JP2008253182A - Improved nitrile hydratase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein having more improved nitrile hydratase activity in heat resistance and/or resistance against amido compounds by improving a wild type nitrile hydratase. <P>SOLUTION: Proteins are represented by (A) or (B): (A) a protein including an amino acid sequence in which a specific amino acid residue in the amino acid sequence of the wild type nitrile hydratase is substituted with another amino acid and having nitrile hydratase activity; and (B) a protein including an amino acid sequence, in which one or several amino acid residues, except the specific amino acid residue, in the amino acid sequence of the protein (A) are deleted, substituted and/or added, and having nitrile hydratase activity. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an improved (mutant) nitrile hydratase and a method for producing the same. Furthermore, the present invention relates to a gene DNA encoding the enzyme, a recombinant vector containing the gene DNA, a transformant having the recombinant vector, and a method for producing an amide compound.

In recent years, nitrile hydratase, an enzyme having nitrile hydration activity that hydrates nitrile groups and converts them into amide groups, was discovered, and corresponding amides from nitrile compounds using the enzyme or microbial cells containing the enzyme. A method for producing the compound is disclosed. This production method is known to have a higher conversion rate and selectivity from a nitrile compound to a corresponding amide compound than conventional chemical synthesis methods.
Examples of microorganisms that produce nitrile hydratase include, for example, the genus Corynebacterium, the genus Pseudomonas, the genus Rhodococcus, the genus Rhizobium, the genus Klebsiella, and the pseudonocardia. Examples include microorganisms belonging to genera and the like. Among them, Rhodococcus rhodochrous J1 strain has been used for industrial production of acrylamide and has proved its usefulness. Moreover, the gene which codes the nitrile hydratase which the strain produces is also clear (refer patent document 1).

  On the other hand, not only using nitrile hydratase and its gene isolated from microorganisms that exist in nature, but also against nitrile hydratase, activity, substrate specificity, Vmax, Km, thermal stability, substrate stability, Attempts have been made to introduce mutations into nitrile hydratase for the purpose of changing the stability of the product (see Patent Documents 2 and 3).

  However, there are few specific examples in which heat resistance or amide compound resistance is improved by introducing a mutation into a specific amino acid residue in the amino acid sequence of wild-type nitrile hydratase. There is no known example of improving both tolerances. Developing these nitrile hydratases with improved heat resistance and / or amide compound resistance and using them in the production of amide compounds is very useful from the viewpoint of production costs such as catalyst costs. Furthermore, the acquisition of an enzyme with improved heat resistance has been desired to be developed from the viewpoint of reducing the amount of enzyme during the reaction and reducing the cost.

Japanese Patent No. 3162091 Japanese Patent Application No. 2004-156593 International Publication No. 2004/056990 Pamphlet

  Accordingly, an object of the present invention is to provide a protein having nitrile hydratase activity with improved heat resistance and / or amide compound resistance by improving wild-type nitrile hydratase, and further, gene DNA encoding the protein, A recombinant vector containing the gene DNA, a transformant containing the recombinant vector, a nitrile hydratase collected from a culture of the transformant, a production method thereof, and the culture or a processed product of the culture It is in providing the manufacturing method of the used amide compound.

  The present inventor has intensively studied to solve the above problems. As a result, in the amino acid sequence of wild-type nitrile hydratase, a protein in which a predetermined amino acid residue is substituted with another amino acid residue has nitrile hydratase activity, and improved heat resistance and / or amide compound resistance. As a result, the present invention has been completed.

That is, the present invention is as follows.
(1) The following protein (A) or (B).
(A) an amino acid sequence in which at least one amino acid residue selected from the group consisting of the following (a) to (g) in the amino acid sequence of wild-type nitrile hydratase is substituted with another amino acid residue; A protein having hydratase activity (a) 53 residues from the most downstream C residue of the amino acid sequence C (S / T) LCSC constituting the prosthetic molecule binding region in the amino acid sequence of the α subunit Downstream amino acid residue (b) Amino acid residue 67 residues downstream from the most downstream C residue of the amino acid sequence C (S / T) LCSC (c) C-terminal amino acid of the β subunit amino acid sequence (D) 97 amino acid residues upstream from the C-terminal amino acid residue (e) The C-terminal amino acid residue (F) 62 amino acid residues upstream from the C-terminal amino acid residue (g) 40 amino acid residues upstream from the C-terminal amino acid residue Amino acid residue (B) In the amino acid sequence of the protein of (A), including an amino acid sequence in which one or several amino acid residues are deleted, substituted and / or added except for the amino acid residue after the substitution, And protein (2) below (A) or (B) protein characterized by having nitrile hydratase activity.
(A) In the amino acid sequence of wild-type nitrile hydratase, the following amino acid residues (a) and (b) and at least one amino acid residue selected from the group consisting of the following (c) to (e) are other amino acids: A protein comprising an amino acid sequence substituted with a residue and having nitrile hydratase activity (a) 63 amino acids upstream from the amino acid residue at the C-terminal in the amino acid sequence of β subunit Amino acid residues (b) 11 amino acid residues upstream from the C-terminal amino acid residue (c) 213 amino acid residues upstream from the C-terminal amino acid residue (d) C 65 amino acid residues upstream from the terminal amino acid residue (e) 62 amino acid residues upstream from the C-terminal amino acid residue (B) (A) The amino acid sequence includes an amino acid sequence in which one or several amino acid residues are deleted, substituted and / or added, except for the amino acid residue after the substitution, and has a nitrile hydratase activity Protein (3) The following (A) or (B) protein.
(A) In the amino acid sequence of wild-type nitrile hydratase, the following amino acid residues (a) and (b) and at least one amino acid residue selected from the group consisting of the following (c) to (f) are other amino acids: A protein comprising an amino acid sequence substituted with a residue and having nitrile hydratase activity (a) 63 amino acids upstream from the amino acid residue at the C-terminal in the amino acid sequence of β subunit Amino acid residues (b) 11 amino acid residues upstream from the C-terminal amino acid residue (c) 97 amino acid residues upstream from the C-terminal amino acid residue (d) C 40 amino acid residues upstream from the terminal amino acid residue (e) Amino acid sequence C (S / T) LCS that constitutes the prosthetic molecule binding region in the amino acid sequence of the α subunit (F) Amino acid residue (B) 67 residues downstream of the most downstream C residue of the amino acid sequence C (S / T) LCSC (B) ) The amino acid sequence of the protein of (A) includes an amino acid sequence in which one or several amino acid residues are deleted, substituted and / or added except for the amino acid residue after substitution, and nitrile hydratase Protein (4) having the activity (4) Protein of (A) or (B) below
(A) In the amino acid sequence of wild-type nitrile hydratase, the following amino acid residues (a), (b) and (c) and at least one amino acid residue selected from the group consisting of the following (d) to (h): A protein having a nitrile hydratase activity, comprising an amino acid sequence substituted with another amino acid residue, and (a) a β-subunit amino acid sequence counted from the C-terminal amino acid residue 63 Amino acid residues upstream of the residues (b) 11 amino acid residues upstream from the C-terminal amino acid residue (c) 173 amino acid residues upstream from the C-terminal amino acid residue ( d) Amino acid residue upstream of 213 residues from the C-terminal amino acid residue (e) Amino acid residue upstream of 118 residues from the C-terminal amino acid residue (f) C 65 amino acid residues upstream from the end amino acid residue (g) 62 amino acid residues upstream from the C-terminal amino acid residue (h) counting from the C-terminal amino acid residue 40 amino acid upstream amino acid residues (B) In the amino acid sequence of the protein of (A), one or several amino acid residues were deleted, substituted and / or added except for the amino acid residues after the substitution. A protein comprising an amino acid sequence and having nitrile hydratase activity
(5) Genetic DNA encoding the protein.
(6) A recombinant vector containing the gene DNA.
(7) A transformant containing the above recombinant vector.
(8) Nitrile hydratase collected from a culture obtained by culturing the transformant.
(9) A method for producing a nitrile hydratase, comprising culturing the transformant and collecting nitrile hydratase from the obtained culture.
(10) An amide characterized by contacting a culture obtained by culturing the protein or transformant or a treated product of the culture with a nitrile compound, and collecting the amide compound produced by the contact. Compound production method.

  In the present specification, unless otherwise specified, “upstream” and “upstream” mean “N-terminal” with respect to the amino acid sequence and “5′-terminal” with respect to the base sequence. . Further, “downstream” and “downstream” mean “C-terminal side” with respect to the amino acid sequence, and “3′-terminal side” with respect to the base sequence.

According to the present invention, a novel improved (mutant) nitrile hydratase having improved heat resistance and / or amide compound resistance can be provided. Among them, the improved nitrile hydratase with improved heat resistance and amide compound resistance is very useful in the production of amide compounds.
According to the present invention, the gene DNA encoding the improved nitrile hydratase, the recombinant vector containing the gene DNA, the transformant containing the recombinant vector, and the culture of the transformant are further collected. Nitrile hydratase and production thereof, and a method for producing an amide compound using the protein (improved nitrile hydratase) or the culture or a processed product of the culture can be provided.

The present invention is described in detail below.
1. Improved nitrile hydratase (a) Wild type nitrile hydratase The improved nitrile hydratase of the present invention is an improved type (mutant) of wild type nitrile hydratase, and its origin is not particularly limited. Here, “wild-type nitrile hydratase” refers to nitrile hydratase that can be isolated from organisms in the natural world (for example, microorganisms such as soil bacteria), an amino acid sequence constituting the enzyme, and a gene encoding the enzyme Is a nitrile hydratase that has not been artificially deleted or inserted, or has not been substituted with other amino acids or bases, and retains its natural properties. Not only known nitrile hydratases derived from microorganisms but also nitrile hydratases derived from unknown organisms are included in the “wild-type nitrile hydratase”.

  “Wild-type nitrile hydratase” has a higher-order structure formed by assembling domains of α subunit and β subunit, and has a non-heme iron atom or a non-choline nuclear cobalt atom as a prosthetic molecule. These nitrile hydratases are distinguished by the names of iron-type nitrile hydratase and cobalt-type nitrile hydratase, respectively.

  Representative examples of iron-type nitrile hydratase include those derived from Rhodococcus genus N-771. This iron-type nitrile hydratase has been subjected to X-ray crystal structure analysis, and its three-dimensional structure has been clarified. As a result, the enzyme is non-heme iron via four amino acid residues in the cysteine cluster (Cys-Ser-Leu-Cys-Ser-Cys (SEQ ID NO: 29)) of the α subunit that forms the active center. Is combined with.

  Examples of the cobalt-type nitrile hydratase include those derived from Rhodococcus rhodochrous J1 strain (hereinafter sometimes referred to as “J1 bacterium”) or those derived from Pseudonocardia thermophila. it can. Cobalt-type nitrile hydratase derived from J1 bacteria has a cobalt atom through a region represented by a cysteine cluster (Cys-Thr-Leu-Cys-Ser-Cys (SEQ ID NO: 30)) of the α subunit that forms the active center. Are connected. Note that the cysteine cluster of Pseudonocardia thermophila-derived cobalt-type nitrile hydratase has a cysteine sulfinic acid (Csi) as the fourth cysteine (Cys) from the upstream side (N-terminal side) of the cysteine cluster derived from the aforementioned J1 bacterium. The 6th cysteine (Cys) on the most downstream side (C-terminal side) is cysteine sulfenic acid (Cse).

  As described above, the prosthetic molecule is bound to the region represented by the cysteine cluster “C (S / T) LCSC (SEQ ID NO: 29 or 30 (hereinafter the same))” in the α subunit. Examples of the nitrile hydratase containing such a prosthetic molecule binding region include Rhodococcus rhodochrous J1 (FERM BP-1478), Rhodococcus rhodochrous M8 (SU1731814), Rhodococcus rhodochrous M33 (VKM Ac-1515D), Rhodococcus rhodochrous. ATCC39484 (JP 2001-292772), Bacillus smithii (JP 9-248188), Pseudocardia thermophila (JP 9-275978) or Geobacillus thermoglucosidasius Examples include nitrile hydratase having an amino acid sequence and nitrile hydratase encoded by a gene sequence. The alignment of the amino acid (single letter code) sequence of the α subunit of wild-type nitrile hydratase derived from various microorganisms including the prosthetic molecule binding region “C (S / T) LCSC” is shown in FIGS. 4-1, 4-2 and 4 -3. The amino acid sequences are connected in the order shown in FIGS. 4-1, 4-2, and 4-3. In each of FIGS. 4-1 to 4-3, the amino acid sequences are shown in SEQ ID NOs: 31 to 53 in this order.

(B) Improved nitrile hydratase The present invention is an improved (mutant) nitrile hydratase obtained by subjecting a wild type nitrile hydratase to amino acid substitution. The amino acid sequence of wild-type nitrile hydratase to be substituted is published in NCBI databases such as GenBank (http://www.ncbi.nlm.nih.gov/).
For example, the α subunit derived from Rhodococcus rhodochrous J1 (FERM BP-1478) has an amino acid sequence represented by SEQ ID NO: 4 and a base sequence represented by SEQ ID NO: 3. The accession number of the β subunit (SEQ ID NOs: 2 and 1) is “P21220”. The accession number of the α subunit derived from Rhodococcus rhodochrous M8 (SU1731814) is “ATT79340”, and the accession number of the β subunit is “AAT79339”. Furthermore, the accession number of the α subunit derived from Pseudomonas thermophila JCM3095 is “1IRE The accession number of the β subunit is “1IRE”. B ".

  In addition, in the amino acid sequence in which a specific amino acid residue is substituted, one or several (for example, about 1 to 10, preferably about 1 to 5) amino acid residues (however, the amino acid after the above substitution) An improved nitrile hydratase having a nitrile hydratase activity consisting of an amino acid sequence deleted, substituted and / or added, and having nitrile hydratase activity is also within the scope of the present invention.

  As the “improved nitrile hydratase” in the present invention, at least one amino acid residue selected from the group consisting of the following (a) to (k) in the amino acid sequence of wild-type nitrile hydratase is substituted with another amino acid residue. And a protein characterized by having a nitrile hydratase activity. In addition, the following amino acid residues (c), (d), (e), (f), (g), (h), (i), (j), and (k) are various wild-type nitriles, respectively. Among the hydratases, an amino acid residue in the amino acid sequence of a wild-type nitrile hydratase derived from a bacterium belonging to Rhodococcus rhodochrous species is preferable.

(A) an amino acid residue that is 53 residues downstream of the most downstream C residue of the amino acid sequence C (S / T) LCSC that constitutes the prosthetic molecule-binding region in the amino acid sequence of the α subunit (b) the amino acid sequence Amino acid residue 67 residues downstream from C residue on the most downstream side of C (S / T) LCSC (c) Amino acid residue 213 residues upstream from the amino acid at the C-terminal in the amino acid sequence of β subunit (D) 97 amino acid residues upstream from the C-terminal amino acid residue (e) 65 amino acid amino acid residues upstream from the C-terminal amino acid residue (f) C-terminal amino acid (G) Amino acid residue 40 residues upstream from the C-terminal amino acid residue (h) 63 residues counted from the C-terminal amino acid residue Upstream amino acid residue (I) 11 amino acid residues upstream from the C-terminal amino acid residue (j) 173 amino acid residues upstream from the C-terminal amino acid residue (k) C-terminal amino acid Amino acid residues 118 residues upstream from the residue

  Further, as the improved nitrile hydratase of the present invention, among the above proteins, those in which the substituted amino acid residues are those listed below are preferably mentioned.

-At least one amino acid residue selected from the group consisting of (a) to (g)-consisting of the amino acid residues (h) and (i) above and (c), (e) and (f) above At least one amino acid residue selected from the group; at least one amino acid residue selected from the group consisting of the amino acid residues (h) and (i) above and (a), (b), (d) and (g) above Amino acid residue ・ At least one selected from the group consisting of the amino acid residues (h), (i) and (j) above and (c), (e), (f), (g) and (k) above Two amino acid residues

  Furthermore, as the improved nitrile hydratase of the present invention, among the above proteins, those in which the substituted amino acid residues are those listed in Table 1 below are more preferable.

As an example of the improved nitrile hydratase of the present invention, in wild-type nitrile hydratase derived from J1 bacteria,
In the amino acid sequence of the β subunit of the nitrile hydratase of the present invention, 63 amino acid residues (asparagine) upstream from the C-terminal amino acid residue, 11 amino acid residues upstream from the C-terminal amino acid residue An amino acid residue (valine) and an amino acid residue (glycine) 40 residues upstream from the C-terminal amino acid residue are substituted with another amino acid (for example, serine), and
Amino acid residue (glycine) 67 residues upstream from the most downstream C residue of the amino acid sequence C (S / T) LCSC constituting the prosthetic molecule binding region in the amino acid sequence of the α subunit of the nitrile hydratase of the present invention Is preferably an enzyme protein containing an amino acid sequence substituted with another amino acid (for example, lysine or alanine) and having nitrile hydratase activity. Examples of such amino acid substitution modes include “Nβ ← 63S, Vβ ← 11A, Gβ ← 40R, Gα67 ↓ E” and the like.

  In addition, the improved nitrile hydratase as an example is the amino acid sequence of the 167th amino acid residue (asparagine) from the N-terminal side in the amino acid sequence of the β subunit of wild type nitrile hydratase derived from J1 bacterium, the amino acid of the β subunit. The 219th amino acid (valine) from the N-terminal side in the sequence and the 190th amino acid (glycine) from the N-terminal side in the amino acid sequence of the β subunit are substituted with other amino acids, and the amino acid of the α subunit It can also be said that the 174th amino acid residue (glycine) from the N-terminal side in the sequence is substituted with another amino acid and has an nitrile hydratase activity.

  In addition, the alphabetical representation of amino acids can be represented by one ordinary letter, and the alphabet displayed on the left side of the number (for example, “26”) indicating the number of amino acid residues up to the substitution site is the one-letter representation of the amino acid before substitution. The alphabet displayed on the right side indicates the one-letter code of the amino acid after substitution.

  Specifically, regarding the amino acid sequence of the α subunit shown in SEQ ID NO: 4, for example, “Sα ↑ 26A”, the most upstream side of the CTLCSC region in the amino acid sequence of the α subunit (SEQ ID NO: 4) This means a mode of amino acid substitution in the improved nitrile hydratase in which serine (S), which is 26 residues upstream of the C residue of, is substituted with alanine (A). Here, the notation “α ↑” means that the substitution position is upstream of the most upstream C residue of CTLCSC (on the N-terminal side without counting the C residue), On the other hand, the notation “α ↓” means that the substitution position is located downstream of the CLCSC region on the most downstream side of the CTLCSC region (the C-terminal side is counted without including the C residue). To do.

  In addition, for example, regarding the amino acid sequence of the β subunit shown in SEQ ID NO: 2, when “Nβ ← 63S” is expressed, it is counted from the C-terminal amino acid residue in the amino acid sequence of the β subunit (SEQ ID NO: 2). This means an aspect of amino acid substitution in the improved nitrile hydratase in which the 63rd asparagine (N) is substituted with serine (S) (counting including the C-terminal amino acid residue). Here, the notation “β ←” means that the substitution position is upstream from the C-terminal amino acid residue (the N-terminal side is counted including the C-terminal amino acid residue).

  The aspect of amino acid substitution in the more preferable improved nitrile hydratase of the present invention shown in Table 1 can be represented by the following notations 1 to 53, respectively.

1. Iα ↓ 53T
2. Gα ↓ 67S
3. Pβ ← 213A
4). Lβ ← 97R
5. Vβ ← 65S
6). Kβ ← 62R
7). Gβ ← 40R
8). Nβ ← 63S, Vβ ← 11A, Pβ ← 213A
9. Nβ ← 63S, Vβ ← 11A, Vβ ← 65S
Ten. Nβ ← 63S, Vβ ← 11A, Kβ ← 62R
11． Nβ ← 63S, Vβ ← 11A, Pβ ← 213A, Vβ ← 65S
12． Nβ ← 63S, Vβ ← 11A, Pβ ← 213A, Kβ ← 62R
13. Nβ ← 63S, Vβ ← 11A, Vβ ← 65S, Kβ ← 62R
14. Nβ ← 63S, Vβ ← 11A, Pβ ← 213A, Vβ ← 65S, Kβ ← 62R
15． Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T
16． Nβ ← 63S, Vβ ← 11A, Gα ↓ 67S
17． Nβ ← 63S, Vβ ← 11A, Lβ ← 97R
18． Nβ ← 63S, Vβ ← 11A, Gβ ← 40R
19. Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Gα ↓ 67S
20． Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Lβ ← 97R
twenty one. Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Gβ ← 40R
twenty two. Nβ ← 63S, Vβ ← 11A, Gα ↓ 67S, Lβ ← 97R
twenty three. Nβ ← 63S, Vβ ← 11A, Gα ↓ 67S, Gβ ← 40R
twenty four. Nβ ← 63S, Vβ ← 11A, Lβ ← 97R, Gβ ← 40R
twenty five. Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Gα ↓ 67S, Lβ ← 97R
26. Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Gα ↓ 67S, Gβ ← 40R
27. Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Lβ ← 97R, Gβ ← 40R
28. Nβ ← 63S, Vβ ← 11A, Gα ↓ 67S, Lβ ← 97R, Gβ ← 40R
29. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A
30. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S
31. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Kβ ← 62R
32. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Gβ ← 40R
33. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Sβ ← 118T
34. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Vβ ← 65S
35. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Kβ ← 62R
36. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Gβ ← 40R
37. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Sβ ← 118 T
38. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Kβ ← 62R
39. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Gβ ← 40R
40. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Sβ ← 118T
41. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Kβ ← 62R, Gβ ← 40R
42. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Kβ ← 62R, Sβ ← 118T
43. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Gβ ← 40R, Sβ ← 118T
44. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Vβ ← 65S, Kβ ← 62R
45. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Vβ ← 65S, Gβ ← 40R
46. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Vβ ← 65S, Sβ ← 118T
47. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Kβ ← 62R, Gβ ← 40R
48. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Kβ ← 62R, Sβ ← 118T
49. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Gβ ← 40R, Sβ ← 118T
50. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Kβ ← 62R, Gβ ← 40R
51. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Kβ ← 62R, Sβ ← 118T
52. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Gβ ← 40R, Sβ ← 118T
53. Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Kβ ← 62R, Gβ ← 40R, Sβ ← 118T

  Among these, the improved nitrile hydratase in which amino acid substitution is performed in the aspect of 8, 9, 10, 23, 26, 27, 29, 31, 32, 33, 34, 38, or 51 is more preferable. Particularly preferred is an improved nitrile hydratase in which amino acid substitution is performed in the form of 26, 27, 29, 31, 32, 33, 34, 38, or 51.

  Preferred examples of the base substitution for causing the amino acid substitution include the following embodiments.

Iα ↓ 53T: The codon “ATC” located 157 to 159 bases downstream (3 ′ end side) from the last C of the base sequence TGCACTCTGTGTTCGTGC (304 to 321st in SEQ ID NO: 3: SEQ ID NO: 54) is ACA, ACC, Replace with ACG or ACT. In particular, it is preferable to substitute T at 158 bases downstream with C (ATC → ACC).
Gα ↓ 67S: The codon “GGT” located 199 to 201 bases downstream (3 ′ end side) from the last C of the base sequence TGCACTCTGTGTTCGTGC (304 to 321st in SEQ ID NO: 3: SEQ ID NO: 54) is TCA, TCC, Replace with TCG, TCT, AGC or AGT. In particular, it is preferable to replace G located downstream of 199 bases with A (GGT → AGT).
Pβ ← 213A: 634 to 636 bases upstream (5 ′ end side) from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) The codon “CCC” located at is replaced with GCA, GCC, GCG or GCT. In particular, it is preferable to replace C located upstream of 636 bases with G (CCC → GCC).
Lβ ← 97R: 286 to 288 bases upstream (5 ′ end side) from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) The codon “CTA” located at is replaced with CGA, CGC, CGG, CGT, AGA or AGG. In particular, it is preferable to replace T located upstream of 287 bases with G (CTA → CGA).
Vβ ← 65S: Base sequence GCG (the 6th to 687th positions in SEQ ID NO: 1 (codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) upstream of 190 to 192 bases (5 ′ end side) The codon “GTG” located in is replaced with TCA, TCC, TCG, TCT, AGC or AGT. In particular, it is preferred that G located upstream of 192 bases is substituted with A and T located upstream of 191 bases is substituted with G (GTG → AGC).
Kβ ← 62R: 181 to 183 bases upstream (5 ′ end side) from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) The codon “AAG” located at is replaced with CGA, CGC, CGG, CGT, AGA or AGG. In particular, it is preferred that A located 183 bases upstream is substituted with C and A located 182 bases upstream is substituted with G (AAG → CGG).
Gβ ← 40R: 115 to 117 bases upstream (5 ′ end side) from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) The codon “GGC” located in is replaced with CGA, CGC, CGG, CGT, AGA or AGG. In particular, it is preferable to substitute G located at 117 bases upstream with C (GGC → CGC).
Nβ ← 63S: Codon “AAC located 184 to 186 bases upstream from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (the codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) Is replaced by AGC, AGT, TCA, TCC, TCG, or TCT. In particular, it is preferable to replace A located 185 bases upstream with G (AAC → AGC).
Vβ ← 11A: Codon “GTC” located 28 to 30 bases upstream from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (the codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) Is replaced by GCA, GCC, GCG or GCT. In particular, it is preferable to substitute T located at the 29 base upstream with C (GTC → GCC).
Sβ ← 173K: Codon “TCG located 514 to 516 bases upstream from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (the codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) Is replaced with AAA or AAG. In particular, it is preferable that T located 516 bases upstream is replaced with A and C located 515 bases upstream is replaced with A (TCG → AAG).
Iβ ← 118T: Codon “TCG” located 349 to 351 bases upstream from the first G of the base sequence GCG (the 685th to 687th positions in SEQ ID NO: 1 (the codon of the C-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2)) Is replaced with ACA, ACC, ACG or ACT. In particular, it is preferable to replace T located upstream of 351 bases with A (TCG → ACG).

  For example, the substitution sites of α ↓ 53 and α ↓ 67 of wild-type nitrile hydratase correspond to the 160th and 174th amino acid residues in the α subunit (SEQ ID NO: 4) of J1 bacteria, respectively. In addition, β ← 213, β ← 97, β ← 65, β ← 62, β ← 40, β ← 63, β ← 11, β ← 173, and β ← 118 are replaced by the β subunit of J1 bacteria, respectively. (SEQ ID NO: 2) corresponds to the 17th, 133rd, 165th, 168th, 190th, 167th, 219th, 57th and 112th amino acid residues.

  The activity of the improved nitrile hydratase of the present invention is improved in heat resistance and / or amide compound resistance with respect to the activity of wild-type nitrile hydratase while retaining the properties derived from nature.

Here, “nitrile hydratase activity” is an enzyme that catalyzes a hydration reaction (RCN + H ₂ O → RCONH ₂ ) that converts a nitrile compound into a corresponding amide compound. The activity measurement can be calculated by bringing a nitrile compound as a substrate into contact with nitrile hydratase and converting it to the corresponding amide compound, and then quantifying the amide compound. As the substrate, any nitrile compound can be used as long as it reacts with nitrile hydratase, but acrylonitrile is preferred. As reaction conditions, the substrate concentration is 2.5%, the reaction temperature is 10 ° C to 30 ° C, and the reaction time is 10 minutes to 30 minutes. The enzymatic reaction is stopped by adding phosphoric acid. Thereafter, the amide compound can be quantified by analyzing the produced acrylamide by HPLC (high performance liquid chromatography).

  “Improving heat resistance” means that the residual activity of the heat-treated mutant strain is 10% or more higher than the residual activity of the parent strain (wild type) subjected to the same treatment. As a method for the heat treatment, after the culture solution or the collected and washed cultured microbial cells are placed in a container, the container is placed in a heating device such as a water bath or an incubator and kept warm for a certain period of time. At this time, a heat treatment may be performed by adding a nitrile compound or an amide compound for enhancing the stability of the enzyme. As the conditions for the heat treatment, it is preferable to appropriately examine the treatment temperature and the treatment time, and to set conditions under which the activity of the parent strain is reduced to 50% or less with respect to that before the heat treatment. Specifically, heat treatment is performed in the range of 50 to 70 ° C. for 5 to 60 minutes. Residual activity refers to the ratio between the amount of amide compound produced by activity measurement using heat-treated cells and the amount of amide compound produced by activity measurement using the same amount of untreated cells. As the untreated microbial cells, a culture solution or a cultivated microbial cell that has been collected and washed is kept cold at 4 ° C.

  Further, “amide compound resistance” means that nitrile hydratase activity can be maintained even in the presence of an amide compound. A culture of a transformant having an improved nitrile hydratase or an improved nitrile hydratase isolated from the transformant in the presence of an amide compound such as acrylamide (for example, a high concentration of 30 to 50%) By analyzing the consumption or consumption rate of nitrile compounds such as acrylonitrile as a substrate, nitrile hydratase whose consumption or consumption rate exceeded 1.1 times compared to the nitrile hydratase derived from the parent strain (wild type) It can be evaluated that the compound is resistant to amide compounds.

Examples of the amide compound include the following general formula (1):
R-CONH ₂ (1)
(Here, R is an optionally substituted linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, an optionally substituted cycloalkyl group having 3 to 18 carbon atoms, or aryl. Or a saturated or unsaturated heterocyclic group which may be substituted.)
The amide compound represented by these is mentioned. In particular, acrylamide in which R is “CH ₂ ═CH—” is preferred.

  The improved nitrile hydratase is obtained by amino acid substitution of wild-type nitrile hydratase. For example, the amino acid sequence of nitrile hydratase derived from Rhodococcus rhodochrous J1 strain (SEQ ID NO: 2 and / or 4) And nitrile hydratase with improved heat resistance and / or improved amide compound resistance can be obtained. In addition, Rhodococcus rhodochrous J1 strain was established as FERM BP-1478 in the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, September 6th, 1987). Deposited internationally by date.

  Also in nitrile hydratases other than J1 bacteria, it is considered that the heat resistance and / or amide compound resistance is improved depending on the position of the mutation, the amino acid species to be mutated, and the DNA sequence. As such bacteria, Rhodococcus rhodochrous M8 (SU1731814), Rhodococcus rhodochrous M33 (VKM Ac-1515D) or Rhodococcus rhodochrous ATCC39484 (JP 2001-292772), Bacillus smithii (specialty) Kaihei 09-248188), Pseudocardia thermophila (JP 09-275978), Geobacillus thermoglucosidasias, and the like. Rhodococcus rhodochrous M33 (VKM Ac-1515D) is a strain selected as a strain that constitutively expresses nitrile hydratase by spontaneous mutation from the M8 strain (SU1731814). There is no mutation in the amino acid sequence and gene sequence of the nitrile hydratase itself (US Pat. No. 5,827,699).

  Wild-type nitrile hydratase can be substituted with amino acids by contacting and acting on a microorganism having nitrile hydratase activity with a drug that acts as a mutation source such as hydroxylamine or nitrous acid, by inducing mutation by ultraviolet irradiation, or nitrile An error prone PCR method or the like in which mutations are randomly introduced into a gene encoding hydratase using PCR can be employed.

(B-1) Error prone PCR
One method for studying the function and properties of proteins using mutants is random mutagenesis. The random mutagenesis method is a method for producing a mutant by introducing a random mutation into a gene encoding a specific protein. In the random mutagenesis method using PCR, base mutation can be introduced (Error prone PCR) by setting conditions with low stringency during DNA amplification. In this error prone PCR, mutations are introduced at arbitrary sites over the entire DNA to be amplified. Then, information on amino acids and domains important for protein-specific functions can be obtained by examining the functions of mutants in which mutations have been introduced at arbitrary sites.

  In the present invention, for example, in the amino acid sequence of the β subunit, an amino acid residue that is 63 residues upstream from the C-terminal amino acid residue, an amino acid residue that is 11 residues upstream from the C-terminal amino acid residue, And the most downstream C residue of C (S / T) LCSC that replaces the amino acid residue 40 residues upstream from the C-terminal amino acid residue and constitutes the prosthetic molecule binding region of the α subunit An improved nitrile hydratase in which an amino acid residue downstream of 67 residues is substituted has higher heat resistance and / or amide compound resistance than wild-type nitrile hydratase, and exhibits high activity.

  The mutation can be obtained by introducing a random mutation into the wild type nitrile hydratase gene by Error prone PCR. The present invention is an improved nitrile hydratase obtained by the error prone PCR method, wherein the host has higher heat resistance and / or amide compound resistance than wild-type nitrile hydratase and has high nitrile hydratase activity. Is also included.

As a nitrile hydratase used as a template for Error prone PCR, a nitrile hydratase gene derived from a wild strain, or a DNA amplified by Error prone PCR can be used.
The reaction conditions for Error prone PCR include, for example, a composition in which the mixing ratio of any one, two, or three of dNTP (dGTP, dCTP, dATP, or dTTP) in the reaction solution is reduced compared to other dNTPs. The conditions to do are mentioned. This increases the possibility that other dNTPs will be mistakenly used at sites where dNTPs with a reduced blending ratio are required during DNA synthesis, and mutations are introduced. As other reaction conditions, conditions may be mentioned preferably a composition with an increased MgCl ₂ and / or MnCl ₂ content in the reaction solution.

(B-2) Improved nitrile hydratase derived from Rhodococcus rhodochrous J1 strain and its gene Examples of the improved nitrile hydratase of the present invention include, for example, Rhodococcus, which is a wild type represented by the nucleotide sequences of SEQ ID NOs: 1 and 3. An amino acid substitution 63 residues upstream from the C-terminal amino acid residue in the amino acid sequence of the β subunit, and an amino acid substitution 11 residues upstream from the C-terminal amino acid residue in the gene derived from Rhodochrous J1 strain; And a mutation corresponding to an amino acid substitution 40 residues upstream from the C-terminal amino acid residue, and the most downstream of C (S / T) LCSCs constituting the prosthetic molecule binding region of the α subunit Those encoded by a gene into which a mutation corresponding to an amino acid substitution 67 residues downstream from the C residue is introduced are also included.

DNA encoding such an improved nitrile hydratase is based on the wild type nitrile hydratase gene, Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John It can be prepared according to the site-specific displacement induction method described in Wiley & Sons (1987-1997) and the like. In order to introduce a mutation into DNA, a mutation introduction kit using site-directed mutagenesis, for example, QuickChange ^™ XL Site-Directed Mutagenesis Kit (manufactured by Stratagene, Inc.) by a known method such as Kunkel method or Gapped duplex method. ), 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.) and the like.

  The gene of the present invention also includes a DNA that hybridizes with a DNA comprising a base sequence complementary to the base sequence of the gene under stringent conditions and encodes a protein having nitrile hydratase activity. .

  Such an improved nitrile hydratase gene can be obtained by introducing a mutation into a wild type gene as described above, and colony hybridization using the gene sequence or a complementary sequence thereof or a fragment thereof as a probe, It can also be obtained from cDNA libraries and genomic libraries by known hybridization methods such as plaque hybridization and Southern blotting. A library prepared by a known method can be used, and a commercially available cDNA library and genomic library can also be used.

  “Stringent conditions” are the conditions at the time of washing after hybridization, wherein the salt concentration is 300 to 2000 mM, the temperature is 40 to 75 ° C., preferably the salt concentration is 600 to 900 mM, and the temperature is 65 ° C. means. For example, 2 × SSC and conditions such as 50 ° C. can be mentioned. Those skilled in the art can code the nitrile hydratase of the present invention in consideration of other conditions such as the concentration of the probe, the length of the probe, and the reaction time in addition to the conditions such as the salt concentration and temperature of the buffer. Conditions for obtaining the DNA to be obtained can be appropriately set.

  For detailed procedures of the hybridization method, Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)) can be referred to. Examples of the DNA that hybridizes include DNA containing a base sequence having at least 40%, preferably 60%, more preferably 90% or more identity to the gene DNA of the present invention, or a partial fragment thereof.

  In the amino acid sequence of wild type nitrile hydratase, any type of amino acid that replaces a specific amino acid residue (substitute amino acid) is a polypeptide (protein) containing the substituted amino acid that exhibits nitrile hydratase activity. It can select suitably in the range which has, and is not limited.

(C) Recombinant vector, transformant The nitrile hydratase gene must be incorporated into a vector so that it can be expressed in the host organism to be transformed. Examples of the vector include plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA, and the like.

  The host that can be used in the present invention is not particularly limited as long as the target nitrile hydratase can be expressed after the introduction of the recombinant vector. For example, bacteria such as Escherichia coli and Bacillus subtilis, yeast, animal cells, insect cells, plant cells and the like can be used. When Escherichia coli is used as a host, it is preferable to use an expression vector having high expression efficiency, for example, an expression vector pkk233-2 (manufactured by Amersham Biosciences) having a trc promoter or pTrc99A (manufactured by Amersham Biosciences).

  In addition to the nitrile hydratase gene, a promoter, terminator, enhancer, splicing signal, poly A addition signal, selection marker, ribosome binding sequence (SD sequence) and the like can be linked to the vector. Examples of the selection marker include kanamycin resistance gene, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.

  When a bacterium is used as a host, for example, Escherichia coli can be mentioned, and examples of Rhodococcus bacteria include Rhodococcus rhodochrous ATCC12674, Rhodococcus rhodochrous ATCC17895, Rhodococcus rhodochrous ATCC19140, and the like. These ATCC strains are available from the American Type Culture Collection.

  The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, etc. are 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 an electroporation method, a spheroplast method, and a lithium acetate method.

  When animal cells are used as hosts, monkey cells COS-7, Vero, CHO cells, mouse L cells, rat GH3, human FL cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.

  When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method, electroporation method and the like are used.

  When plant cells are used as hosts, tobacco BY-2 cells and the like can be mentioned, but are not limited thereto. As a method for introducing a recombinant vector into a plant cell, for example, an Agrobacterium method, a particle gun method, a PEG method, an electroporation method, or the like is used.

(D) Production method of culture and improved nitrile hydratase In the present invention, the improved nitrile hydratase can be produced by culturing the transformant and collecting it from the resulting culture.
The present invention also includes a method for producing an improved nitrile hydratase, wherein the improved nitrile hydratase is collected from the culture.
In the present invention, the “culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host. The desired improved nitrile hydratase accumulates in the culture.

  The medium for culturing the transformant of the present invention contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host fungus, and any natural medium can be used as long as the transformant can be cultured efficiently. Either a medium or a synthetic medium may be used. Examples of the carbon source include 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. Examples of the nitrogen source include inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or ammonium salts of organic acids, and other nitrogen-containing compounds. In addition, peptone, yeast extract, meat extract, corn steep liquor, various amino acids, and the like may be used. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, and calcium carbonate. Moreover, you may add an antifoamer in order to prevent foaming during culture | cultivation as needed. Further, cobalt ions and iron ions, which are nitrile hydratase prosthetic molecules, may be added to the medium, and nitriles and amides serving as enzyme inducers may be added.

  During the culture, the vector and the target gene may be cultured under selective pressure in order to prevent the loss of the vector and the target gene. That is, a drug corresponding to the case where the selection marker is a drug resistance gene may be added to the medium, or a nutrient factor corresponding to the case where the selection marker is an auxotrophic complementary gene may be removed from the medium. When the selectable marker is an assimilation gene, a corresponding assimilation factor can be added as a sole factor as necessary. For example, when culturing Escherichia coli transformed with a vector containing an ampicillin resistance gene, ampicillin may be added as needed during the culture.

  When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a transformant transformed with an expression vector having a promoter inducible with isopropyl-β-D-thiogalactoside (IPTG), IPTG or the like can be added to the medium. In addition, when cultivating a transformant transformed with an expression vector using a trp promoter inducible with indoleacetic acid (IAA), IAA or the like can be added to the medium.

  The culture conditions for the transformant are not particularly limited as long as the productivity of the desired improved nitrile hydratase and the growth of the host are not hindered, but are usually 10 ° C to 40 ° C, preferably 20 ° C. C. to 37.degree. C. for 5 to 100 hours. The pH is adjusted using an inorganic or organic acid, an alkaline solution or the like. For example, in the case of Escherichia coli, the pH is adjusted to 6-9. Examples of the culture method include solid culture, stationary culture, shaking culture, and aeration and agitation culture. In particular, when culturing E. coli transformants, aerobic by shaking culture or aeration and agitation culture (jar fermenter). It is preferable to culture under typical conditions.

  When cultured under the above culture conditions, the improved nitrile hydratase of the present invention is produced in a high yield in the above culture, that is, at least one of a culture supernatant, cultured cells, cultured cells, or cells or disrupted cells. Can accumulate.

  When improved nitrile hydratase is produced in cells or cells after culturing, the desired improved nitrile hydratase can be collected by disrupting the cells or cells. As a method for disrupting cells or cells, high-pressure treatment using a French press or homogenizer, ultrasonic treatment, grinding treatment using glass beads, enzyme treatment using lysozyme, cellulase, pectinase, etc., freeze-thawing treatment, hypotonic solution treatment, It is possible to use a lysis inducing treatment with a phage or the like. After crushing, the cells or cell crushing residues (including the cell extract insoluble fraction) can be removed as necessary. Examples of the method for removing the residue include centrifugation and filtration. If necessary, the residue removal efficiency can be increased by using a flocculant or a filter aid. The supernatant obtained after removing the residue is a soluble fraction of the cell extract and can be a crude purified modified nitrile hydratase solution.

  In addition, when improved nitrile hydratase is produced in cells or cells, the cells or cells themselves can be recovered by centrifugation, membrane separation, etc., and used without being crushed.

  When the improved nitrile hydratase is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or filtration. Then, if necessary, the improved nitrile hydratase is collected from the culture by extraction with ammonium sulfate precipitation, and further, if necessary, dialysis, various chromatography (gel filtration, ion exchange chromatography, affinity chromatography, etc.) Can also be isolated and purified.

  The production yield of nitrile hydratase obtained by culturing the transformant is, for example, SDS-PAGE (polyacrylamide) in units such as per culture broth, per microbial wet weight or dry weight per crude enzyme solution protein, etc. It can be confirmed by gel electrophoresis) or nitrile hydratase activity measurement, but is not particularly limited. SDS-PAGE can be performed by those skilled in the art using a known method. Moreover, the value of the activity mentioned above can be applied to the nitrile hydratase activity.

  In the present invention, an improved nitrile hydratase can be produced by employing a cell-free protein synthesis system without using any living cells.

  The cell-free protein synthesis system is a system that synthesizes a protein in an artificial container such as a test tube using a cell extract. The cell-free protein synthesis system used in the present invention includes a cell-free transcription system that synthesizes RNA using DNA as a template.

  In this case, the organism corresponding to the host corresponds to the organism from which the following cell extract is derived. Here, eukaryotic cell-derived or prokaryotic cell-derived extracts such as wheat germ, Escherichia coli and the like can be used as the cell extract. Note that these cell extracts may be concentrated or not concentrated.

The cell extract can be obtained, for example, by ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like. Furthermore, in the present invention, cell-free protein synthesis can also be performed using a commercially available kit. Examples of such kits include reagent kits PROTEIOS ^™ (Toyobo), TNT ^™ System (Promega), synthesizer PG-Mate ^™ (Toyobo), RTS (Roche Diagnostics) and the like.

  The improved nitrile hydratase obtained by cell-free protein synthesis as described above can be purified by appropriately selecting chromatography as described above.

2. Method for Producing Amide Compound The improved nitrile hydratase produced as described above can be used for substance production as an enzyme catalyst. For example, an amide compound is produced by contacting the nitrile compound with the improved nitrile hydratase. And the amide compound produced | generated by contact is extract | collected. Thereby, an amide compound can be manufactured.

  As the enzyme catalyst, nitrile hydratase separated and purified as described above can be used. Further, as described above, a culture after introducing a gene so that the improved nitrile hydratase gene is expressed in an appropriate host and culturing the host, or a processed product of the culture can be used. Examples of the treated product include those in which the cultured cells are included in a gel such as acrylamide, those treated with glutaraldehyde, and those supported on an inorganic carrier such as alumina, silica, zeolite, and diatomaceous earth.

  Here, “contact” means that the improved nitrile hydratase and the nitrile compound are present in the same reaction system or culture system. For example, the separated and purified improved nitrile hydratase and nitrile compound are mixed. , Adding a nitrile compound to a cell culture vessel expressing the improved nitrile hydratase gene, culturing the cell in the presence of the nitrile compound, mixing the cell extract with the nitrile compound, etc. It is.

The nitrile compound used as the substrate is selected in consideration of the substrate specificity of the enzyme, the stability of the enzyme to the substrate, and the like. As the nitrile compound, acrylonitrile is preferable.
The reaction method and the method for collecting the amide compound after completion of the reaction are appropriately selected depending on the characteristics of the substrate and the enzyme catalyst.

  The enzyme catalyst is preferably recycled as long as its activity is not deactivated. In view of preventing deactivation and facilitating recycling, the enzyme catalyst is preferably used in the form of a treated product.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

Example 1
Acquisition of improved nitrile hydratase gene (1) Construction of mutant gene library As a template plasmid, 63 residues upstream from the C-terminal amino acid residue of the amino acid sequence of the β subunit (SEQ ID NO: 2) The amino acid residue was mutated from asparagine (N) to serine (S), and the amino acid residue 11 residues upstream from the C-terminal amino acid residue was mutated from valine (V) to alanine (A). Plasmid pAB002 (FIG. 1) was used.
Here, as a method for preparing plasmid pAB002, a site-directed mutagenesis method was used, and a commercially available kit: QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) was used. Plasmid pJH601 (FIG. 2) was used as a template for introducing mutations.
The plasmid pJH601 is an expression plasmid for wild-type nitrile hydratase, and was assigned to the Patent Organism Depositary at the National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, Japan) as accession number FERM BP-10314. Deposited internationally on December 26, 2002.

Two kinds of primers for introducing mutations were synthesized. The underlined portion indicates the mutation introduction site.
β ← 63-F: ccgaaatatgtgcgg AGC aagatcggggaaatc (SEQ ID NO: 5)
β ← 63-R: gatttccccgatctt GCT ccgcacatatttcgg (SEQ ID NO: 6)

PCR for introducing the mutation was performed under the following conditions using GeneAmp9700 (PE Bioscience).
<Composition of PCR reaction solution>
Template plasmid pJH601 (10ng) 2μl
10 × Reaction Buffer 5 μl
Primer β ← 63-F (100ng / μl) 1μl
Primer β ← 63-R (100ng / μl) 1μl
2.5 mM dNTPmix 1 μl
Sterile water 36μl
QuickSolution 3μl
Pfu Turbo DNA Polymerase 1μl
<Reaction conditions>
1 minute at 95 ° C
(50 seconds at 95 ° C, 50 seconds at 60 ° C, 14 minutes at 68 ° C) x 18 cycles
7 minutes at 68 ° C

After completion of PCR, 10 μl of the reaction solution was subjected to 0.7% agarose gel electrophoresis, and an amplified fragment of about 11 kb was detected. After confirming the amplified fragment, 1 μl of Dpn I (supplied with the kit) was added to the PCR reaction solution and reacted at 37 ° C. for 1 hour to remove the template plasmid. Next, transformation was performed using XL10-GOLD ultracompetent cells (included in the kit). 2 μl of DpnI-treated PCR reaction solution and 45 μl of competent cells were mixed and incubated at 4 ° C. for 30 minutes. Next, heat shock was performed at 42 ° C. for 30 seconds, and 0.5 ml of NZY ⁺ medium (1% NZ amine, 0.5% yeast extract, 0.5% NaCl, 12.5 mM MgCl ₂ , 12.5 mM MgSO ₄ , 0.4% glucose, pH 7) .5) was added. After the addition, it was cultured at 37 ° C. for 1 hour. 250 μl of this culture solution was plated on an LB plate (1% NaCl, 1% tryptone, 0.5% yeast extract, 2% agar, 50 mg / L ampicillin) and cultured at 37 ° C. for 1 day.
After culturing, several colonies grown on the plate were each cultured in 1.5 ml of LB medium (50 mg / L ampicillin), and then the plasmid was extracted using FlexiPrep Kit (Amersham Bioscience). After extraction, the base sequence of the obtained plasmid was determined, and it was confirmed that the target mutation was introduced. In this manner, a mutation-introduced plasmid: p52 into which a base substitution that causes an amino acid substitution of Nβ ← 63S was introduced was obtained.

Subsequently, two kinds of primers for further introducing mutations were synthesized. The underlined portion indicates the mutation introduction site.
β ← 11-F: gaaagacgtagtgtgc GCC gatctctgggaaccg (SEQ ID NO: 7)
β ← 11-R: cggttcccagagatc GGC gcacactacgtctttc (SEQ ID NO: 8)
PCR and plasmid extraction for introducing mutations were performed in the same manner as described above, and the nucleotide sequence of the obtained plasmid was determined to confirm that the target mutation was introduced. In this way, a mutation-introduced plasmid: pAB002 into which a base substitution for causing amino acid substitution of Nβ ← 63S and Vβ ← 11A was introduced was obtained.

Next, random mutation was introduced into the nitrile hydratase gene.
Random mutagenesis utilized base substitution by error incorporation of nucleotides in PCR (Error prone PCR).

Error prone PCR for the nitrile hydratase gene was performed using GeneAmp9700 (PE Bioscience) under the following conditions.
<Composition of PCR reaction solution>
Template plasmid (pAB002) (10ng) 1μl
10 × PCR Buffer (GIBCO) 10 μl
50 mM MgCl ₂ (GIBCO) 3 μl
Primer Trc-02 (100ng / μl) 1μl
Primer Trc-03 (100ng / μl) 1μl
2.5mM dNTPmix 8μl
10 mM dITP 2 μl
10 mM dBraUTP 2 μl
Sterile water 71 μl
Taq DNA polymerase (GIBCO) 1μl
<Primer>
Trc-02: ggaattcgtataatgtgtggaattgtgagc (SEQ ID NO: 9)
Trc-03: ggctgaaaatcttctctcatccgcc (SEQ ID NO: 10)
<Reaction conditions>
(30 seconds at 94 ° C, 30 seconds at 65 ° C, 3 minutes at 72 ° C) x 30 cycles

After completion of PCR, 5 μl of the reaction solution was subjected to 0.7% agarose gel electrophoresis to detect a 3 kb PCR amplification product. The reaction solution containing the PCR product was purified with GFX column (Amersham Bioscience) and cleaved with restriction enzymes NcoI and HindIII. The restriction enzyme-treated PCR product was subjected to 0.7% agarose gel electrophoresis, and a band around 3 Kb was recovered. The collected PCR product was ligated with the vector pTrc99A (NcoI-HindIII site) using Ligation Kit (Takara Shuzo). This ligation product was used to transform E. coli JM109. The bound product and 100 μl of E. coli JM109 competent cell were mixed and incubated at 4 ° C. for 30 minutes, and heat shock was performed at 42 ° C. for 40 seconds. Thereafter, 0.5 ml of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 20 mM MgCl ₂ , 20 mM MgSO ₄ , 0.2% glucose, pH 7.0) was added thereto, and the mixture was added at 37 ° C. for 1 Incubate for hours. 250 μl of this culture solution was plated on an LB plate (1% NaCl, 1% tryptone, 0.5% yeast extract, 2% agar, 50 mg / L ampicillin) and cultured at 37 ° C. for 1 day. An E. coli JM109 transformant containing the nitrile hydratase gene was obtained.

(2) Screening of improved nitrile hydratase and identification of substitution site For screening, E. coli JM109 transformant containing the nitrile hydratase gene obtained in (1) above and E. coli JM producing wild type nitrile hydratase were used. 109 / pJH601 was used. Each of the above strains was inoculated into a 96-well deep well plate containing 1 ml of LB-Amp medium (1 mM IPTG, containing 5 μg / ml CoCl ₂ ), followed by liquid culture at 37 ° C. for 12 hours. Next, the culture solution was subjected to a heat treatment at 60 ° C. for 30 minutes, and then the residual nitrile hydratase activity was measured by the following method. Screening was performed on tens of thousands of transformants having nitrile hydratase genes mutated randomly.

  For the activity measurement, an equal amount of 5% acrylonitrile / 50 mM phosphate buffer pH 7.0 was added to the bacterial solution and reacted at 30 ° C. for 30 minutes. After completion of the reaction, 0.1M phosphoric acid was added in an amount equivalent to the reaction solution and centrifuged. The supernatant after centrifugation was appropriately diluted and subjected to HPLC, and the resulting acrylamide concentration was analyzed (WAKOSIL 5C8 (Wako Pure Chemical Industries, Ltd.) 250 mm, 10% acetonitrile containing 5 mM phosphoric acid, mobile phase flow rate 1 ml / min And ultraviolet absorption detector wavelength 260 nm). As a comparative control, the activity was measured using each of the untreated bacteria kept at 4 ° C. without heat treatment, and the residual activity (%) after the heat treatment was determined based on the obtained activity value.

  A transformant producing an improved nitrile hydratase having a higher residual activity compared to a cell producing wild type (wt) nitrile hydratase (pJH601) was cultured and the plasmid was recovered. The recovered plasmids were named pE306, pE316, pE317, pE414, pE415, pE416, pE418, pE419, pE420, pE516, pE536, pE539 and pE601. The results of residual activity are shown in Table 2. The base sequence of each plasmid was determined by BeckmanCEQ-2000XL. These plasmids are found to have high residual activity even when compared with plasmid pAB002.

The substitution mode of amino acid residues in the amino acid sequence encoded by the mutated gene sequence of each plasmid is shown below.
pE306: Nβ ← 63S, Vβ ← 11A, Pβ ← 213A
pE316: Nβ ← 63S, Vβ ← 11A, Vβ ← 65S
pE317: Nβ ← 63S, Vβ ← 11A, Kβ ← 62R
pE414: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Kβ ← 62R
pE415: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A
pE416: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Sβ ← 118T
pE418: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Kβ ← 62R
pE419: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Vβ ← 65S
pE420: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Kβ ← 62R, Sβ ← 118T
pE516: Nβ ← 63S, Vβ ← 11A, Gα ↓ 67S, Gβ ← 40R
pE536: Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Gα ↓ 67S, Gβ ← 40R
pE539: Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Lβ ← 97R, Gβ ← 40R
pE601: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Gβ ← 40R

(Example 2)
Preparation and Evaluation of Rhodococcus Transformant (1) Plasmid Construction In the amino acid sequence of the α subunit by the following method, 60 residues upstream of the C residue at the most upstream of the CTLCSC region constituting the prosthetic molecule binding region A plasmid for Rhodococcus having a mutation (Nα ↑ 60D) in the residue was prepared. The mutation was introduced by site-directed mutagenesis using a commercially available kit: QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). The experiment followed the operation manual. PSJ034 was used as a template plasmid for introducing the mutation. pSJ034 is a plasmid that expresses nitrile hydratase in Rhodococcus. pSJ034 was produced from pSJ023 by the method described in JP-A-10-337185.
PSJ023 is a transformant “R. rhodochrous ATCC12674 / pSJ023”, and under the accession number FERM BP-6232, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center ) Was deposited internationally on March 4, 1997.

Two kinds of primers for introducing mutations were synthesized. The underlined portion indicates the mutation introduction site.
β ← 63-F: ccgaaatatgtgcgg AGC aagatcggggaaatc (SEQ ID NO: 5)
β ← 63-R: gatttccccgatctt GCT ccgcacatatttcgg (SEQ ID NO: 6)

Position-directed mutagenesis PCR was performed using GeneAmp9700 (PE Bioscience) under the following conditions.
<Composition of PCR reaction solution>
Template plasmid pSJ034 (10ng) 2μl
10 × Reaction Buffer 5 μl
Primer β ← 63-F (100ng / μl) 1μl
Primer β ← 63-R (100ng / μl) 1μl
2.5 mM dNTPmix 1 μl
Sterile water 36μl
QuickSolution 3μl
Pfu Turbo DNA Polymerase 1μl
<Reaction conditions>
1 minute at 95 ° C
(50 seconds at 95 ° C, 50 seconds at 60 ° C, 14 minutes at 68 ° C) x 18 cycles
7 minutes at 68 ° C

  After completion of PCR, 10 μl of the reaction solution was subjected to 0.7% agarose gel electrophoresis, and an amplified fragment of about 11 kb was detected. After confirming the amplified fragment, 1 μl of Dpn I (supplied with the kit) was added to the PCR reaction solution and reacted at 37 ° C. for 1 hour to remove the template plasmid.

Next, transformation was performed using XL10-GOLD ultracompetent cells (included in the kit). 2 μl of DpnI-treated PCR reaction solution and 45 μl of competent cells were mixed and incubated at 4 ° C. for 30 minutes, followed by heat shock at 42 ° C. for 30 seconds. Thereafter, 0.5 ml of NZY ⁺ medium (1% NZ amine, 0.5% yeast extract, 0.5% NaCl, 12.5 mM MgCl ₂ , 12.5 mM MgSO ₄ , 0.4% glucose, pH 7.5) is added to the bacterial solution, and 37 Incubation was performed at 0 ° C. for 1 hour. 250 μl of this culture solution was plated on an LB plate (1% NaCl, 1% tryptone, 0.5% yeast extract, 2% agar, 50 mg / L ampicillin) and cultured at 37 ° C. for 1 day.

  Several colonies obtained were cultured in 1.5 ml of LB medium (50 mg / L ampicillin), and a plasmid was prepared using FlexiPrep Kit (Amersham Bioscience). Finally, the nucleotide sequence of the obtained plasmid was determined, and it was confirmed that the target mutation was introduced. In this way, a mutation-introduced plasmid: p52R into which a base substitution causing an amino acid substitution of Nβ ← 63S was introduced was obtained.

Next, mutation was introduced in a position-specific manner using the following two kinds of primers as described above using p52R as a template plasmid.
β ← 11-F: gaaagacgtagtgtgc GCC gatctctgggaaccg (SEQ ID NO: 7)
β ← 11-R: cggttcccagagatc GGC gcacactacgtctttc (SEQ ID NO: 8)

  In this way, a mutation-introduced plasmid: pAR002 (FIG. 3) into which a base substitution that causes amino acid substitution of Nβ ← 63S and Vβ ← 11A was introduced was obtained.

  Subsequently, mutation was introduced site-specifically using the following two kinds of primers in the same manner as described above using pAR002 as a template plasmid. Specifically, any one or more of Iα ↓ 53T, Gα ↓ 67S, Gβ ← 40R, Kβ ← 62R, Vβ ← 65S, Lβ ← 97R, Sβ ← 118T, and Sβ ← 173M, Pβ ← 213A A plasmid into which a base substitution that causes an amino acid substitution was introduced was prepared.

Primer sequences for introducing each base substitution are as follows.
α ↓ 53-F: aggacactccgaa ACC cgctacatcgtcatcccg (SEQ ID NO: 11)
α ↓ 53-R: cgggatgacgatgtagcg GGT ttcggagctgct (SEQ ID NO: 12)

α ↓ 67-F: gccggcaccgac AGT tggtccgaggag (SEQ ID NO: 13)
α ↓ 67-R: ctcctcggacca ACT gtcggtgccggc (SEQ ID NO: 14)

β ← 40-F: gagcagctccgccggcctc CGC gacgatcctcg (SEQ ID NO: 15)
β ← 40-R: cgaggatcgtc GCG gaggccggcggagctgctc (SEQ ID NO: 16)

β ← 62-F: cgaaatatgtgcggaac CGG atcggggaaatcg (SEQ ID NO: 17)
β ← 62-R: cgatttccccgat CCG gttccgcacatatttcg (SEQ ID NO: 18)

β ← 65-F: ggtgcccgaaatat AGC cggaacaagatcgggg (SEQ ID NO: 19)
β ← 65-R: ccccgatcttgttccg GCT atatttcgggcacc (SEQ ID NO: 20)

β ← 97-F: acgagccccactcc CGA gcgcttccaggagcg (SEQ ID NO: 21)
β ← 97-R: cgctcctggaagcgc TCG ggagtggggctcgt (SEQ ID NO: 22)

β ← 118-F: cacggacaggaagccg ACG cggaagttcgatccg (SEQ ID NO: 23)
β ← 118-R: cggatcgaacttccg CGT cggcttcctgtccgtg (SEQ ID NO: 24)

β ← 173-F: cggttcttccgggag AAG atggggaacgaaaac (SEQ ID NO: 25)
β ← 173-R: gttttcgttccccat CTT ctcccggaagaaccg (SEQ ID NO: 26)

β ← 213-F: gatacggaccggtc GCC tatcagaaggacgagccc (SEQ ID NO: 27)
β ← 213-R: gggctcgtccttctgata CGC gaccggtccgtatc (SEQ ID NO: 28)

The obtained plasmids were named pER306, pER316, pER317, pER414, pER415, pER416, pER418, pER419, pER420, pER516, pER536, pER539 and pER601, respectively. The substitution mode of amino acid residues in the amino acid sequence encoded by the mutated gene sequence of each plasmid is shown below.
pER306: Nβ ← 63S, Vβ ← 11A, Pβ ← 213A
pER316: Nβ ← 63S, Vβ ← 11A, Vβ ← 65S
pER317: Nβ ← 63S, Vβ ← 11A, Kβ ← 62R
pER414: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Kβ ← 62R
pER415: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A
pER416: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Sβ ← 118T
pER418: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Kβ ← 62R
pER419: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Pβ ← 213A, Vβ ← 65S
pER420: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Vβ ← 65S, Kβ ← 62R, Sβ ← 118T
pER516: Nβ ← 63S, Vβ ← 11A, Gα ↓ 67S, Gβ ← 40R
pER536: Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Gα ↓ 67S, Gβ ← 40R
pER539: Nβ ← 63S, Vβ ← 11A, Iα ↓ 53T, Lβ ← 97R, Gβ ← 40R
pER601: Nβ ← 63S, Vβ ← 11A, Sβ ← 173M, Gβ ← 40R

(2) Production of transformants of Rhodococcus rhodochrous ATCC 12674 strain cells were collected in a logarithmic growth phase using a centrifuge, washed three times with ice-cooled sterilized water, and suspended in sterilized water. 1 μl of the plasmid prepared in (1) above and 10 μl of the cell suspension were mixed and ice-cooled. A suspension of DNA and bacterial cells was placed in a cuvette and subjected to electric pulse treatment at 2.0 KV and 200 OHMS using a gene introduction device Gene Pulser (BIO RAD). The cuvette containing the electric pulse treatment solution was allowed to stand for 10 minutes under ice-cooling, and heat shocked at 37 ° C. for 10 minutes. Then add 500μl of MYK medium (0.5% polypeptone, 0.3% bacto yeast extract, 0.3% bacto malt extract, 0.2% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ ) to the cuvette, and leave it at 30 ° C for 5 hours. And 50 μg / ml kanamycin-containing MYK agar medium. Colonies after 3 days of culture at 30 ° C. were used as transformants.

(3) Evaluation of heat resistance of transformants of Rhodococcus The residual nitrile hydratase activity after heat treatment was examined using the obtained transformants of Rhodococcus. As a comparative control, Rhodococcus rhodochrous ATCC12674 / pSJ034 (hereinafter referred to as ATCC12674 / pSJ034) having wild type nitrile hydratase was used.
The nitrile hydratase activity of the cell suspension was measured by the following method.
Mix 0.2 ml of bacterial cell solution and 4.8 ml of 50 mM phosphate buffer (pH 7.0), and add 5 ml of 50 mM phosphate buffer (pH 7.0) containing 5.0% (w / v) acrylonitrile to the mixture. And allowed to react at 10 ° C. with shaking for 10 minutes.
Subsequently, the cells were separated by filtration, and the amount of acrylamide produced was quantified using gas chromatography.

<Analysis conditions>
Analytical instrument: Gas chromatograph GC-14B (manufactured by Shimadzu Corporation)
Detector: FID (detection 200 ° C)
Column: 1m glass column packed with Polapack PS (Waters column filler) Column temperature: 190 ° C
Nitrile hydratase activity was converted from the amount of acrylamide. Here, the nitrile hydratase activity is defined as 1 U for the amount of enzyme that produces 1 μmol of acrylamide per minute.
ATCC12674 / pSJ034 and each transformant obtained in the step (2) above were inoculated into MYK medium (50 μg / ml kanamycin), shake-cultured at 30 ° C. for 2 days, and GGPK medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K ₂ HPO ₄ , 0.05% KH ₂ PO ₄ , 0.05% MgSO ₄ · 7H ₂ O, 1% CoCl ₂ , 0.1% urea, 50 μg / ml kanamycin, pH 7.2) % Inoculation. The cells were cultured with shaking at 30 ° C. for 3 days and collected by centrifugation. Thereafter, the cells were washed with 100 mM phosphate buffer (pH 7.0) and finally suspended in a small amount of the same buffer.

  In the heat treatment, 0.5 ml of an appropriately diluted cell suspension was placed in a test tube, kept in a water bath at a temperature of 65 ° C. for 60 minutes, and then cooled in ice. As a comparative control, the activity was measured using each of the untreated bacteria kept at 4 ° C. without heat treatment, and the residual activity (%) after the heat treatment was determined based on the obtained activity value. The results of residual activity are shown in Table 3.

  ATCC12674 / pSJ034 containing wild-type nitrile hydratase has a residual activity of 3% after heat treatment at 65 ° C. for 60 minutes, and ATCC12674 / pAB002 has 36%. In contrast, Rhodococcus rhodochrous ATCC 12674 / pER306, ATCC 12674 / pER316, ATCC 12674 / pER317, ATCC 12674 / pER414, ATCC 12674 / pER415, ATCC 12674 / pER416, ATCC 12674 / pER418, containing an improved nitrile hydratase, ATCC 12674 / pER419, ATCC 12674 / pER420, ATCC 12674 / pER516, ATCC 12674 / pER536, ATCC 12674 / pER539 and ATCC 12674 / pER601 all retained a residual activity of 40% or more. Therefore, the improved nitrile hydratase has improved heat resistance.

(Example 3)
Evaluation of Amide Compound Resistance Using the transformant obtained in Example 2, amide compound resistance was evaluated. The said evaluation was performed with the following reaction liquid composition and reaction conditions. Each cell suspension used in the reaction was measured with a spectrophotometer for the turbidity of the cultured cell solution, and appropriately diluted with 100 mM phosphate buffer (pH 7.0) so as to obtain the same cell concentration. Prepared. As a control, ATCC12674 / pSJ034 with wild type nitrile hydratase was used.

<Reaction solution composition>
94g of 50% acrylamide solution
Acrylonitrile 4g
1M phosphate buffer 1g
Bacterial liquid (same cell concentration) 1g
<Reaction conditions>
Reaction for 6 hours with stirring (30 ℃)
1 ml of the reaction solution was sampled before starting the reaction (0 hour), 0.5 hour and 6 hours later, and filtered using a 0.45 μm filter. The obtained filtrate was subjected to gas chromatography. The analysis conditions were the same as the analysis conditions for acrylamide determination shown in (2) of Example 2. Table 4 shows the analysis result of the ratio (%) of the remaining acrylonitrile.

  The improved nitrile hydratase of the example contained less acrylonitrile than the wild-type nitrile hydratase (pSJ034) or qAR002. Therefore, the improved nitrile hydratase maintained nitrile hydratase activity even in the presence of a high concentration of acrylamide, and improved resistance to acrylamide. Combining the results of Example 2, it can be said that the improved nitrile hydratase has improved heat resistance and amide compound resistance.

  According to the present invention, an improved nitrile hydratase is provided. The improved nitrile hydratase of the present invention has improved heat resistance and / or amide compound resistance. For this reason, an amide compound can be efficiently produced from a nitrile compound by using the improved nitrile hydratase of the present invention.

It is a block diagram of plasmid pAB002. It is a block diagram of plasmid pJH601. It is a block diagram of plasmid pAR002. It is a figure which shows the amino acid sequence (part of N terminal side) of alpha subunit of wild type nitrile hydratase derived from various microorganisms including prosthetic molecule binding region “C (S / T) LCSC”. It is a figure which shows the amino acid sequence similar to FIG. 4-1, and has shown the arrangement | sequence following the amino acid sequence of FIG. 4-1. It is a figure which shows the amino acid sequence similar to FIG. 4-1, and has shown the arrangement | sequence (part by the side of C terminal) following the amino acid sequence of FIG.

Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA
SEQ ID NO: 7: synthetic DNA
Sequence number 8: Synthetic DNA
SEQ ID NO: 9: synthetic DNA
SEQ ID NO: 10: synthetic DNA
SEQ ID NO: 11: synthetic DNA
SEQ ID NO: 12: synthetic DNA
SEQ ID NO: 13: synthetic DNA
SEQ ID NO: 14: synthetic DNA
SEQ ID NO: 15: synthetic DNA
SEQ ID NO: 16: synthetic DNA
SEQ ID NO: 17: synthetic DNA
Sequence number 18: Synthetic DNA
Sequence number 19: Synthetic DNA
SEQ ID NO: 20: synthetic DNA
SEQ ID NO: 21: synthetic DNA
SEQ ID NO: 22: synthetic DNA
SEQ ID NO: 23: synthetic DNA
Sequence number 24: Synthetic DNA
SEQ ID NO: 25: synthetic DNA
SEQ ID NO: 26: synthetic DNA
SEQ ID NO: 27: synthetic DNA
SEQ ID NO: 28: synthetic DNA

Claims (10)

The following protein (A) or (B).
(A) an amino acid sequence in which at least one amino acid residue selected from the group consisting of the following (a) to (g) in the amino acid sequence of wild-type nitrile hydratase is substituted with another amino acid residue; A protein having hydratase activity (a) 53 residues from the most downstream C residue of the amino acid sequence C (S / T) LCSC constituting the prosthetic molecule binding region in the amino acid sequence of the α subunit Downstream amino acid residue (b) Amino acid residue 67 residues downstream from the most downstream C residue of the amino acid sequence C (S / T) LCSC (c) C-terminal amino acid of the β subunit amino acid sequence (D) 97 amino acid residues upstream from the C-terminal amino acid residue (e) C-terminal amino acid residue (F) 62 amino acid residues upstream from the C-terminal amino acid residue (g) 40 amino acid residues upstream from the C-terminal amino acid residue Amino acid residue (B) In the amino acid sequence of the protein of (A), including an amino acid sequence in which one or several amino acid residues are deleted, substituted and / or added except for the amino acid residue after the substitution, And a protein having nitrile hydratase activity The following protein (A) or (B).
(A) In the amino acid sequence of wild-type nitrile hydratase, the following amino acid residues (a) and (b) and at least one amino acid residue selected from the group consisting of the following (c) to (e) are other amino acids: A protein comprising an amino acid sequence substituted with a residue and having nitrile hydratase activity (a) 63 amino acids upstream from the amino acid residue at the C-terminal in the amino acid sequence of β subunit Amino acid residues (b) 11 amino acid residues upstream from the C-terminal amino acid residue (c) 213 amino acid residues upstream from the C-terminal amino acid residue (d) C 65 amino acid residues upstream from the terminal amino acid residue (e) 62 amino acid residues upstream from the C-terminal amino acid residue (B) (A) The amino acid sequence includes an amino acid sequence in which one or several amino acid residues are deleted, substituted and / or added, except for the amino acid residue after the substitution, and has a nitrile hydratase activity Protein The following protein (A) or (B).
(A) In the amino acid sequence of wild-type nitrile hydratase, the following amino acid residues (a) and (b) and at least one amino acid residue selected from the group consisting of the following (c) to (f) are other amino acids: A protein comprising an amino acid sequence substituted with a residue and having nitrile hydratase activity (a) 63 amino acids upstream from the amino acid residue at the C-terminal in the amino acid sequence of β subunit Amino acid residues (b) 11 amino acid residues upstream from the C-terminal amino acid residue (c) 97 amino acid residues upstream from the C-terminal amino acid residue (d) C 40 amino acid residues upstream from the terminal amino acid residue (e) Amino acid sequence C (S / T) LCS that constitutes the prosthetic molecule binding region in the amino acid sequence of the α subunit (F) Amino acid residue (B) 67 residues downstream of the most downstream C residue of the amino acid sequence C (S / T) LCSC (B) ) The amino acid sequence of the protein of (A) includes an amino acid sequence in which one or several amino acid residues are deleted, substituted and / or added except for the amino acid residue after substitution, and nitrile hydratase Protein characterized by having activity The following protein (A) or (B).
(A) In the amino acid sequence of wild-type nitrile hydratase, the following amino acid residues (a), (b) and (c) and at least one amino acid residue selected from the group consisting of the following (d) to (h): A protein having a nitrile hydratase activity, comprising an amino acid sequence substituted with another amino acid residue, and (a) a β-subunit amino acid sequence counted from the C-terminal amino acid residue 63 Amino acid residues upstream of the residues (b) 11 amino acid residues upstream from the C-terminal amino acid residue (c) 173 amino acid residues upstream from the C-terminal amino acid residue ( d) Amino acid residue upstream of 213 residues from the C-terminal amino acid residue (e) Amino acid residue upstream of 118 residues from the C-terminal amino acid residue (f) C 65 amino acid residues upstream from the end amino acid residue (g) 62 amino acid residues upstream from the C-terminal amino acid residue (h) counting from the C-terminal amino acid residue 40 amino acid upstream amino acid residues (B) In the amino acid sequence of the protein of (A), one or several amino acid residues were deleted, substituted and / or added except for the amino acid residues after the substitution. A protein comprising an amino acid sequence and having nitrile hydratase activity   A genetic DNA encoding the protein according to any one of claims 1 to 4.   A recombinant vector comprising the gene DNA according to claim 5.   A transformant comprising the recombinant vector according to claim 6.   A nitrile hydratase collected from a culture obtained by culturing the transformant according to claim 7.   A method for producing nitrile hydratase, comprising culturing the transformant according to claim 7 and collecting nitrile hydratase from the resulting culture.   A culture obtained by culturing the protein according to any one of claims 1 to 4 or the transformant according to claim 7 or a treated product of the culture is brought into contact with a nitrile compound, and produced by the contact. A method for producing an amide compound, comprising collecting the amide compound to be obtained.

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