Claims
- 1. A decarbamylase having improved thermostability wherein said decarbamylase comprises an amino acid sequence as shown in the even-numbered SEQ IDs selected from the group consisting of SEQ ID Nos. 4 to 68.
- 2. A decarbamylase according to claim 1, wherein said decarbamylase is produced by a microorganism transformed with a DNA sequence encoding said decarbamylase wherein said DNA sequence is derived from Pseudomonas species KNK003A (FERM BP3181) or Agrobacterium species KNK712 (FERM BP-1900), said DNA sequence being modified to encode a decarbamylase having an amino acid sequence which differs from native decarbamylase by at least one amino acid among amino acids at one or more thermostability-related sites so as to permit improved thermostability and repeated usability of said decarbamylase; said microorganism being E. coli JM109 pAD402 (FERM BP-3912), E. coli JM109 pAD404(FERM BP-3913), E. coli JM109 pAD406 (FERM BP-3914), E. coliJM109 pAD416 (FERM BP-3915), E. coli JM109 pAD429 (FERM BP-4035), E. coli JM109 pAD424 (FERM BP-4034), E. coli JM109 pAD455 (FERM BP-4036) or E. coli HB101 pNT4553 (FERM BP-4368).
- 3. An immobilized enzyme preparation comprising:
- the decarbamylase of claim 2;
- 5 to 10 mg of at least one antioxidant selected from the group consisting of dithiothreitol, 2-mercaptoethanol, L-cystein hydrochloride, cysteamine hydrochloride, dithioerythritol and reduced glutathione per 1 g of the immobilized enzyme preparation; and
- a support for immobilization which is an anion exchange resin,
- wherein said immobilized enzyme can be used repeatedly at 30 times or more for converting a renewed N-carbamyl-D-.alpha.-amino acid into D-.alpha.-amino acid.
- 4. A process for preparing a D-.alpha.-amino acid comprising reacting the decarbamylase of claim 3 with an N-carbamyl-D-.alpha.-amino acid and repeatedly at 30 times or more reacting said decarbamylase with a renewed N-carbamyl-D-.alpha.-amino acid.
- 5. An immobilized enzyme preparation which comprises the decarbamylase of claim 1
- 5 to 10 mg of at least one antioxidant selected from the group consisting of dithiothreitol, 2-mercaptoethanol, L-cystein hydrochloride, cysteamine hydrochloride, dithioerythritol and reduced glutathione for each 1 g of the immobilized enzyme preparation; and
- an anion exchange resin as a supporting material for immobilization, wherein said immobilized enzyme can be used repeatedly at 30 times or more for converting a renewed N-carbamyl-D-.alpha.-amino acid into D-.alpha.-amino acid.
- 6. A process for preparing a D-.alpha.-amino acid comprising reacting the decarbamylase of claim 5 with an N-carbamyl-D-.alpha.-amino acid; and repeatedly at 30 times or more reacting said decarbamylase with a renewed N-carbamyl-D-.alpha.-amino acid.
- 7. A decarbamylase having an amino acid sequence of amino acids No. 1 to No. 311 of SEQ ID 70.
- 8. A decarbamylase according to claim 7 capable of producing D-.mu.-amino acids by removing the carbamoyl group of corresponding N-carbamyl-D-.mu.-amino acids, wherein said decarbamylase is produced by a microorganism transformed with a DNA sequence encoding said decarbamylase wherein said DNA sequence is derived from Pseudomonas species KNK003A (FERM BP3181) and wherein said microorganism is Escherichia coli JM109pPD304 (FERM BP-3183).
- 9. An immobilized enzyme preparation comprising:
- the decarbamylase of claim 8;
- 5 to 10 mg of at least one antioxidant selected from the group consisting of dithiothreitol, 2-mercaptoethanol, L-cystein hydrochloride, cysteamine hydrochloride, dithioerythritol and reduced glutathione per 1 g of the immobilized enzyme preparation; and
- a support for immobilization which is an anion exchange resin,
- wherein said immobilized enzyme can be used repeatedly at 30 times or more for converting a renewed N-carbamyl-D-.alpha.-amino acid into D-.alpha.-amino acid.
- 10. A process for preparing a D-.alpha.-amino acid comprising reacting the decarbamylase of claim 9 with an N-carbamyl-D-.alpha.-amino acid; and
- repeatedly at 30 times or more reacting said decarbamylase with a renewed N-carbamyl-D-.alpha.-amino acid.
- 11. An immobilized enzyme preparation comprising:
- the decarbamylase of claim 7;
- 5 to 10 mg of at least one antioxidant selected from the group consisting of dithiothreitol, 2-mercaptoethanol, L-cystein hydrochloride, cysteamine hydrochloride, dithioerythritol and reduced glutathione per 1 g of the immobilized enzyme preparation; and
- a support for immobilization which is an anion exchange resin,
- wherein said immobilized enzyme can be used repeatedly at 30 times or more for converting a renewed N-carbamyl-D-.alpha.-amino acid into D-.alpha.-amino acid.
- 12. A process for preparing a D-.alpha.-amino acid comprising reacting the decarbamylase of claim 11 with an N-carbamyl-D-.alpha.-amino acid; and repeatedly at 30 times or more reacting said decarbamylase with a renewed N-carbamyl-D-.alpha.-amino acid.
Priority Claims (6)
Number |
Date |
Country |
Kind |
2-400848 |
Dec 1990 |
JPX |
|
2-407922 |
Dec 1990 |
JPX |
|
3-078840 |
Apr 1991 |
JPX |
|
3-140051 |
Jun 1991 |
JPX |
|
4-212692 |
Aug 1992 |
JPX |
|
4-340078 |
Dec 1992 |
JPX |
|
CROSS REFERENCE
This is a continuation-in-part application of application Ser. No. 07/971,758 filed Apr. 12, 1993, now abandoned; application Ser. No. 07/917,111 filed Aug. 7, 1992, now U.S. Pat. No. 5,565,344; and application Ser. No. 08/211,641 filed Apr. 11, 1994, now abandoned.
US Referenced Citations (8)
Foreign Referenced Citations (8)
Number |
Date |
Country |
515698 |
Dec 1992 |
EPX |
0 677 584 |
Oct 1995 |
EPX |
0 677 585 |
Oct 1995 |
EPX |
0018793 |
Apr 1982 |
JPX |
63-20520 |
Aug 1988 |
JPX |
1-320991 |
Dec 1989 |
JPX |
2042531 |
Sep 1980 |
GBX |
WO 9400577 |
Jan 1994 |
WOX |
Related Publications (2)
|
Number |
Date |
Country |
|
917111 |
Aug 1992 |
|
|
211641 |
Apr 1994 |
|
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
971758 |
Apr 1993 |
|