cysteine ​​(Cys) at the 6th position on the most downstream side (C-terminal side) is cysteine ​​sulfenic acid (Cse).

[0080] As described above, the prosthetic group is bound to the region indicated by the cysteine ​​cluster "C(S / T)LCSC" in the α subunit. Examples of nitrile hydratases containing such a prosthetic group-binding region include nitrile hydratases having amino acid sequences derived from Rhodococcus rhodochrous J1 (FERM BP-1478), Rhodococcus rhodochrous M8 (Soviet Union Patent No. 1731814 (SU1731814)), Rhodococcus rhodochrous M33 (VKM Ac-1515D), Rhodococcus rhodochrous ATCC39484 (Japanese Patent Laid-Open No. 2001-292772), Bacillus smithii (Japanese Patent Laid-Open No. 9-254188), Pseudonocardia thermophila (Japanese Patent Laid-Open No. 9-275978) or Geobacillus thermoglucosidasius, and nitrile hydratases encoded by gene sequences derived from the above-mentioned bacteria. On the other hand, it is believed that the β subunit is related to the stability of the structure.

[0081] The GenBank accession number of the nitrile hydratase from Rhodococcus rhodochrous J1 (FERM BP-1478) is "P21220". In addition, the GenBank accession number of the α subunit from Rhodococcus rhodochrous M8 (SU1731814) is "ATT79340", and the GenBank accession number of the β subunit is "AAT79339". The GenBank accession number of the nitrile hydratase gene from Rhodococcus pyridinophilus MW3 is AJ582605, the GenBank accession number of the nitrile hydratase gene from Rhodococcus pyridinophilus S85-2 is AJ582605, and the nitrile hydratase gene from Rhodococcus erythrophilus TH (CGMCC No. 2380) is recorded in Chinese Patent No. 101463358 (CN1463358). Furthermore, the GenBank accession number of the nitrile hydratase gene derived from Nocardia sp. YS-2002 is "X86737", and the GenBank accession number of the nitrile hydratase gene derived from Nocardia sp. JBRs is "AY141130".

[0082] SEQ ID NOs. 1 to 19 in the sequence listing describe the amino acid sequences and base sequences of known nitrile hydratases.

[0083] SEQ ID NO. 1: Base sequence of the β subunit from Rhodococcus rhodochrous J1

[0084] SEQ ID NO. 2: Amino acid sequence of the β subunit from Rhodococcus rhodochrous J1

[0085] SEQ ID NO: 3: Base sequence of the α subunit from Rhodococcus rhodochrous J1

[0086] SEQ ID NO. 4: Amino acid sequence of the α subunit from Rhodococcus rhodochrous J1

[0087] SEQ ID NO: 5: Amino acid sequence of the α subunit of Rhodococcus rhodochrous M8

[0088] SEQ ID NO: 6: Amino acid sequence of the α subunit of Rhodococcus erythrocyte TH

[0089] SEQ ID NO: 7: Amino acid sequence of the α subunit of Rhodococcus pyridinophilus MW33

[0090] SEQ ID NO: 8: Amino acid sequence of the α subunit of Rhodococcus pyridinophilus S85-2

[0091] SEQ ID NO: 9: Amino acid sequence of the α subunit of Rhodococcus pyridinophilus MS-38

[0092] SEQ ID NO: 10: Amino acid sequence of the α subunit of Nocardia sp. JBRs

[0093] SEQ ID NO: 11: Amino acid sequence of the α subunit of Nocardia sp. YS-2002

[0094] SEQ ID NO: 12: Amino acid sequence of the α subunit of uncultured bacteria SP1

[0095] SEQ ID NO: 13: Amino acid sequence of the α subunit of uncultured bacteria BD2

[0096] SEQ ID NO: 14: Amino acid sequence of the α subunit of Rhodococcus rhodochrous ATCC39484

[0097] SEQ ID NO: 15: Amino acid sequence of the α subunit of Sinorhizobium meliloti WSM419

[0098] SEQ ID NO: 16: Amino acid sequence of the α subunit of the thermoglucosidase Geobacillus sp. Q6

[0099] SEQ ID NO: 17: Amino acid sequence of the α subunit of Pseudonocardia thermophila JCM3095

[0100] SEQ ID NO: 18: Amino acid sequence of the α subunit of Rhodococcus rhodochrous Cr4

[0101] SEQ ID NO: 19: Amino acid sequence of the α subunit of Comamonas testosteroni

[0102] In addition, the alignment of the amino acid sequences (single letter symbol) of the α subunits of known nitrile hydratase derived from various microorganisms is shown in Figure 2-1 and 2-2 It should be noted that Figure 2-1 , 2-2 In each of them, the amino acid sequences correspond to SEQ ID NOs. 4, 5 to 19 from top to bottom.

[0103] The nitrile hydratase of the present invention is not limited to the enzyme having the above sequence, and proteins having nitrile hydratase activity are also included in the nitrile hydratase of the present invention, which comprises an amino acid sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more, further preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more homology or identity with the amino acid sequence of any one of SEQ ID NOs. 1 to 19.

[0104] In addition, the nitrile hydratase of the present invention includes an amino acid sequence in which one or more, specifically 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2 amino acids are deleted, substituted, or added to the amino acid sequence of any one of SEQ ID NOs. 1 to 19, and a protein having nitrile hydratase activity is also included in the nitrile hydratase of the present invention.

[0105] 1.2 Improved nitrile hydratase

[0106] The improved nitrile hydratase of the present invention is a novel improved nitrile hydratase having improved tolerance to amide compounds at high temperatures.

[0107] The source of the improved nitrile hydratase of the present invention is not particularly limited, and for example, nitrile hydratase registered in the GenBank database (http: / / www.ncbi.nlm.nih.gov / entrez / query.fcgi?CMD=search&DB=protein) provided by the National Center for Biotechnology Information (NCBI) of the United States or nitrile hydratase described in known literature can be used.

[0108] Specifically, for example, nitrile hydratases described in WO2005 / 116206, Japanese Patent Application Laid-Open No. 2007-143409, Japanese Patent Application Laid-Open No. 2007-43910, Japanese Patent Application Laid-Open No. 2008-253182, and Japanese Patent Application Laid-Open No. 2010-172295 (incorporated herein by reference) can be shown. These nitrile hydratases have heat resistance and acrylamide resistance, and by further adding the amino acid substitution of the present invention, the property of improving the resistance to amide compounds at high temperatures can be imparted.

[0109] As an example of the improved nitrile hydratase of the present invention, the α subunit may be Figure 3 An enzyme having the amino acid sequence shown (SEQ ID NO: 50). Figure 3In the amino acid sequence shown, the amino acid sequence shown in sequence number 46 exists at the 8th to 19th positions from the N-terminus, the amino acid sequence shown in sequence number 47 exists at the 88th to 105th positions, and the amino acid sequence shown in sequence number 48 or sequence number 49 exists at the 153rd to 165th positions.

[0110] As one embodiment of the present invention, there can be mentioned nitrile hydratase (X1 to X2) having at least one amino acid mutation selected from the group consisting of (a) to (d) in the amino acid sequence shown in SEQ ID NO: 50. 29 represents an independent arbitrary amino acid residue).

[0111] (a) X1 is glycine or valine

[0112] (b) X9 is valine or threonine

[0113] (c)X 23 is an amino acid selected from the group consisting of isoleucine, leucine, methionine and threonine

[0114] (d)X 24 Leucine

[0115] As another embodiment, in the improved nitrile hydratase having the amino acid sequence shown in SEQ ID NO: 50, X2 is T (threonine), X3 is E (glutamate), X4 is Y (tyrosine), X5 is E (glutamate), X6 is A (alanine), X7 is T (threonine), X8 is I (isoleucine), X9 is Y (tyrosine), X10 is Y (glutamate), X11 is Y (tyrosine), X12 is Y (glutamate), X13 is Y (glutamate), X14 is Y (tyrosine), X15 is Y (glutamate), X16 is Y (alanine), X17 is Y (threonine), X18 is Y (isoleucine), X19 is Y (tyrosine), X20 is Y (tyrosine), X21 is Y (glutamate), X22 is Y (tyrosine), X23 is Y (glutamate), X24 is Y (tyrosine), X25 is Y (glutamate), X26 is Y (alanine), X27 is Y (threonine), X28 is Y (isoleucine), X29 is Y (tyrosine), X30 is Y (glutamate), X31 is Y (tyrosine), X32 is Y (glutamate), X33 is Y (tyrosine), X34 is Y (tyrosine), X35 is Y (tyrosine), X36 is Y (tyrosine), 10 A (alanine), X 11 V (valine), X 12 F (phenylalanine), X 13 D (aspartic acid), X 14 is S (serine), X 15 Q (glutamine), X 16 is T (threonine), X 17 H (histidine), X 18 H (histidine), X 19 V (valine), X 20 V (valine), X 25 is S (serine), X 26 is S (serine), X 27 I (isoleucine), X 28 Y (tyrosine), X 29 A nitrile hydratase which is I (isoleucine) and has at least one characteristic selected from the above (a) to (d).

[0116] It should be noted that a nitrile hydratase having an amino acid sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, further preferably about 95% or more, and particularly preferably about 98% or more homology or identity with the amino acid sequence shown in SEQ ID NO: 50 except for the above-mentioned substitution site and having the same heat resistance and / or amide compound resistance is also included in the improved nitrile hydratase of the present invention.

[0117] In addition, a nitrile hydratase having an amino acid sequence in which 1 to 10, preferably 1 to 5, and more preferably 1 to 2 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 50 other than the above-mentioned substitution site and having the same heat resistance and / or amide compound resistance is also included in the improved nitrile hydratase of the present invention.

[0118] Another example of the improved nitrile hydratase of the present invention is a nitrile hydratase having at least one feature selected from the following (e) to (h) in the amino acid sequence of the known nitrile hydratase shown in SEQ ID NO: 4.

[0119] (e) Substitution of the amino acid residue (tyrosine) at position 8 of the α subunit with glycine or valine

[0120] (f) Substituting the amino acid residue (serine) at position 88 of the α subunit with valine or threonine

[0121] (g) replacing the amino acid residue (valine) at position 153 of the α subunit with an amino acid selected from the group consisting of isoleucine, leucine, methionine and threonine

[0122] (h) Substitution of amino acid residue 154 (tryptophan) of the α subunit with leucine

[0123] It should be noted that a nitrile hydratase having an amino acid sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, further preferably about 95% or more, and particularly preferably about 98% or more homology or identity with the amino acid sequence shown in SEQ ID NO: 4 except for the above-mentioned substitution site and having the same heat resistance and / or amide compound resistance is also included in the improved nitrile hydratase of the present invention.

[0124] In addition, a nitrile hydratase having an amino acid sequence in which 1 to 10, preferably 1 to 5, and more preferably 1 to 2 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 4 other than the above-mentioned substitution site and having the same heat resistance and / or amide compound resistance is also included in the improved nitrile hydratase of the present invention.

[0125] The amino acid substitutions of (e) to (h) above are represented as "Yα8G, Sα88V, Vα153I, Wα154L", etc. Standard amino acids are represented by single letters, and amino acid substitutions can be represented by showing the single letter symbol of the amino acid before substitution on the left side of the number representing the substitution position (the number of amino acid residues up to the substitution position) and showing the single letter symbol of the amino acid after substitution on the right side.

[0126] Specifically, regarding the amino acid sequence of the α subunit shown in SEQ ID NO: 4, when expressed as "Yα8G", it means a mode of amino acid substitution in the improved nitrile hydratase in which the tyrosine (Y) at the 8th position from the number of amino acid residues at the N-terminus (counting including the amino acid residue at the N-terminus) in the amino acid sequence of the α subunit (SEQ ID NO: 4) is replaced with glycine (G).

[0127] More preferred amino acid substitution aspects in the improved nitrile hydratase of the present invention can be represented by the following symbols 1 to 8, respectively.

[0128] 1.Yα8G

[0129] 2.Yα8V

[0130] 3. Sα88V

[0131] 4.Sα88T

[0132] 5.Vα153I

[0133] 6.Vα153L

[0134] 7. Vα153M

[0135] 8.Vα153T

[0136] 9.Wα154L

[0137] Preferred examples of base substitution for producing the above-mentioned amino acid substitution include the following.

[0138] [Table 1]

[0139]

[0140] The activity of the improved nitrile hydratase of the present invention improves the tolerance to amide compounds at high temperatures compared to the activity of the wild-type nitrile hydratase that maintains the naturally derived properties.

[0141] Here, "nitrile hydratase activity" refers to the enzyme activity that catalyzes the hydration reaction (RCN+H2O→RCONH2) of converting a nitrile compound into a corresponding amide compound. The activity can be measured by contacting a nitrile compound as a substrate with a nitrile hydratase, converting it into a corresponding amide compound, and then quantifying the amide compound and calculating it. As a substrate, any nitrile compound can be used as long as the nitrile hydratase reacts, and acrylonitrile is preferred.

[0142] Regarding the reaction conditions, the substrate concentration is 2.5%, the reaction temperature is 10°C to 30°C, and the reaction time is in the range of 10 minutes to 30 minutes. The enzyme reaction is stopped by adding phosphoric acid. Afterwards, the generated acrylamide can be analyzed by HPLC (high performance liquid chromatography) or gas chromatography to quantify the amide compound.

[0143] "High temperature amide compound resistance" means that the nitrile hydratase activity can be maintained in the presence of an amide compound at high temperature. "High temperature" specifically means 40°C to 60°C, more preferably 45°C to 55°C.

[0144] "High temperature amide compound resistance" can be evaluated by analyzing the consumption or consumption rate of a nitrile compound such as acrylonitrile as a substrate in the presence of an amide compound such as acrylamide (e.g., a high concentration of 30 to 50%) at high temperature for a culture of a transformant having the improved nitrile hydratase or the improved nitrile hydratase isolated from the transformant. For example, when the improved nitrile hydratase is brought into contact with an amide compound in the range of 40°C to 60°C, a nitrile hydratase with a consumption or consumption rate of 1.1 times or more, preferably 1.15 times or more, and more preferably 1.2 times or more relative to that of a comparative example (nitrile hydratase not subjected to mutation) can be evaluated as having high temperature amide compound resistance.

[0145] Examples of the "amide compound" include amide compounds represented by the following general formula (1).

[0146] R-CONH2 (1)

[0147] (Here, 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 an optionally substituted saturated or unsaturated heterocyclic group.)

[0148] Acrylamide wherein R is "CH2=CH-" is particularly preferred.

[0149] The improved nitrile hydratase of the present invention is obtained by subjecting known nitrile hydratase to amino acid substitution, for example, by introducing the above mutation into the amino acid sequence (SEQ ID NO: 4) of nitrile hydratase derived from Rhodococcus rhodochrous J1 strain and selecting a nitrile hydratase having improved resistance to amide compounds at high temperatures.

[0150] For nitrile hydratases other than J1 bacteria, the same mutation can be introduced into the corresponding modified position to improve the amide compound resistance at high temperature. For example, as nitrile hydratase producing bacteria, there can be listed Rhodococcus rhodochus M8 (sequence number 5), Rhodococcus erythrocytes TH (sequence number 6), Rhodococcus rhodochus M33 (VKM Ac-1515D), Rhodococcus pyridinophilus MW3 (sequence number 7), Rhodococcus pyridinophilus S85-2 (sequence number 8), Nocardia sp. JBRs (sequence number 10), Nocardia · YS-2002 (sequence number 11), etc. It should be noted that Rhodococcus rhodochus M33 (VKM Ac-1515D) is a strain selected as a strain expressing nitrile hydratase in structure by natural mutation from the above-mentioned M8 bacteria, and there is no mutation in its amino acid sequence and gene sequence (U.S. Patent No. 5,827,699).

[0151] The improved nitrile hydratase of the present invention can be obtained, for example, by randomly or site-specifically introducing mutations into a gene encoding a known nitrile hydratase by a known method, and selecting an enzyme having a desired function, that is, resistance to amide compounds at high temperatures.

[0152] Examples of methods for introducing mutations include random mutation introduction methods such as Error prone PCR and site-directed mutagenesis (site-specific mutation introduction methods) such as the Kunkel method and the Gapped duplex method.

[0153] [Error prone PCR]

[0154] As one of the methods for studying the function and properties of proteins using variants, there is a random mutation introduction method. The random mutation introduction method refers to a method of introducing random mutations into a gene encoding a specific protein to produce a variant. In the random mutation introduction method using PCR, base mutations can be introduced by setting low stringency conditions during DNA amplification (Error prone PCR; error prone polymerase chain reaction).

[0155] In the error-prone polymerase chain reaction, mutations are introduced at arbitrary positions for all regions of the amplified DNA. In this way, by studying the functions of the obtained variants with mutations introduced at arbitrary positions, information on amino acids and domains important for the intrinsic functions of the protein can be obtained. The nitrile hydratase as a template for the error-prone polymerase chain reaction can use a nitrile hydratase gene derived from a wild strain or a DNA as an amplification product obtained by the error-prone polymerase chain reaction.

[0156] As the reaction conditions of the error-prone polymerase chain reaction, for example, the conditions of setting the mixing ratio of any one, two or three kinds of dNTPs (dGTP, dCTP, dATP or dTTP) in the reaction solution to a composition reduced compared with other dNTPs can be listed. Thus, during DNA synthesis, the possibility of using other dNTPs by mistake at the position where the dNTP whose mixing ratio has been reduced is required increases, thereby introducing mutations. In addition, as other reaction conditions, the conditions of setting the composition to increase the amount of MgCl2 and / or MnCl2 in the reaction solution are also preferably listed.

[0157] Site-directed Mutagenesis

[0158] In the method of introducing mutations at a specific site, generally, the following steps are performed: dissociating a DNA strand containing a target gene into single strands, annealing an oligonucleotide strand containing a target mutation, extending the strands with a DNA polymerase to form two strands, introducing the two strands into Escherichia coli and replicating them, and selecting clones containing the target mutation (see Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc.). In addition to the Kunkel method, various methods such as the Gapped duplex method are known, and can be easily performed using commercially available mutation introduction kits, such as QuickChange™ XL Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailor™ Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: manufactured by TAKARA BIO INC.), etc.

[0159] The improved nitrile hydratase of the present invention can be obtained by metagenomic screening from environmental DNA in addition to the method of introducing mutations into known nitrile hydratase genes as described above.

[0160] 1.3 DNA encoding improved nitrile hydratase

[0161] The present invention also provides a DNA encoding the improved nitrile hydratase of the present invention.

[0162] The "DNA encoding improved nitrile hydratase" of the present invention also includes a DNA which hybridizes with a DNA having a base sequence complementary to the above-mentioned DNA encoding improved nitrile hydratase under stringent conditions and encodes a protein having nitrile hydratase activity that is resistant to amide compounds at high temperatures.

[0163] "Stringent conditions" means conditions for washing after hybridization, and are salt concentrations of 300 to 2000 mM and temperatures of 40 to 75°C; preferably conditions of salt concentrations of 600 to 900 mM and temperatures of 65°C. For example, conditions such as 2×SSC at 50°C can be cited. A person skilled in the art can appropriately set conditions for obtaining the DNA encoding the nitrile hydratase of the present invention, taking into account various conditions such as probe concentration, probe length, reaction time, etc., based on such conditions such as salt concentration and temperature of the buffer.

[0164] For detailed procedures of the hybridization method, reference may be made to Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)), etc. Examples of hybridized DNA include DNA or partial fragments thereof containing a base sequence having a sequence identity of at least 40%, preferably 60%, and more preferably 90% or more with the gene DNA of the present invention.

[0165] 1.4 Recombinant vectors and transformants

[0166] The DNA encoding the improved nitrile hydratase needs to be recombined into a vector so as to be expressed in the transformed host organism. Examples of the vector used include plasmid DNA, phage DNA, retrotransposon DNA, and artificial chromosome DNA.

[0167] In addition to the nitrile hydratase gene, the vector may also be connected with a promoter, a terminator, an enhancer, a splicing signal, a poly-A additional signal, a selection marker, a ribosome binding sequence (SD sequence), etc. Examples of the selection marker include a kanamycin resistance gene, a dihydrofolate reductase gene, an ampicillin resistance gene, and a neomycin resistance gene.

[0168] The host that can be used for the transformant of the present invention is not particularly limited as long as it can express the target nitrile hydratase after the introduction of the above-mentioned recombinant vector, and for example, bacteria such as Escherichia coli and Bacillus subtilis, yeast, animal cells, insect cells, plant cells, etc. can be used.

[0169] When Escherichia coli is used as a host, it is preferable to use an expression vector with high expression efficiency, for example, expression vector pkk233-2 (manufactured by Amersham Biosciences) or pTrc99A (manufactured by Amersham Biosciences) having a trc promoter.

[0170] When using bacteria as hosts, for example, Escherichia coli can be listed, and as Rhodococcus, for example, Rhodococcus rhodococcus ATCC12674, Rhodococcus rhodococcus ATCC17895, Rhodococcus rhodococcus ATCC19140, etc. can be listed. These ATCC strains can be purchased from the American Type Culture Collection. As a method for introducing a recombinant vector into bacteria, as long as it is a method for introducing DNA into bacteria, there is no particular limitation. For example, a method using calcium ions, an electroporation method, etc. can be listed.

[0171] When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, etc. can be used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include electroporation, protoplast method, lithium acetate method, etc.

[0172] When animal cells are used as hosts, monkey cells COS-7, Vero, CHO cells, mouse L cells, rat GH3, human FL cells, etc. can be used. Methods for introducing recombinant vectors into animal cells include, for example, electroporation, calcium phosphate method, liposome method, etc.

[0173] When insect cells are used as hosts, Sf9 cells, Sf21 cells, etc. can be used. As a method for introducing a recombinant vector into insect cells, for example, a calcium phosphate method, a liposome method, an electroporation method, etc. can be used.

[0174] When plant cells are used as hosts, examples thereof include tobacco BY-2 cells, but are not limited thereto. Examples of methods for introducing recombinant vectors into plant cells include the Agrobacterium method, the gene gun method, the PEG method, and the electroporation method.

[0175] When Escherichia coli is used as the host, most of the expressed nitrile hydratase becomes inclusion bodies and does not dissolve, so a transformant with low bacterial activity is obtained. On the other hand, when Rhodococcus is used as the host, nitrile hydratase is present in the soluble fraction, so a highly active transformant can be obtained. The host can be selected according to the purpose, and when selecting an improved enzyme under severe conditions, it is preferred to use a transformant of Rhodococcus with high activity.

[0176] 1.5 Method for producing improved nitrile hydratase

[0177] The improved nitrile hydratase can be produced by culturing the above-mentioned transformant and extracting a protein having nitrile hydratase activity from the obtained culture. The present invention also provides a method for producing such an improved nitrile hydratase.

[0178] In the present invention, the term "culture" includes any of culture supernatant, cultured cells, cultured bacterial bodies, or disrupted products of cells or bacterial bodies.

[0179] The culture of the transformant is carried out according to the method commonly used in host culture. As long as the culture medium for culturing the transformant of the present invention contains a carbon source, a nitrogen source, an inorganic salt, etc. that can be assimilated by the host bacteria, and can effectively culture the transformant, any of natural culture medium and synthetic culture medium can be used. As the carbon source, carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be listed. As the nitrogen source, inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate or ammonium salts of organic acids or other nitrogen-containing compounds can be listed.

[0180] In addition, peptone, yeast extract, meat extract, corn steep liquor, various amino acids, etc. can also be used. As inorganic substances, potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium carbonate, etc. can be listed. In addition, defoamers can also be added as needed to prevent foaming during culture. Furthermore, cobalt ions and iron ions as cofactors of nitrile hydratase can also be added to the culture medium to become nitriles and amides as inducers of the enzyme.

[0181] In order to prevent the vector and the target gene from falling off during the culture, the culture may be carried out under the condition of applying selection pressure. That is, when the selection marker is a drug resistance gene, the corresponding drug may be added to the culture medium; or when the selection marker is a nutrient deficiency complementation gene, the corresponding nutritional factor may be removed from the culture medium.

[0182] In addition, when the selection marker is a gene that confers assimilation, the corresponding assimilation factor can be added as a sole factor as needed. For example, when culturing Escherichia coli transformed with a vector containing an ampicillin resistance gene, ampicillin can be added during the culture as needed.

[0183] In the case of transformants made by transforming a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the culture medium as needed. For example, when transformants are made by transforming an expression vector having a promoter induced by isopropyl-β-D-thiogalactoside (IPTG), IPTG etc. may be added to the culture medium. In addition, when transformants are made by transforming an expression vector using a trp promoter induced by indoleacetic acid (IAA), IAA etc. may be added to the culture medium.

[0184] The culture conditions of the transformant are not particularly limited as long as they do not hinder the productivity of the target improved nitrile hydratase and the growth of the host, and are usually carried out at 10° C. to 40° C., preferably 20° C. to 37° C., for 5 to 100 hours. The pH is adjusted using an inorganic or organic acid, an alkaline solution, etc., and for example, in the case of Rhodococcus, the pH is adjusted to 6 to 9.

[0185] Examples of the culture method include solid culture, static culture, shaking culture, and aeration stirring culture. In particular, when culturing a transformant of Rhodococcus, it is preferably cultured under aerobic conditions by shaking culture or aeration stirring culture (small fermenter).

[0186] When culture is carried out under the above culture conditions, the improved nitrile hydratase of the present invention can be accumulated in the above culture, that is, in at least one of the culture supernatant, cultured cells, cultured bacterial bodies, or cell or bacterial body fragments at a high yield.

[0187] After the culture, when the improved nitrile hydratase is produced in the bacteria or cells, the target improved nitrile hydratase can be extracted by disrupting the bacteria or cells. As a method for disrupting the bacteria or cells, high-pressure treatment with a French press or a homogenizer, ultrasonic treatment, grinding treatment with glass beads, etc., enzyme treatment using lysozyme, cellulase, or pectinase, freeze-thaw treatment, hypotonic solution treatment, lysis induction treatment with bacteriophage, etc. can be used.

[0188] After the disruption, the disrupted residues of the bacteria or cells (including the insoluble fraction of the cell extract) can be removed as needed. As a method for removing the residue, for example, centrifugation, filtration, etc. can be cited, and flocculants, filter aids, etc. can also be used as needed to improve the efficiency of residue removal. The supernatant obtained after removing the residue is a cell extract soluble fraction, which can be used as a crudely purified improved nitrile hydratase solution.

[0189] When the improved nitrile hydratase is produced in bacteria or cells, the bacteria or cells themselves may be recovered by centrifugation, membrane separation or the like and used in an undisrupted state.

[0190] When the improved nitrile hydratase is produced outside the bacteria or outside the cells, the culture solution is used as it is, or the bacteria or cells are removed by centrifugation, filtration, etc. Thereafter, the improved nitrile hydratase may be extracted from the culture by extraction using ammonium sulfate precipitation, etc., as required, and then separated and purified by dialysis or various chromatography (gel filtration, ion exchange chromatography, affinity chromatography, etc.) as required.

[0191] The yield of the nitrile hydratase obtained by culturing the transformant can be confirmed by SDS-PAGE (polyacrylamide gel electrophoresis) or nitrile hydratase activity assay, etc., for example, per culture solution, per wet weight or dry weight of the bacterial cells, per crude enzyme solution protein, etc., without particular limitation. SDS-PAGE can be performed using a method known to those skilled in the art. In addition, the nitrile hydratase activity can be applied to the above-mentioned activity value.

[0192] In addition to the above methods, a cell-free protein synthesis system can also be used to produce improved nitrile hydratase. The cell-free protein synthesis system refers to a system that uses a cell extract to synthesize protein in an artificial container such as a test tube. It should be noted that the cell-free protein synthesis system used in the present invention also includes a cell-free transcription system that uses DNA as a template to synthesize RNA.

[0193] At this time, the organism corresponding to the host described above corresponds to the organism from which the cell extract described below is derived. Here, the cell extract described above can be an extract derived from eukaryotic cells or an extract derived from prokaryotic cells, such as an extract from wheat germ, Escherichia coli, etc. It should be noted that these cell extracts may be concentrated or not.

[0194] Cell extracts can be obtained by, for example, ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation, etc. Furthermore, in the present invention, cell-free protein synthesis can also be performed using commercially available kits. Examples of such kits include: TM(TOYOBO CO.,LTD.), TNT TM System (Promega KK.), PG-Mate for synthesis equipment TM (TOYOBO CO., LTD.), RTS (Roche Diagnostics KK), etc.

[0195] As described above, the improved nitrile hydratase obtained by cell-free protein synthesis can be purified by selecting an appropriate chromatography as described above.

[0196] 2. Method for producing amide compound

[0197] The improved nitrile hydratase of the present invention can be used as an enzyme catalyst for material production. For example, an amide compound can be produced by contacting a nitrile compound with the improved nitrile hydratase and collecting the generated amide compound.

[0198] As the enzyme catalyst, in addition to the isolated and purified nitrile hydratase, a culture obtained by culturing the transformant of the present invention or a treated product of the culture may be used. Examples of the treated product include: a treated product obtained by embedding the cultured cells (transformant) in a gel such as acrylamide; a treated product obtained by treating with glutaraldehyde; a treated product obtained by supporting the cultured cells (transformant) on an inorganic carrier such as alumina, silica, zeolite, and diatomaceous earth; and the like.

[0199] Here, "contact" means that the improved nitrile hydratase and the nitrile compound are present in the same reaction system or culture system, for example, including: mixing the separated and purified improved nitrile hydratase with the nitrile compound; adding the nitrile compound to the culture container of the cells (transformants) expressing the improved nitrile hydratase gene; culturing the cells in the presence of the nitrile compound; mixing the extract of the cells with the nitrile compound, etc.

[0200] The nitrile compound used as the substrate is selected in consideration of the substrate specificity of the enzyme, the stability of the enzyme relative to the substrate, etc. Acrylonitrile is preferred as the nitrile compound. The reaction method and the method for extracting the amide compound after the reaction are appropriately selected according to the characteristics of the substrate and the enzyme catalyst.

[0201] The enzyme catalyst is preferably used in a recycled form as long as its activity is not deactivated. From the viewpoint of preventing deactivation and facilitating recycling, the enzyme catalyst is preferably used in the form of a treated product.

[0202] Example

[0203] The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples. The "%" described here means mass %.

[0204] [Example 1] Preparation of Plasmid for Expressing Modified Nitrile Hydratase

[0205] A plasmid serving as a template for introducing the amino acid substitution of the present invention was prepared by the following method.

[0206] The plasmid having the nitrile hydratase gene of strain J1 uses pSJ034( Figure 1 ). pSJ034 is a plasmid for expressing nitrile hydratase in Rhodococcus. pJD034 was prepared from pSJ023 by the method described in Japanese Patent Laid-Open No. 10-337185. That is, the plasmid pSJ023 was partially digested with XbaI and ligated with an Sse8387I linker to produce a plasmid pSJ033 in which one XbaI site was replaced with Sse8387I. Subsequently, pSJ033 was partially digested with Sse8387I, the ends were blunted using Klenow fragment, and the plasmid pSJ034 was prepared by self-ligation.

[0207] It should be noted that Rhodococcus rhodochrous J-1 strain was deposited at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology (1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan (now NITE Patent Biological Depositary Center: Room 120, 2-5-8 Kamigeneral Kuzushi, Kisarazu, Chiba 292-0818, Japan)) under the accession number FERM BP-1478. (Original deposit date: September 18, 1987).

[0208] In addition, pSJ023 is the transformant "R. rhodochrous ATCC12674 / pSJ023", which was internationally deposited at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology (1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan (now NITE Patent Biological Depositary Center: Room 120, 2-5-8 Kamigeneral Kuzushi, Kisarazu, Chiba 292-0818, Japan)) under the accession number FERM BP-6232 on March 4, 1997.

[0209] [Example 2] Preparation of Modified Nitrile Hydratase

[0210] Using the plasmid pSJ034 prepared in Example 1, amino acid substitution was performed. PCR was carried out using the following reaction solution composition, reaction conditions, and primers.

[0211] <PCR Reaction Solution Composition>

[0212]

[0213] <PCR Reaction Conditions>

[0214] (98°C for 10 seconds, 55°C for 5 seconds, 72°C for 90 seconds) × 30 cycles

[0215] [Table 2]

[0216] <Primer>

[0217]

[0218] After the PCR was completed, 5 μl of the reaction solution was subjected to 0.7% agarose gel electrophoresis to confirm the amplified fragment of 11 kb, and 1 μl of Dpn I (attached to the kit) was added to the PCR reaction solution and reacted at 37°C for 1 hour to remove the template plasmid. The reaction solution was purified using Wizard SV Gel and PCR Clean-Up System (Promega KK.), and the purified PCR reaction product was used to transform JM109. From the obtained culture, plasmid DNA was extracted using QIAprep Spin Miniprep Kit (QIAGEN), and the base sequence of nitrile hydratase was confirmed using the automatic sequencer CEQ8000 (Beckman Coulter, Inc.). The obtained plasmids were named as shown in Table 3.

[0219] [Table 3]

[0220]

[0221] [Example 3] Preparation of Rhodococcus transformants

[0222] The cells of the logarithmic growth phase of Rhodococcus rhodochrous ATCC12674 strain were collected by centrifugation, washed three times in ice-cold sterile water, and suspended in sterile water. 1 μl of the plasmid prepared in Example 2 and 10 μl of the bacterial suspension were mixed and ice-cooled. DNA and bacterial suspension were added to the cuvette, and electric pulse treatment was performed at 2.0 kV and 200 OHMS using the gene introduction device Gene Pulser (BIORAD). The electric pulse treated liquid was allowed to stand for 10 minutes under ice-cooling, heat-shocked at 37°C for 10 minutes, and MYK medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) 500 μl was added and allowed to stand at 30°C for 5 hours. Then, the cells were spread on MYK agar medium containing 50 μg / ml kanamycin and cultured for 3 days at 30° C. Colonies obtained after culture at 30° C. for 3 days were designated as transformants.

[0223] Each transformant obtained in the above steps was inoculated in MYK medium (50 μg / ml kanamycin) and cultured with shaking at 30°C for 2 days. 1% inoculation was performed in GGPK medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HPO4, 0.05% KH2PO4, 0.05% Mg2O4·7H2O, 1% CoCl2, 0.1% urea, 50 μg / ml kanamycin, pH7.2). Cultured with shaking at 30°C for 3 days, the bacteria were collected by centrifugation. After that, the bacteria were washed with 100 mM phosphate buffer (pH7.0) to prepare a bacterial suspension.

[0224] [Example 4] Evaluation of amide compound resistance at high temperature

[0225] The amide compound resistance of the improved nitrile hydratase obtained in Example 3 was measured by the following method.

[0226] 0.2 ml of the bacterial cell liquid and 4.8 ml of 50 mM phosphate buffer (pH 7.0) were mixed, and 5 ml of 50 mM phosphate buffer (pH 7.0) containing 5.0% (w / v) acrylonitrile was further added to the mixed solution, and the mixture was reacted for 10 minutes while shaking at 10° C. Then, the bacterial cells were filtered, and the amount of acrylamide generated was quantified using gas chromatography.

[0227] <Analysis conditions>

[0228] Analytical instrument: Gas chromatograph GC2014 (manufactured by Shimadzu Corporation)

[0229] Detector: FID (detection 200℃)

[0230] Column: 1 m glass column filled with Porapak PS (column filler manufactured by Nihon Waters KK)

[0231] Column temperature: 190°C

[0232] The nitrile hydratase activity was calculated from the amount of acrylamide. Here, regarding the nitrile hydratase activity, the amount of enzyme that generates 1 μmol of acrylamide in 1 minute is defined as 1 U.

[0233] Next, the experiment was conducted with the following reaction solution composition and reaction conditions. It should be noted that each bacterial suspension used in the reaction was appropriately diluted with 100 mM phosphate buffer (pH 7.0) in advance to achieve the same amount of activity according to the measured enzyme activity. As a comparative control, ATCC12674 / pSJ034 was used as a comparative strain.

[0234] <Reaction Solution Composition>

[0235]

[0236] <Reaction Conditions>

[0237] Reaction temperature 45℃

[0238] Reaction time 3 hours

[0239] 1 ml of the reaction solution was collected before the start of the reaction (0 hour) and 3 hours later, filtered using a 0.45 μm filter, and the obtained filtrate was supplied to a gas chromatograph. Table 4 shows the analysis results of the ratio (%) of residual acrylonitrile.

[0240] [Table 4]

[0241] Plasmid name Consumption of acrylonitrile (%) Relative ratio (%) pSJ034 (Comparative Example) 0.8 100％ pSJH001 1.0 125％ pSJH002 1.4 163％ pSJH064 0.88 110％ pSJH004 1.8 225％ pSJH005 1.1 138％ pSJH022 2.3 288％ pSJH023 2.6 325％ pSJH024 1.3 163％ pSJH025 2.3 288％ pSJH026 1.7 213％ pSJH027 1.5 188％ pSJH028 1.6 200％ pSJH030 2.4 300％ pSJH032 2.6 325％

[0242] According to the above results, the consumption rate of acrylonitrile by all the improved nitrile hydratases was 110% or more compared to pSJ034 as a comparative example. In the synthesis reaction of amide compounds using nitrile hydratase, inactivation due to exposure to high temperature / high concentration of product and reaction inhibition by the product amide compound become problems. The improved nitrile hydratase of the present invention maintains nitrile hydratase activity even at high temperature and in the presence of high concentration of acrylamide, so it can be said that acrylamide tolerance at high temperature is improved.

[0243] [Example 5] Preparation of improved nitrile hydratase

[0244] Using the nitrile hydratase (pSJ306A) described in WO2012 / 164933, amino acid substitution was performed in the same manner as in Example 2. Table 5 shows the prepared plasmids.

[0245] [Table 5]

[0246]

[0247] Rhodococcus rhodochrous ATCC12674 transformants were obtained from the plasmids shown in Table 5 in the same manner as in Example 3 and cultured in MYK medium. Using the obtained cultured cells, amide compound resistance at high temperature was evaluated under the following conditions.

[0248] <Reaction Solution Composition>

[0249]

[0250] <Reaction Conditions>

[0251] Reaction temperature 45℃

[0252] Reaction time 5 hours

[0253] [Table 6]

[0254] Plasmid name Consumption of acrylonitrile (%) Consumption rate of acrylonitrile (%) pSJ306A (Comparative Example) 1.63 100％ pSJA006 2.16 133％ pSJA018 1.99 122％ pSJB018 1.79 110％ pSJC008 2.20 135％ pSJC010 2.41 148％ pSJC011 2.26 139％ pSJC017 2.42 148％ pSJD010 2.31 142％ pSJG001 2.26 139％ pSJG002 2.63 161％ pSJG003 2.68 164％ pSJG004 2.57 158％ pSJG005 2.38 146％

[0255] Based on the above results, compared with pSJ306A as a comparative example, the acrylonitrile consumption rate of all the improved nitrile hydratases was 110% or more. Therefore, the improved nitrile hydratase of the present invention maintained the nitrile hydratase activity even in the presence of high temperature and high concentration of acrylamide, and thus it can be said that the acrylamide tolerance at high temperature was improved.

[0256] [Example 6] Preparation of improved nitrile hydratase (JBRs) An expression plasmid of a nitrile hydratase gene (GenBank accession number AY141130) derived from Nocardia sp. JBRs was prepared by the following method.

[0257] Regarding the vector fragment, PCR was performed using pSJ034 as a template, and it was prepared by Wizard SV Gel and PCR Clean-Up Syste (Promega KK.).

[0258] <Composition of PCR reaction solution>

[0259]

[0260] <PCR reaction conditions>

[0261] (98°C for 10 seconds, 55°C for 5 seconds, 72°C for 90 seconds) × 30 cycles

[0262] NH-F: GAAGTGATCG TATGAGTGAA GACACACTCA CTG (SEQ ID NO: 51)

[0263] NH-R: GTGGATACCA TCCATTTCCT CATTCCTTTC ATC (SEQ ID NO: 52)

[0264] For the above-prepared vector fragment and the artificially synthesized nitrile hydratase gene (SEQ ID NO: 44) derived from Nocardia sp. JBRs, cloning was performed using the In-Fusion cloning kit (TAKARA Bio), and it was transformed into Escherichia coli HST08 (TAKARA Bio). The plasmid was recovered from the obtained colonies, the DNA sequence was confirmed, and a nitrile hydratase expression plasmid (pSJ-JBRs) derived from Nocardia sp. JBRs was obtained.

[0265] Furthermore, using pSJ-JBRs as a template, amino acid substitution was carried out by the same method as in Example 2. The prepared plasmids are shown in Table 7.

[0266] [Table 7]

[0267]

[0268] Rhodococcus rhodochrous ATCC12674 transformants were obtained from the plasmids described in Table 7 by the same method as in Example 3 and cultured in MYK medium. Using the obtained cultured cells, amide compound resistance at high temperature was evaluated under the conditions of Example 4. The results are shown in Table 8.

[0269] [Table 8]

[0270]

[0271] According to the above results, the acrylonitrile consumption rate of all the improved nitrile hydratases was 108% or more compared to pSJ-JBRs as a comparative example. Therefore, the improved nitrile hydratase of the present invention maintains nitrile hydratase activity even at high temperature and in the presence of high concentration of acrylamide, and thus it can be said that the acrylamide tolerance at high temperature is improved.

[0272] [Example 7] Preparation of improved nitrile hydratase (S85-2)

[0273] An expression plasmid of the nitrile hydratase gene (GenBank accession number AJ582605) derived from Rhodococcus pyridinophilus S85-2 was prepared using the artificially synthesized nitrile hydratase gene (SEQ ID NO: 45) by the same method as in Example 6. The obtained plasmid was named pSJ-S85-2.

[0274] Furthermore, using pSJ-S85-2 as a template, amino acid substitution was carried out in the same manner as in Example 2. The prepared plasmids are shown in Table 9.

[0275] [Table 9]

[0276]

[0277] Rhodococcus rhodochrous ATCC12674 transformants were obtained from the plasmids described in Table 9 by the same method as in Example 3 and cultured in MYK medium. Using the obtained cultured cells, amide compound resistance at high temperature was evaluated under the conditions of Example 4. The results are shown in Table 10.

[0278] [Table 10]

[0279]

[0280] According to the above results, the acrylonitrile consumption rate of all the improved nitrile hydratases was 130% or more compared to pSJ-S85-2 as a comparative example. Therefore, the improved nitrile hydratase of the present invention maintains nitrile hydratase activity even at high temperature and in the presence of high concentration of acrylamide, and thus it can be said that the acrylamide tolerance at high temperature is improved.

[0281] [Example 8] Preparation of improved nitrile hydratase (M8)

[0282] The plasmid pSJ-NO1A described in JP-A-2011-200132 was used as the expression plasmid for the nitrile hydratase gene derived from Rhodococcus rhodochrous M8 (GenBank Accession Nos. ATT79340, AAT79339), and amino acid substitution was performed in the same manner as in Example 2. The prepared plasmids are shown in Table 11.

[0283] [Table 11]

[0284]

[0285] Rhodococcus rhodochrous ATCC12674 transformants were obtained from the plasmids described in Table 11 by the same method as in Example 3 and cultured in MYK medium. Using the obtained cultured cells, amide compound resistance at high temperature was evaluated under the conditions of Example 4. The results are shown in Table 12.

[0286] [Table 12]

[0287]

[0288] According to the above results, the acrylonitrile consumption rate of all the improved nitrile hydratases was 110% or more compared to pSJ-NO1A as a comparative example. Therefore, the improved nitrile hydratase of the present invention maintains nitrile hydratase activity even at high temperature and in the presence of high concentration of acrylamide, and thus it can be said that the acrylamide tolerance at high temperature is improved.

[0289] [Example 9] Rhodococcus rhodochrous bacteria (Mitsui bacteria)

[0290] The plasmid pSJ-NO2A described in JP-A-2011-200132 was used as an expression plasmid for the nitrile hydratase gene derived from Pseudonocardia thermophila JCM3095 (GenBank Accession Nos. DD028560 and DD028561), and amino acid substitution was performed in the same manner as in Example 2. The prepared plasmids are shown in Table 10.

[0291] [Table 13]

[0292] Plasmid name Amino acid substitutions from wild-type strains pSJ-NO2A (Comparative Example) pSJH021 <![CDATA[Wα160L (equivalent to X of serial number 49 22 )]]>

[0293] Rhodococcus rhodochrous ATCC12674 transformants were obtained from the plasmids described in Table 13 by the same method as in Example 3 and cultured in MYK medium. Using the obtained cultured cells, amide compound resistance at high temperature was evaluated under the conditions of Example 4. The results are shown in Table 14.

[0294] [Table 14]

[0295] Plasmid name Consumption of acrylonitrile (%) Consumption rate of acrylonitrile (%) pSJ-NO2A (Comparative Example) 0.50 100％ pSJH021 1.20 240％

[0296] According to the above results, the acrylonitrile consumption rate of all the improved nitrile hydratases was 240% compared to pSJ-NO2A as a comparative example. Therefore, the improved nitrile hydratase of the present invention maintains the nitrile hydratase activity even at high temperature and in the presence of high concentration of acrylamide, and thus it can be said that the acrylamide tolerance at high temperature is improved.

[0297] Industrial Applicability

[0298] The improved nitrile hydratase of the present invention has improved acrylamide tolerance at high temperatures, and therefore can efficiently produce an amide compound corresponding to a nitrile compound, and is useful in the industrial production of amide compounds.

[0299] All publications, patents and patent applications cited in this specification are incorporated herein by reference as if they were directly incorporated by reference.

[0300] Accession number

[0301] Rhodococcus rhodochrous J1 strain: FERM BP-1478

[0302] Rhodococcus rhodochrous ATCC12674 / pSJ023: FERM BP-6232

[0303] SEQ ID NO: 20: α8G-F primer

[0304] SEQ ID NO: 21: α8G-R primer

[0305] SEQ ID NO: 22: α8V-F primer

[0306] SEQ ID NO: 23: α8V-R primer

[0307] SEQ ID NO: 24: α88V-F primer

[0308] SEQ ID NO: 25: α88V-R primer

[0309] SEQ ID NO: 26: α153I-F primer

[0310] SEQ ID NO: 27: α153I-R primer

[0311] SEQ ID NO: 28: α153L-F primer

[0312] SEQ ID NO: 29: α153L-R primer

[0313] SEQ ID NO: 30: α153M-F primer

[0314] SEQ ID NO: 31: α153M-R primer

[0315] SEQ ID NO: 32: α153T-F primer

[0316] SEQ ID NO: 33: α153T-R primer

[0317] SEQ ID NO: 34: α154L-F primer

[0318] SEQ ID NO: 35: α154L-R primer

[0319] SEQ ID NO: 36: α153I·α154L-F primer

[0320] SEQ ID NO: 37: α153I·α154L-R primer

[0321] SEQ ID NO: 38: α153L·α154L-F primer

[0322] SEQ ID NO: 39: α153L·α154L-R Primer

[0323] SEQ ID NO: 40: α153M·α154L-F Primer

[0324] SEQ ID NO: 41: α153M·α154L-R Primer

[0325] SEQ ID NO: 42: α153T·α154L-F Primer

[0326] SEQ ID NO: 43: α153T·α154L-R Primer

[0327] SEQ ID NO: 46: Specific amino acid of the present invention

[0328] SEQ ID NO: 47: Specific amino acid of the present invention

[0329] SEQ ID NO: 48: Specific amino acid of the present invention

[0330] SEQ ID NO: 49: Specific amino acid of the present invention

[0331] SEQ ID NO: 50: Amino Acids of the α Subunit of the Present Invention

[0332] SEQ ID NO: 51: NH-F primer

[0333] SEQ ID NO: 52: NH-R primer

[0334] SEQ ID NO: 53: α88T-F primer

[0335] SEQ ID NO: 54: α88T-R primer Sequence Listing <110> Mitsubishi Chemical Corporation <120> Improved nitrile hydratase <130> PMR-9014WO <150> JP2014-118041 <151> 2014-06-06 <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 690 <212> DNA <213> Rhodococcus rhodochrous J-1 <400> 1 atg gat ggt atc cac gac aca ggc ggc atg acc gga tac gga ccg gtc 48 ccc tat cag aag gac gag ccc ttc ttc cac tac gag tgg gag ggt cgg 96 acc ctg tca att ctg act tgg atg cat ctc aag ggc ata tcg tgg tgg 144 gac aag tcg cgg ttc ttc cgg gag tcg atg ggg aac gaa aac tac gtc 192 aac gag att cgc aac tcg tac tac acc cac tgg ctg agt gcg gca gaa 240 cgt atc ctc gtc gcc gac aag atc atc acc gaa gaa gag cga aag cac 288 cgt gtg caa gag atc ctt gag ggt cgg tac acg gac agg aag ccg tcg 336 cgg aag ttc gat ccg gcc cag atc gag aag gcg atc gaa cgg ctt cac 384 gag ccc cac tcc cta gcg ctt cca gga gcg gag ccg agt ttc tct ctc 432 ggt gac aag atc aaa gtg aag agt atg aac ccg ctg gga cac aca cgg 480 tgc ccg aaa tat gtg cgg aac aag atc ggg gaa atc gtc gcc tac cac 528 ggc tgc cag atc tat ccc gag agc agc tcc gcc ggc ctc ggc gac gat 576 cct cgc ccg ctc tac acg gtc gcg ttt tcc gcc cag gaa ctg tgg ggc 624 gac gac gga aac ggg aaa gac gta gtg tgc gtc gat ctc tgg gaa ccg 672 tac ctg atc tct gcg tga 690 <210> 2 <211> 229 <212> PRT <213> Rhodococcus rhodochrous <400> 2 Met Asp Gly Ile His Asp Thr Gly Gly Met Thr Gly Tyr Gly Pro Val 1 5 10 15 Pro Tyr Gln Lys Asp Glu Pro Phe Phe His Tyr Glu Trp Glu Gly Arg 20 25 30 Thr Leu Ser Ile Leu Thr Trp Met His Leu Lys Gly Ile Ser Trp Trp 35 40 45 Asp Lys Ser Arg Phe Phe Arg Glu Ser Met Gly Asn Glu Asn Tyr Val 50 55 60 Asn Glu Ile Arg Asn Ser Tyr Tyr Thr His Trp Leu Ser Ala Ala Glu 65 70 75 80 Arg Ile Leu Val Ala Asp Lys Ile Ile Thr Glu Glu Glu Arg Lys His 85 90 95 Arg Val Gln Glu Ile Leu Glu Gly Arg Tyr Thr Asp Arg Lys Pro Ser 100 105 110 Arg Lys Phe Asp Pro Ala Gln Ile Glu Lys Ala Ile Glu Arg Leu His 115 120 125 Glu Pro His Ser Leu Ala Leu Pro Gly Ala Glu Pro Ser Phe Ser Leu 130 135 140 Gly Asp Lys Ile Lys Val Lys Ser Met Asn Pro Leu Gly His Thr Arg 145 150 155 160 Cys Pro Lys Tyr Val Arg Asn Lys Ile Gly Glu Ile Val Ala Tyr His 165 170 175 Gly Cys Gln Ile Tyr Pro Glu Ser Ser Ser Ala Gly Leu Gly Asp Asp 180 185 190 Pro Arg Pro Leu Tyr Thr Val Ala Phe Ser Ala Gln Glu Leu Trp Gly 195 200 205 Asp Asp Gly Asn Gly Lys Asp Val Val Cys Val Asp Leu Trp Glu Pro 210 215 220 Tyr Leu Ile Ser Ala 225 <210> 3 <211> 612 <212> DNA <213> Rhodococcus rhodochrous J-1 <400> 3 gtg agc gag cac gtc aat aag tac acg gag tac gag gca cgt acc aag 48 gcg atc gaa acc ttg ctg tac gag cga ggg ctc atc acg ccc gcc gcg 96 gtc gac cga gtc gtt tcg tac tac gag aac gag atc ggc ccg atg ggc 144 ggt gcc aag gtc gtg gcc aag tcc tgg gtg gac cct gag tac cgc aag 192 tgg ctc gaa gag gac gcg acg gcc gcg atg gcg tca ttg ggc tat gcc 240 ggt gag cag gca cac caa att tcg gcg gtc ttc aac gac tcc caa acg 288 cat cac gtg gtg gtg tgc act ctg tgt tcg tgc tat ccg tgg ccg gtg 336 ctt ggt ctc ccg ccc gcc tgg tac aag agc atg gag tac cgg tcc cga 384 gtg gta gcg gac cct cgt gga gtg ctc aag cgc gat ttc ggt ttc gac 432 atc ccc gat gag gtg gag gtc agg gtt tgg gac agc agc tcc gaa atc 480 cgc tac atc gtc atc ccg gaa cgg ccg gcc ggc acc gac ggt tgg tcc 528 gag gag gag ctg acg aag ctg gtg agc cgg gac tcg atg atc ggt gtc 576 agt aat gcg ctc aca ccg cag gaa gtg atc gta tga 612 <210> 4 <211> 203 <212> PRT <213> Rhodococcus rhodochrous <400> 4 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 5 <211> 203 <212> PRT <213> Rhodococcus rhodochrous M8 <400> 5 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 6 <211> 203 <212> PRT <213> Rhodococcus ruber TH <400> 6 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 7 <211> 203 <212> PRT <213> Rhodococcus pyridinivorans_MW3 <400> 7 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 8 <211> 203 <212> PRT <213> Rhodococcus pyridinivorans S85-2 <400> 8 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 9 <211> 203 <212> PRT <213> Rhodococcus pyridinivorans MS-38 <400> 9 Val Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 10 <211> 203 <212> PRT <213> Nocardia JBRs <400> 10 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 11 <211> 203 <212> PRT <213> Nocardia YS - 2002 <400> 11 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 12 <211> 180 <212> PRT <213> Uncultured Bacterium SP1 <400> 12 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Val Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr 85 90 95 Pro Trp Pro Val Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu 100 105 110 Tyr Arg Ser Arg Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp 115 120 125 Phe Gly Phe Asp Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser 130 135 140 Ser Ser Glu Ile Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr 145 150 155 160 Asp Gly Trp Ser Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser 165 170 175 Ile Ile Gly Val 180 <210> 13 <211> 203 <212> PRT <213> Uncultured bacterium BD2 <400> 13 Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 14 <211> 203 <212> PRT <213> Rhodococcus rhodochrous ATCC39484 <400> 14 Val Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys 1 5 10 15 Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr 85 90 95 His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile 145 150 155 160 Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 15 <211> 213 <212> PRT <213> Sinorhizobium meliloti WSM419 <400> 15 Met Ser Glu His Arg His Gly Pro Gly Glu Glu His Gly His His His 1 5 10 15 Asp Asn His Leu Thr Asp Met Glu Ala Arg Val Lys Ala Leu Glu Thr 20 25 30 Val Leu Thr Glu Lys Gly Leu Ile Asp Pro Ala Ala Ile Asp Ala Ile 35 40 45 Val Asp Thr Tyr Glu Thr Lys Val Gly Pro Arg Asn Gly Ala Arg Val 50 55 60 Val Ala Lys Ala Trp Ser Asp Pro Asp Phe Ala Asp Trp Leu Arg Arg 65 70 75 80 Asp Ala Thr Ala Ala Ile Ala Ser Leu Gly Phe Thr Gly Arg Gln Gly 85 90 95 Glu His Met Arg Ala Val Phe Asn Thr Ser Glu Thr His Asn Leu Ile 100 105 110 Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Ala Val Leu Gly Leu Pro 115 120 125 Pro Val Trp Tyr Lys Ala Pro Pro Tyr Arg Ser Arg Ala Val Ile Asp 130 135 140 Pro Arg Gly Val Leu Ala Glu Phe Gly Leu Asn Leu Pro Ala Glu Lys 145 150 155 160 Lys Ile Arg Val Trp Asp Ser Thr Ala Glu Leu Arg Tyr Leu Val Val 165 170 175 Pro Glu Arg Pro Ala Ala Thr Asp Asp Leu Gly Glu Asp Ala Leu Ala 180 185 190 Lys Leu Val Thr Arg Asp Ser Met Ile Gly Thr Gly Leu Ala Leu Ser 195 200 205 Pro Glu Ala Phe Arg 210 <210> 16 <211> 205 <212> PRT <213> Geobacillus thermoglucosidasius Q6 <400> 16 Met Ser Val Gln Lys Val His His Asn Val Leu Pro Glu Lys Pro Ala 1 5 10 15 Gln Thr Arg Thr Lys Ala Leu Glu Ser Leu Leu Ile Glu Ser Gly Leu 20 25 30 Val Ser Thr Asp Ala Leu Asp Ala Ile Ile Glu Ala Tyr Glu Asn Asp 35 40 45 Ile Gly Pro Met Asn Gly Ala Lys Val Val Ala Lys Ala Trp Val Asp 50 55 60 Pro Asp Tyr Lys Glu Arg Leu Leu Arg Asp Gly Thr Ser Ala Ile Ala 65 70 75 80 Glu Leu Gly Phe Leu Gly Leu Gln Gly Glu His Met Val Val Val Glu 85 90 95 Asn Thr Pro Lys Val His Asn Val Val Val Cys Thr Leu Cys Ser Cys 100 105 110 Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Ser Trp Tyr Lys Ser Ala 115 120 125 Ser Tyr Arg Ala Arg Ile Val Ser Glu Pro Arg Thr Val Leu Lys Glu 130 135 140 Phe Gly Leu Glu Leu Asp Asp Asp Val Glu Ile Arg Val Trp Asp Ser 145 150 155 160 Ser Ala Glu Ile Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr 165 170 175 Glu Gly Trp Ser Glu Glu Glu Leu Ala Lys Leu Val Thr Arg Asp Ser 180 185 190 Met Ile Gly Val Ala Lys Ile Lys Ser Pro Val Lys Lys 195 200 205 <210> 17 <211> 205 <212> PRT <213> Thermopseudonocardia thermophila JCM3095 <400> 17 Met Thr Glu Asn Ile Leu Arg Lys Ser Asp Glu Glu Ile Gln Lys Glu 1 5 10 15 Ile Thr Ala Arg Val Lys Ala Leu Glu Ser Met Leu Ile Glu Gln Gly 20 25 30 Ile Leu Thr Thr Ser Met Ile Asp Arg Met Ala Glu Ile Tyr Glu Asn 35 40 45 Glu Val Gly Pro His Leu Gly Ala Lys Val Val Val Lys Ala Trp Thr 50 55 60 Asp Pro Glu Phe Lys Lys Arg Leu Leu Ala Asp Gly Thr Glu Ala Cys 65 70 75 80 Lys Glu Leu Gly Ile Gly Gly Leu Gln Gly Glu Asp Met Met Trp Val 85 90 95 Glu Asn Thr Asp Glu Val His His Val Val Val Cys Thr Leu Cys Ser 100 105 110 Cys Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Asn Trp Phe Lys Glu 115 120 125 Pro Gln Tyr Arg Ser Arg Val Val Arg Glu Pro Arg Gln Leu Leu Lys 130 135 140 Glu Glu Phe Gly Phe Glu Val Pro Pro Ser Lys Glu Ile Lys Val Trp 145 150 155 160 Asp Ser Ser Ser Glu Met Arg Phe Val Val Leu Pro Gln Arg Pro Ala 165 170 175 Gly Thr Asp Gly Trp Ser Glu Glu Glu Leu Ala Thr Leu Val Thr Arg 180 185 190 Glu Ser Met Ile Gly Val Glu Pro Ala Lys Ala Val Ala 195 200 205 <210> 18 <211> 207 <212> PRT <213> Rhodococcus rhodochrous Cr4 <400> 18 Met Thr Ala His Asn Pro Val Gln Gly Thr Phe Pro Arg Ser Asn Glu 1 5 10 15 Glu Ile Ala Ala Arg Val Lys Ala Met Glu Ala Ile Leu Val Asp Lys 20 25 30 Gly Leu Ile Ser Thr Asp Ala Ile Asp Tyr Met Ser Ser Val Tyr Glu 35 40 45 Asn Glu Val Gly Pro Gln Leu Gly Ala Lys Ile Ala Ala His Ala Trp 50 55 60 Val Asp Pro Glu Phe Lys Gln Arg Leu Leu Ala Asp Ala Thr Gly Ala 65 70 75 80 Cys Lys Glu Met Gly Val Gly Gly Met Gln Gly Glu Glu Met Val Val 85 90 95 Leu Glu Asn Thr Asp Thr Val Asn Asn Met Val Val Cys Thr Leu Cys 100 105 110 Ser Cys Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Asn Trp Tyr Lys 115 120 125 Tyr Pro Ala Tyr Arg Ala Arg Ala Ala Arg Asp Pro Arg Gly Val Met 130 135 140 Ala Glu Phe Gly Tyr Thr Pro Ala Ser Asp Val Glu Ile Arg Val Trp 145 150 155 160 Asp Ser Ser Ala Glu Leu Arg Tyr Trp Val Leu Pro Gln Arg Pro Ala 165 170 175 Gly Thr Glu Asn Phe Thr Glu Glu Gln Leu Ala Ala Leu Val Thr Arg 180 185 190 Asp Ser Leu Ile Gly Val Ser Val Pro Thr Ala Pro Asn Lys Ala 195 200 205 <210> 19 <211> 210 <212> PRT <213> Comamonas testosteroni <400> 19 Met Gly Gln Ser His Thr His Asp His His His Asp Gly Tyr Gln Ala 1 5 10 15 Pro Pro Glu Asp Ile Ala Leu Arg Val Lys Ala Leu Glu Ser Leu Leu 20 25 30 Ile Glu Lys Gly Leu Val Asp Pro Ala Ala Met Asp Leu Val Val Gln 35 40 45 Thr Tyr Glu His Lys Val Gly Pro Arg Asn Gly Ala Lys Val Val Ala 50 55 60 Lys Ala Trp Val Asp Pro Ala Tyr Lys Ala Arg Leu Leu Ala Asp Gly 65 70 75 80 Thr Ala Gly Ile Ala Glu Leu Gly Phe Ser Gly Val Gln Gly Glu Asp 85 90 95 Met Val Ile Leu Glu Asn Thr Pro Ala Val His Asn Val Val Val Cys 100 105 110 Thr Leu Cys Ser Cys Tyr Pro Trp Pro Thr Leu Gly Leu Pro Pro Ala 115 120 125 Trp Tyr Lys Ala Pro Pro Tyr Arg Ser Arg Met Val Ser Asp Pro Arg 130 135 140 Gly Val Leu Ala Glu Phe Gly Leu Val Ile Pro Ala Lys Glu Ile Arg 145 150 155 160 Val Trp Asp Thr Thr Ala Glu Leu Arg Tyr Met Val Leu Pro Glu Arg 165 170 175 Pro Ala Gly Thr Glu Ala Tyr Ser Glu Glu Gln Leu Ala Glu Leu Val 180 185 190 Thr Arg Asp Ser Met Ile Gly Thr Gly Leu Pro Ile Gln Pro Thr Pro 195 200 205 Ser His 210 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> α8G-F Primer <400> 20 aataagggca cggagtacga ggcacgt 27 <210> 21 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α8G-R Primers <400> twenty one ctccgtgccc ttattgacgt gctcgct 27 <210> twenty two <211> 27 <212> DNA <213> Artificial sequence <220> <223> α8V-F Primer <400> twenty two aataaggtca cggagtacga ggcacgt 27 <210> twenty three <211> 27 <212> DNA <213> Artificial sequence <220> <223> α8V-R Primer <400> twenty three ctccgtgacc ttattgacgt gctcgct 27 <210> twenty four <211> 27 <212> DNA <213> Artificial sequence <220> <223> α88V-F Primer <400> twenty four ccaaattgtc gcggtcttca acgactc 27 <210> 25 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α88V-R Primer <400> 25 gaccgcgaca atttggtgtg cctgctc 27 <210> 26 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153I-F primer <400> 26 gtcaggatct gggacagcag ctccgaa 27 <210> 27 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153I-R primer <400> 27 gtcccagatc ctgacctcca cctcatc 27 <210> 28 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153L-F Primer <400> 28 gtcaggctct gggacagcag ctccgaa 27 <210> 29 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153L-R Primer <400> 29 gtcaggggagt gggacagcag ctccgaa 27 <210> 30 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153M-F Primer <400> 30 gtcaggatgt gggacagcag ctccgaa 27 <210> 31 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153M-R Primer <400> 31 gtcaggcatt gggacagcag ctccgaa 27 <210> 32 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153T-F Primer <400> 32 gtcaggacct gggacagcag ctccgaa 27 <210> 33 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153T-R Primer <400> 33 gtcaggggtt gggacagcag ctccgaa 27 <210> 34 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α154L-F Primer <400> 34 agggttctcg acagcagctc cgaaatc 27 <210> 35 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α154L-R Primer <400> 35 gctgtcgaga accctgacctccacctc 27 <210> 36 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153I / α154L-F primers <400> 36 gtcaggatcc tcgacagcag ctccgaa 27 <210> 37 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153I / α154L-R primers <400> 37 gctgtcgagg atcctgacctccacctc 27 <210> 38 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153L / α154L-F primers <400> 38 gtcaggctcc tcgacagcag ctccgaa 27 <210> 39 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153L / α154L-R primers <400> 39 gctgtcgagg agcctgacctccacctc 27 <210> 40 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153M / α154L-F primers <400> 40 gtcaggatgc tcgacagcag ctccgaa 27 <210> 41 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153M / α154L-R Primers <400> 41 gctgtcgagc atcctgacctccacctc 27 <210> 42 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153T / α154L-F primers <400> 42 gtcaggaccc tcgacagcag ctccgaa 27 <210> 43 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α153T / α154L-R Primers <400> 43 gctgtcgagg agcctgacctccacctc 27 <210> 44 <211> 1315 <212> DNA <213> Nocardia sp. JBRs <400> 44 atggatggta tccacgacac aggcggcatg accggatacg gaccggtccc ctatcagaag 60 gacgagccct tcttccacta cgagtgggag ggtcggaccc tgtcgattct gacctggatg 120 catctcaagg gcatgtcgtg gtgggacaag tcgcggttct tccggggagtc gatggggaac 180 gaaaactacg tcaacgagat tcgcaactcg tactacaccc actggctgag tgcggcagaa 240 cgtatcctcg tcgccgacaa gatcatcacc gaagaagagc gaagcaccg tgtgcaggag 300 atcctcgagg gtcggtacac ggaggaac ccgtcgcgga agttcgatcc ggccgagatc 360 gagaaggcga tcgaacggct tcacgagccc cactccctag cacttccagg agcggagccg 420 agtttctccc tcggtgacaa ggtcaaagtg aagaatatga acccgctggg acacacacgg 480 540 tatcccgaga gcagctccgc cggcctcggc gacgatcccc gcccgctcta cacggtcgcg 600 ttttccgccc aggaactgtg gggcgacgac ggaacggga aagacgtagt gtgcgtcgat 660 ctctgggaac cgtacctgat ctctgcgtga aaaggaatacg atagtgagcg agcacgtcaa 720 tagtacacg gagtacgagg cacgtaccaa ggcaatcgaa actttgctgt acgagcgagg 780 gctcatcacg cccgccgcgg tcgaccgagt cgtttcgtac tacgagaacg agatcggccc 840 gatgggcggt gccaaggtcg tggcgaagtc ctgggtggac cctgagtacc gcaagtggct 900 cgaagaggac gcgacggccg cgatggcgtc attgggctat gccggtgagc aggcacacca 960 cgaagaggac gcgacggccg cgatggcgtc attgggctat gccggtgagc aggcacacca 960 aatttcggcg gtcttcaacg actcccaaac gcatcacgtg gtggtgtgca ctctgtgttc 1020 aatttcggcg gtcttcaacg actcccaaac gcatcacgtg gtggtgtgca ctctgtgttc 1020 gtgctatccg tggccggtgc ttggtctccc gcccgcctgg tacaagagca tggagtaccg 1080 gtgctatccg tggccggtgc ttggtctccc gcccgcctgg tacaagagca tggagtaccg 1080 gtcccgagtg gtagcggacc ctcgtggagt gctcaagcgc gatttcggtt tcgacatccc 1140 gtcccgagtg gtagcggacc ctcgtggagt gctcaagcgc gatttcggtt tcgacatccc 1140 cgatgaggtg gaggtcaggg tttgggacag cagctccgaa atccgctaca tcgtcatccc 1200 cgatgaggtg gaggtcaggg tttgggacag cagctccgaa atccgctaca tcgtcatccc 1200 ggaacggccg gccggcaccg acggttggtc cgaggacgag ctggcgaagc tggtgagccg 1260 ggaacggccg gccggcaccg acggttggtc cgaggacgag ctggcgaagc tggtgagccg 1260 ggactcgatg atcggtgtca gtaatgcgct cacaccccag gaagtgatcg tatga 1315 ggactcgatg atcggtgtca gtaatgcgct cacaccccag gaagtgatcg tatga 1315 <210> 45<210> 45 <211> 1315<211> 1315 <212> DNA <212> DNA <213> 嗜吡啶红球菌 S85-2 <213> Rhodococcus pyridinivorans S85-2 <400> 45 <400> 45 atggatggta tccacgacac aggcggcatg accggatacg gaccggtccc ctatcagaag 60 atggatggta tccacgacac aggcggcatg accggatacg gaccggtccc ctatcagaag 60 gacgagccct tcttccacta cgagtgggag ggtcggaccc tgtcgattct gacttggatg 120 gacgagccct tcttccacta cgagtgggag ggtcggaccc tgtcgattct gacttggatg 120 catctcaagg gcatatcgtg gtgggacaag tcgcggttct tccgggagtc gatggggaac 180 catctcaagg gcatatcgtg gtgggacaag tcgcggttct tccgggagtc gatggggaac 180 gaaaactacg tcaacgagat tcgcaactcg tactacaccc actggctgag tgcggcagaa 240 cgtatcctcg tcgccgacaa gatcatcacc gaagaagagc gaagcaccg tgtgcaagag 300 atccttgagg gtcggtacac ggaggaag ccgtcgcgga agttcgatcc ggcccagatc 360 gagaaggcga tcgaacggct tcacgagccc cactccctag cacttccagg agcggagccg 420 agtttctctc tcggtgacaa gatcaaagtg aagagtatga acccgctggg acacacacgg 480 540 tatcccgaga gcagctccgc cggcctcggc gacgatcctc gcccgctcta cacggtcgcg 600 ttttccgccc aggaactgtg gggcgacgac ggaacggga aagacgtagt gtgcgtcgat 660 ctctgggaac cgtacctgat ctctgcgtga aaaggaatacg atagtgagcg agcacgtcaa 720 tagtacacg gagtacgagg cacgtaccaa ggcgatcgaa accttgctgt acgagcgagg 780 gctcatcacg cccgccgcgg tcgaccgagt cgtttcgtac tacgagaacg agatcggccc 840 gatgggcggt gccaaggtcg tggccaagtc ctgggtggac cctgagtacc gcaagtggct 900 cgaagaggac gcgacggccg cgatggcgtc attgggctat gccggtgagc aggcacacca 960 aatttcggcg gtcttcaacg actcccaaac gcatcacgtg gtggtgtgca ctctgtgttc 1020 gtgctatccg tggccggtgc ttggtctccc gcccgcctgg tacaagagca tggagtaccg 1080 gtcccgagtg gtagcggacc ctcgtggagt gctcaagcgc gatttcggtt tcgacatccc 1140 cgatgaggtg gaggtcaggg tttggggacag cagctccgaa atccgctaca tcgtcatccc 1200 ggaacggccg gccggcaccg acggttggtc cgaggaggag ctgacgaagc tggtgagccg 1260 ggactcgatg atcggtgtca gtaatgcgct cacaccgcag gaagtgatcg tatga 1315 <210> 46 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Specific sequences according to the present invention <220> <221> misc_feature <222> (1)..(6) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (8) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be any natural amino acid <400> 46 Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Lys Ala Xaa Glu 1 5 10 <210> 47 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Specific sequences according to the present invention <220> <221> misc_feature <222> (1)..(4) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (6)..(14) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (16) <223> Xaa can be any natural amino acid <400> 47 Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Leu Cys <210> 48 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Specific sequences according to the present invention <220> <221> misc_feature <222> (1) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (8) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (10)..(11) <223> Xaa can be any natural amino acid <400> 48 Xaa Trp Asp Ser Xaa Xaa Glu Xaa Arg Xaa Xaa Val 1 5 10 <210> 49 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Specific sequences according to the present invention <220> <221> misc_feature <222> (2)..(2) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (8) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (10)..(11) <223> Xaa can be any natural amino acid <400> 49 Val Xaa Asp Ser Xaa Xaa Glu Xaa Arg Xaa Xaa Val 1 5 10 <210> 50 <211> 203 <212> PRT <213> Artificial sequence <220> <223> The alpha subunit amino acids according to the present invention <220> <221> misc_feature <222> (8)..(13) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (15) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (18) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (88)..(91) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (93)..(101) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (103)..(103) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (153)..(154) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (157)..(158) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (160)..(160) <223> Xaa can be any natural amino acid <220> <221> misc_feature <222> (162)..(163) <223> Xaa can be any natural amino acid <400> 50 Met Ser Glu His Val Asn Lys Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Lys 1 5 10 15 Ala Xaa Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala 20 25 30 Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly 35 40 45 Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys 50 55 60 Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala 65 70 75 80 Gly Glu Gln Ala His Gln Ile Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Cys Xaa Leu Cys Ser Cys Tyr Pro Trp Pro Val 100 105 110 Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg 115 120 125 Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp 130 135 140 Ile Pro Asp Glu Val Glu Val Arg Xaa Xaa Asp Ser Xaa Xaa Glu Xaa 145 150 155 160 Arg Xaa Xaa Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser 165 170 175 Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val 180 185 190 Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 195 200 <210> 51 <211> 33 <212> DNA <213> Artificial sequence <220> <223> NH-F primer <400> 51 gaagtgatcg tatgagtgaa gacacactca ctg 33 <210> 52 <211> 33 <212> DNA <213> Artificial sequence <220> <223> NH-R Primer <400> 52 gtggatacca tccatttcct cattcctttc atc 33 <210> 53 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α88T-F primer <400> 53 ccaaattacc gcggtcttca acgactc 27 <210> 54 <211> 27 <212> DNA <213> Artificial sequence <220> <223> α88T-R Primer <400> 54 gaccgcggta atttggtgtg cctgctc 27 <210> 55 <211> 6 <212> PRT <213> Rhodococcus rhodochrous <220> <221> MISC_FEATURE <222> (2)..(2) <223> Replace Xaa with Ser or Thr <400> 55 Cys Xaa Leu Cys Ser Cys 1 5

Claims

1. An improved nitrile hydratase having improved tolerance to amide compounds at high temperatures compared to a wild type, The characteristic is that the tyrosine at position 8 of the α subunit is replaced by glycine or valine, The nitrile hydratase is derived from Rhodococcus or Nocardia, The wild type contains α subunit and β subunit. The α subunit is composed of the amino acid sequence shown in sequence number 4, and the β subunit is composed of the amino acid sequence shown in sequence number 2; The α subunit is composed of the amino acid sequence shown in SEQ ID NO: 5, and the amino acid sequence of the β subunit is the amino acid sequence shown in GenBank Accession No. AAT79339; The α subunit consists of the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence of the β subunit is the amino acid sequence of the β subunit shown in GenBank Accession No. AJ582605; or The α subunit consists of the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence of the β subunit is the amino acid sequence of the β subunit shown in GenBank Accession No. AY141130; The α subunit further has one or more substitutions selected from the following (i) to (iii): (i) substitution of serine at position 88 with valine or threonine; (ii) substitution of valine at position 153 with isoleucine, leucine, methionine, or threonine; (iii) substitution of tryptophan at position 154 with leucine, The α subunits have the following common sequence: (a) YTEYEARTKAIE (8th to 19th digits) (b) SAVFNDSQTHHVVVCTLC (position 88 to 105) (c) VWDSSSEIRYIV (153rd to 164th position) There are no substitutions in the β subunit.

2. The improved nitrile hydratase according to claim 1, wherein The nitrile hydratase is derived from Rhodococcus rhodochrous J1, Rhodococcus rhodochrous M8, Rhodococcus pyridinophilus S85-2 or Nocardia sp. JBRs.

3. An improved nitrile hydratase, which further has substitutions of Gα174L, Pβ17G, Sβ57M, Tβ107K, Kβ114Y, Nβ167S, Cβ218H and Vβ219A in the improved nitrile hydratase according to claim 1, and is derived from Rhodococcus rhodochrous J1, and has improved amide compound tolerance at high temperatures compared to the wild type.

4. The improved nitrile hydratase according to claim 1, which is any of the following: 1) Derived from Rhodococcus rhodochrous J1, having the following substitutions in the wild-type sequence: 2) Derived from Rhodococcus rhodochrous M8, with the following substitutions in the wild-type sequence: 3) from Rhodococcus pyridinophilus S85-2, having the following substitutions in the wild-type sequence: 4) from Nocardia sp. JBRs, with the following substitutions in the wild-type sequence: 。 5 . A DNA encoding the improved nitrile hydratase according to claim 1 . A recombinant vector comprising the DNA according to claim 5.

7. A transformant comprising the recombinant vector according to claim 6; The transformant is selected from at least one of bacteria, yeast, animal cells, and insect cells; the animal cells are selected from at least one of monkey cells COS-7, Vero, CHO cells, mouse L cells, rat GH3, or human FL cells.

8. A method for producing nitrile hydratase, comprising culturing the transformant according to claim 7 and extracting nitrile hydratase from the obtained culture.

9. A method for producing an amide compound, characterized in that: The improved nitrile hydratase according to any one of claims 1 to 4, or a culture obtained by culturing the transformant according to claim 7, or a processed product of the culture is contacted with a nitrile compound.

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