D-N-carbamoyl-amino acid amidohydrolase and hydantoinase
AssigneeSmithKline Beecham Corporation
InventorsRobert John Neal, Alison Michelle Griffin, Hazel Claire Gorham
Life of this patent
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Abstract
Processes are provided for the production of a carbamoylase enzyme which has the capability of converting a D-N-carbamoyl (optionally substituted phenyl) glycine by expressing recombinant DNA encoding a carbamoylase in a homologous host. Also provided are specific recombinant DNA vectors producing high levels of expression of the carbamoylase enzyme and/or hydantoinase enzyme in homologous and heterologous hosts and their use in the production of D-.alpha. amino acids.
Claims45
An isolated DNA endogenous to Agrobacterium 80/44-2A, said DNA encoding a carbamoylase enzyme said enzyme having the capability of converting a D-N-carbamoyl (optionally substituted phenyl) glycine into the corresponding D-(optionally substituted phenyl) glycine and encoding a hydantoinase enzyme which enzyme has the ability to convert a D,L-(optionally substituted phenyl) hydantoin into the corresponding D-N-carbamoyl (optionally substituted phenyl) glycine.
An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:
- 2.
An isolated DNA molecule comprising the coding region of SEQ ID NO:
- 1.
A recombinant DNA vector selected from the group consisting of:
- pCAR1 (having the configuration of restriction sites in FIG. 3),
- pCAR6 (having the configuration of restriction sites in FIG. 4),
- pCAR12 (having the configuration of restriction sites in FIG. 6),
- pCAR21 (having the configuration of restriction sites in FIG. 7),
- pCAR26 (having the configuration of restriction sites in FIG. 14),
- pCAR27 (having the configuration of restriction sites in FIG. 8 ),
- pCAR28 (having the configuration of restriction sites in FIG. 9),
- pCAR29 (having the configuration of restriction sites in FIG. 12),
- pCAR31 (having the configuration of restriction sites in FIG. 22),
- pCAR32 (having the configuration of restriction sites in FIG. 23),
- pCAR36 (having the configuration of restriction sites in FIG. 24),
- pCAR44 (having the configuration of restriction sites in FIG. 17), and
- pCAR46 (having the configuration of restriction sites in FIG. 21).
A recombinant DNA vector selected from the group consisting of pCAR1, pCAR6, pCAR31, pCAR32, and pCAR36.
A recombinant DNA vector selected from the group consisting of pDAN3 (having the configuration of restriction sites in FIG. 25) and pDAN4 (having the configuration of restriction sites in FIG. 26).
A process for the production of a carbamoylase enzyme, said process comprising the step of:
- expressing in an Agrobacterium host recombinant DNA endogenous to Agrobacterium 80/44-2A, said DNA encoding a carbamoylase enzyme.
A process for the production of a carbamoylase enzyme, said process comprising the step of:
- expressing in a host cell DNA comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:
- 2.
A method of producing carbamoylase activity in a host cell, said method comprising the steps of:
- (a) introducing into said host cell DNA comprising the coding region of SEQ ID NO:
- 1 or a carbamoylase-encoding portion thereof in a vector selected from the group consisting of pWOR902 (NCIMB 40451) having the configuration of restriction sites in FIG. 16, pWOR903 having the configuration of restriction sites in FIG. 18, pWOR904 having the configuration of restriction sites in FIG. 19, pWOR905 having the configuration of restriction sites in FIG. 20 wherein said DNA is positioned for expression within said vector;
- and
- (b) culturing said host cell transformed in step (a) under conditions sufficient to produce said carbamoylase activity.
A process for the production of a carbamoylase enzyme, said process comprising the step of:
- expressing in a host cell DNA comprising the coding region of SEQ ID NO:
- 1.
An isolated carbamoylase enzyme comprising the amino acid sequence set forth in SEQ ID NO:
- 2.
A polypeptide sequence comprising the amino acid sequence set forth in SEQ ID NO:
- 2.
An isolated carbamoylase enzyme encoded for by a nucleotide sequence comprising the coding region of SEQ ID NO:
- 1.
An isolated hydantoinase enzyme encoded by a vector selected from the group consisting of pDAN3 and pDAN4.
An isolated DNA comprising a hydantoinase coding portion of a vector selected from the group consisting of pDAN3 and pDAN4.
A method of producing hydantoinase activity in a host cell, said method comprising the steps of:
- (a) introducing into said host cell DNA comprising the hydantoinase encoding portion of a vector selected from the group consisting of pDAN3 and pDAN4 in a vector selected from the group consisting of pWOR902 (NCIMB 40451) having the configuration of restriction sites in FIG. 16, pWOR903 having the configuration of restriction sites in FIG. 18, pWOR904 having the configuration of restriction sites in FIG. 19, PWOR905 having the configuration of restriction sites in FIG. 20 wherein said DNA is positioned for expression within said vector;
- and
- (b) culturing said host cell transformed in step (a) under conditions sufficient to produce said hydantoinase activity.
A vector selected from the group consisting of pWOR902 (NCIMB 40451) having the configuration of restriction sites in FIG. 16, pWOR903 having the configuration of restriction sites in FIG. 18, pWOR904 having the configuration of restriction sites in FIG. 19, pWOR905 having the configuration of restriction sites in FIG. 20. thereof.
Dependencies are inferred from the claim text.