# Patent

## Title
Nucleic acid fragments encoding nitrile hydratase and amidase enzymes from Comamonas testosteroni  5-MGAM-4D and recombinant organisms expressing those enzymes useful for the production of amides and acids

## Abstract

The invention relates to the isolation, sequencing, and recombinant expression of genes encoding either a nitrile hydratase (NHase) or amidase (Am) from Comamonas testosteroni 5-MGAM-4D, where the NHase is useful for catalyzing the hydration of nitriles to the corresponding amides, and the amidase is useful for hydrolysis of amides to the corresponding carboxylic acids. Also provided are transformed host cells containing polynucleotides for expressing the nitrile hydratase or amidase enzymes from Comamonas testosteroni 5-MGAM-4D.

## Background
This application claims the benefit of U.S. application Ser. No. 10/431,966 filed May 8, 2003 now abandoned.


FIELD OF THE INVENTION
The present invention relates to the field of molecular biology and methods for the isolation and expression of foreign genes in recombinant microorganisms. More specifically, the invention relates to the isolation, sequencing, and recombinant expression of nucleic acid fragments (genes) encoding either a nitrile hydratase (NHase) or amidase (Am) from Comamonas testosteroni 5-MGAM-4D, where the NHase is useful for catalyzing the hydration of nitrites to the corresponding amides, and the amidase is likewise useful for hydrolysis of amides to the corresponding carboxylic acids.
BACKGROUND OF THE INVENTION
Nitrile hydratases catalyze the addition of one molecule of water to the nitrile, resulting in the formation of the corresponding amide according to Reaction 1:

R—CN+H2O→RCONH2  Reaction 1

Similarly, methods for producing carboxylic acids are known and use microorganisms that produce an enzyme that possesses amidase (Am) activity. In general, amidases convert the amide product of Reaction 1 to the corresponding carboxylic acid plus ammonia according to Reaction 2

RCONH2→RCOOH+NH3  Reaction 2

A wide variety of bacterial genera are known to possess a diverse spectrum of nitrile hydratase and amidase activities, including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus (Martinkova and Kren, Biocatalysis and Biotransformation, 20:73–93 (2002); Cowan et al., Extremophiles, 2:207–216 (1998)). For example, nitrile hydratase enzymes have been isolated from Pseudomonas chlororaphis B23 (Nishiyama et al., J. Bacteriol., 173:2465–2472 (1991)), Rhodococcus rhodochrous J1 (Kobayashi et al., Biochem. Biophys. Acta, 1129:23–33 (1991)), Brevibacterium sp. 312 (Mayaux et al., J. Bacteriol., 172:6764–6773 (1990)), Rhodococcus sp. N-774 (Ikehata et al., Eur. J. Biochem., 181:563–570 (1989)), and Pseudomonas putida 5B NRRL-18668 (Payne et al., Biochemistry, 36:5447–5454 (1997)).
Wild-type microorganisms known to possess nitrile hydratase activity have been used to convert nitriles to the corresponding amides. Nagasawa et al. (Appl. Microbiol. Biotechnol., 40:189–195 (1993)) have compared three microbial nitrile hydratase catalysts which have been used for commercial production of acrylamide from acrylonitrile; the nitrile hydratase activities of Brevibacterium R312 and Pseudomonas chlororaphis B23 were not stable above 10° C., compared to the nitrile hydratase activity of Rhodococcus rhodochrous J1. Cowan et al. (supra) reported that many mesophilic nitrile hydratases are remarkably unstable, having very short enzyme activity half-lives in the growth temperature range of 20–35° C. In addition to temperature instability, microbial catalysts containing a nitrile hydratase can be susceptible to inactivation by high concentrations of certain substrates such as acrylonitrile. In commercial use, the concentration of acrylonitrile was maintained at 1.5–2 wt % when using Brevibacterium R312 and P. chlororaphis B23 catalysts, while a concentration of up to 7 wt % was used with R. rhodochrous J1 (Nagasawa et al., supra). Similarly, Padmakumar and Oriel (Appl. Biochem. Biotechnol., 77–79:671–679 (1999)) reported that Bacillus sp. BR449 expresses a thermostable nitrile hydratase, but when used for hydration of acrylonitrile to acrylamide, inactivation of the enzyme occurred at concentration of acrylonitrile of only 2 wt %, making this catalyst unsuitable for commercial applications. Webster et al. (Biotechnology Letters, 23:95–101 (2001)) compare two Rhodococcus isolates as catalysts for ammonium acrylate production (one with only a nitrilase activity, and one with only a combination of nitrile hydratase and amidase activities), and concluded that the catalyst having a combination of nitrile hydratase and amidase activities was less preferred due to (a) difficulty in inducing the two enzymes in the required ratio, (b) the susceptibility of the two enzymes (nitrile hydratase and amidase) to deactivation by acrylonitrile, and (c) inhibition of the two enzymes by the respective products.
The hydration of aromatic and heteroaromatic nitriles to the corresponding amides has been reported using the nitrile hydratase activity of Rhodococcus rhodochrous AJ270 (A. Meth-Cohn and M. Wang, J. Chem Soc., Perkin Trans. 1, (8):1099–1104 (1997)), where significant subsequent conversion of the amide to the corresponding acid by amidase was also observed. The nitrile hydratase activity of Rhodococcus rhodochrous J1 was used to convert a variety of aromatic and heteroaromatic nitriles to the corresponding amides with 100% molar conversion (J. Mauger et al., Tetrahedron, 45:1347–1354 (1989); J. Mauger et al., J. Biotechnol., 8:87–96 (1988)); an inhibitory affect of certain nitrites on the nitrile hydratase was overcome by maintaining a low concentration of the nitrile over the course of the reaction. U.S. 20040142447 describes the use of several Rhodococcus strains for the conversion of 3-cyanopyridine to nicotinamide, where the Rhodococcus strains were relatively stable and had a relatively low Km value for 3-cyanopyridine when compared to previously-reported microbial cell catalysts.
In addition to the use of wild-type organisms, recombinant organisms containing heterologous genes for the expression of nitrile hydratase are also known for the conversion of nitrites. For example, Cerebelaud et al. (WO 9504828) teach the isolation and expression in E. coli of nitrile hydratase genes isolated from C. testosteroni. The transformed hosts effectively convert nitriles to amides, including substrates which consist of one nitrile and one carboxylate group. Endo et al. disclose the production of an E. coli transformant which expresses the nitrile hydratase of Rhodococcus N-771 (U.S. Pat. No. 6,316,242 B1). Similarly, Beppu et al., (EP 5024576) disclose plasmids carrying both nitrile hydratase and amidase genes from Rhodococcus capable of transforming E. coli where the transformed host is then able to use isobutyronitrile and isobutyramide as enzymatic substrates. A stereoselective nitrile hydratase from Pseudomonas putida 5B has been overproduced in E. coli (Wu et al., Appl. Microbiol. Biotechnol., 48:704–708 (1997); U.S. Pat. No. 5,811,286).
Genes encoding enzymes having amidase activity have also been cloned, sequenced, and expressed in recombinant organisms. For example, Azza et al., (FEMS Microbiol. Lett., 122:129 (1994)) disclose the cloning and over-expression in E. coli of an amidase gene from Brevibacterium sp. R312 under the control of the native promoter. Similarly, Kobayashi et al., (Eur. J. Biochem., 217:327 (1993)) teach the cloning of both a nitrile hydratase and amidase gene from R. rhodococcus J1 and their co-expression in E. coli. Wu et al. (DNA Cell Biol., 17:915-920 (1998); U.S. Pat. No. 6,251,650) report the cloning and overexpressing of a gene for amidase from Pseudomonas putida 5B in E. coli. 
Applicants have previously isolated Comamonas testosteroni 5-MGAM-4D (ATCC 55744; U.S. Pat. No. 5,858,736 and U.S. Pat. No. 5,922,589). Comamonas testosteroni 5-MGAM-4D has been shown to contain thermally-stable, regiospecific nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities useful in the conversion of a variety of nitriles to their corresponding amides and carboxylic acids. Methods illustrating the utility of the Comamonas testosteroni 5-MGAM-4D nitrile hydratase and amidase activities have been described previously by the Applicants. These uses include regio-selective preparation of lactams from aliphatic α,ω-dinitriles (U.S. Pat. No. 5,858,736), bioconversion of 3-hydroxynitriles to 3-hydroxyacids (US 2002/0039770 A1), and bioconversion of methacrylonitrile and acrylonitrile to their corresponding carboxylic acids (U.S. Ser. No. 10/067,652), hereby incorporated by reference in their entirety. However, the isolation and recombinant expression of the nucleic acid fragments encoding the nitrile hydratase and amidase from Comamonas testosteroni 5-MGAM-4D has been elusive.
The problem to be solved is to provide the genes and encoding for the thermally-stable, regio-selective nitrile hydratase and amidase enzymes from Comamonas testosteroni 5-MGAM-4D and to provide transformants expressing these catalysts.
Additionally, the development of industrial processes which employ microbial catalysts having nitrile hydratase/amidase activities to efficiently manufacture amides or carboxylic acids has proved difficult. Many methods using enzyme catalysts to prepare these products from the corresponding nitriles do not produce and accumulate the product at a sufficiently high concentration to meet commercial needs, or are subject to enzyme inactivation (requiring a low concentration of nitrile over the course of the reaction) or product inhibition during the course of the reaction.
The additional problem to be solved continues to be the lack of facile microbial catalysts to convert nitriles to the corresponding amides or acids in a process characterized by high yield, high concentration, and high selectivity, and with the added advantages of low temperature and energy requirements and low waste production when compared to known chemical methods of nitrile hydrolysis. Comamonas testosteroni 5-MGAM-4D expresses a thermally-stable, regio-selective nitrile hydratase as well as a thermally-stable amidase. An enzyme catalyst having only the nitrile hydratase activity of Comamonas testosteroni 5-MGAM-4D would be highly useful in applications where only the amide product from nitrile hydration is desired.
SUMMARY OF THE INVENTION
The Applicants have isolated and sequenced the genes necessary to express thermally-stable, regio-selective nitrile hydratase and amidase from Comamonas testosteroni 5-MGAM-4D. The corresponding amino acid sequences for each enzyme are also disclosed. The invention also encompasses 1) an isolated polynucleotide encoding a polypeptide having at least 98% identity to a polypeptide alpha-subunit of the nitrile hydratase enzyme from Comamonas testosteroni 5-MGAM-4D as represented by SEQ ID No:4; 2) an isolated polynucleotide encoding a polypeptide having at least 95% identity to a polypeptide beta-subunit of the nitrile hydratase enzyme from Comamonas testosteroni 5-MGAM-4D as represented by SEQ ID No:6; and 3) an isolated polynucleotide encoding a polypeptide having amidase activity and having at least 95% identity to the polypeptide from Comamonas testosteroni 5-MGAM-4D as represented by SEQ ID No:17.
The invention further provides a region of the Comamonas testosteroni 5-MGAM-4D genome encompassed within a 0.9 kb fragment (SEQ ID NO:9) which encodes a polypeptide (designated “PK7”; and represented by SEQ ID NO:14) that is necessary for optimum activity of the nitrile hydratase enzyme.
Transformants are provided that express either the nitrile hydratase or amidase enzymes separately or that co-express both enzymes. Also provided are methods to produce the nitrile hydratase and amidase catalysts in a recombinant host. The present invention further provides recombinant hosts, transformed with the polynucleotides encoding the amidase and/or the nitrile hydratase in combination with the PK7 accessory protein.
A particular embodiment of the invention is Escherichia coli transformed with the nucleic acid sequence represented by SEQ ID NO:11.
The Applicants also provide methods for converting a variety of aliphatic nitrites, aromatic nitriles, heterocyclic aromatic nitriles, unsaturated nitriles, aliphatic dinitriles, and 2-, 3-, or 4-hydroxynitriles to the corresponding amides using a transformed host cell expressing the nitrile hydratase from Comamonas testosteroni 5-MGAM-4D. Comamonas testosteroni 5-MGAM-4D expresses a thermally-stable, regioselective nitrile hydratase as well as a thermally-stable amidase. The present application describes the preparation of microbial transformants that have only the nitrile hydratase activity of Comamonas testosteroni 5-MGAM-4D for use in applications where the production of only the amide product from nitrile hydration would be desirable.



BRIEF DESCRIPTION OF THE DRAWINGS SEQUENCE DESCRIPTIONS, AND BIOLOGICAL DEPOSITS
The invention can be more fully understood from the Figure, the Sequence Listing, the Biological Deposits, and the detailed description that together form this application.
FIG. 1 shows the nucleic acid fragments inserted in several plasmids created for recombinant expression of genes cloned from Comamonas testosteroni 5-MGAM-4D (ATCC 55744).



The following sequences comply with 37 C.F.R. 1.821–1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
SEQ ID NO:1 is the nucleic acid sequence encoding the α-subunit of a nitrile hydratase from Pseudomonas putida 5B (NRRL-18668) used to probe genomic DNA fragments from Comamonas testosteroni 5-MGAM-4D (ATCC 55744).
SEQ ID NO:2 is the nucleic acid sequence encoding the β-subunit of a nitrile hydratase from Pseudomonas putida 5B (NRRL-18668) used to probe genomic DNA fragments from Comamonas testosteroni 5-MGAM-4D (ATCC 55744).
SEQ ID NO:3 is the nucleic acid sequence encoding the α-subunit of a nitrile hydratase from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:4 is the deduced amino acid sequence for the β-subunit of a nitrile hydratase from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:5 is the nucleic acid sequence encoding the β-subunit of a nitrile hydratase from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:6 is the deduced amino acid sequence for the β-subunit of a nitrile hydratase from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:7 is the nucleic acid sequence encoding the α- and β-subunits of the nitrile hydratase from Comamonas testosteroni 5-MGAM-4D used in the creation of pSW131.
SEQ ID NO:8 is the first of two primers (“Primer 1”) useful for amplifying a nucleic acid fragment (SEQ ID NO:7) for creation of plasmid pSW131 and for amplifying a nucleic acid fragment (SEQ ID NO:11) for creation of pSW137.
SEQ ID NO:9 is the second of two primers (“Primer 2”) useful for amplifying a nucleic acid fragment (SEQ ID NO:7) for creation of plasmid pSW131.
SEQ ID NO:10 is the nucleic acid sequence of a 0.9 kb nucleic acid fragment from Comamonas testosteroni 5-MGAM-4D containing a small open reading frame (ORF) which encodes an accessory protein (denoted as “P7K”) useful in the expression of active nitrile hydratase.
SEQ ID NO:11 is the nucleic acid sequence encoding the α- and β-subunits of the nitrile hydratase plus 0.9 kb of downstream DNA (SEQ ID NO. 10) encoding the accessory protein P7K from Comamonas testosteroni 5-MGAM-4D used in the creation of pSW132 and pSW137.
SEQ ID NO:12 is the second of two primers (“Primer 3”) useful for amplifying a nucleic acid fragment (SEQ ID NO:11) for creation of pSW137 and for amplifying a nucleic acid fragment (SEQ ID NO:23) for creation of pSW136.
SEQ ID NO:13 is the nucleic acid sequence encoding the accessory protein P7K, and found within the 0.9 kb downstream DNA sequence (SEQ ID NO:10) from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:14 is the deduced amino acid sequence for the accessory protein P7K useful in the recombinant expression of Comamonas testosteroni 5-MGAM-4D nitrile hydratase.
SEQ ID NO:15 is the nucleic acid sequence of a nucleic acid fragment comprising the first 0.6 kb of the pKP57 insert useful as a probe to identify an amidase from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:16 is the nucleic acid sequence encoding an amidase from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:17 is the deduced amino acid sequence of an amidase from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:18 is the nucleic acid sequence of 7.4 kb nucleic acid fragment comprising the complete coding sequences for an amidase and a nitrile hydratase and the P7K accessory protein from Comamonas testosteroni 5-MGAM-4D.
SEQ ID NO:19 is the first of two primers (“Primer 4”) useful for amplifying a nucleic acid fragment encoding an amidase from Comamonas testosteroni 5-MGAM-4D for creation of plasmid pKP60.
SEQ ID NO:20 is the second of two primers (“Primer 5”) useful for amplifying a nucleic acid fragment encoding an amidase from Comamonas testosteroni 5-MGAM-4D for creation of plasmid pKP60.
SEQ ID NO:21 is the first of two primers (“Primer 6”) useful for amplifying a nucleic acid fragment encoding an amidase from Comamonas testosteroni 5-MGAM-4D for creation of plasmid pSW133 and for amplifying a nucleic acid fragment (SEQ ID NO:23) for creation of pSW136.
SEQ ID NO:22 is the second of two primers (“Primer 7”) useful for amplifying a nucleic acid fragment encoding an amidase from Comamonas testosteroni 5-MGAM-4D for creation of plasmid pSW133.
SEQ ID NO:23 is the nucleic acid fragment encoding an amidase, a nitrile hydratase (α- and β-subunits), and the accessory protein P7K from Comamonas testosteroni 5-MGAM-4D used in the creation of plasmid pSW136.
Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:















Depositor Identification
Int'l. Depository




Reference
Designation
Date of Deposit









Comamonas testosteroni
ATCC 55744
Mar. 8, 1996



5-MGAM-4D



Pseudomonas putida 5B
NRRL 18668
Jul. 6, 1990



Escherichia coli SW132
ATCC PTA-5073
Mar. 21, 2003



Escherichia coli SW137
ATCC PTA-5074
Mar. 21, 2003










As used herein, “ATCC” refers to the American Type Culture Collection International Depository Authority located at ATCC, 10801 University Blvd., Manassas, Va. 20110–2209, USA. The “International Depository Designation” is the accession number to the culture on deposit with ATCC.
As used herein, “NRRL” refers to the Northern Regional Research Laboratory, Agricultural Research Service Culture Collection International Depository Authority located at 11815 N. University Street, Peoria, Ill. 61604 U.S.A. The “NRRL No.” is the accession number to cultures on deposit at the NRRL.
The listed deposits will be maintained in the indicated international depository for at least thirty (30) years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides isolated polynucleotides and the nucleic acid sequences that encode three polypeptides (α- and β-subunits of a nitrile hydratase and an amidase) from Comamonas testosteroni 5-MGAM-4D (ATCC 55744) that act as catalysts. When coexpressed, the α- and β-subunits of the nitrile hydratase selectively hydrate nitriles into the corresponding amides, and the amidase hydrolyzes amides into the corresponding carboxylic acids. The invention also provides transformed microbial host cells expressing the polypeptides. The invention further provides a method for producing the polypeptide catalysts using the transformed microbes and a method for using the catalysts for converting nitriles to the corresponding amides and/or carboxylic acids, or for converting amides to the corresponding carboxylic acids.
Definitions:
In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.
The terms “catalyst”, “enzyme catalyst” or “microbial cell catalyst” refer to polypeptides (or proteins) having a nitrile hydratase activity, an amidase activity, or having a combination of nitrile hydratase and amidase activities. The catalyst may be in the form of an intact microbial cell, permeabilized microbial cell(s), one or more cell components of a microbial cell extract, partially purified enzyme(s), or purified enzyme(s).
As used herein, the term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, and it does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.
The term “thermally-stable” characterizes an enzyme that retains activity despite exposure to a given temperature.
The terms “Comamonas testosteroni” and “C. testosteroni” are used interchangeably and refer to Comamonas testosteroni 5-MGAM-4D (ATCC 55744).
The terms “Psuedomonas putida 5B” and “P. putida 5B” are used interchangeably and refer to Psuedomonas putida NRRL-18668.
The terms “Escherichia coli SW132” and “E. coli SW132” are used interchangeably and refer to an E. coli strain transformed with plasmid pSW132 and having ATCC accession number PTA-5073.
The term “pSW132” refers to a plasmid containing a DNA fragment encoding the C. testosteroni 5-MGAM-4D nitrile hydratase α- and β-subunits plus 0.9 kb of downstream DNA under the control of the T7 promoter. The 0.9 kb of downstream DNA encodes an accessory protein (“P7K”) useful in recombinant expression of nitrile hydratase. E. coli strain SW132 harbors plasmid pSW132 and has ATCC accession number PTA-5073.
The terms “Escherichia coli SW137” and “E. coli SW137” are used interchangeably and refer to an E. coli strain transformed with plasmid pSW137 and having ATCC accession number PTA-5074.
The term “pSW137” refers to a plasmid containing a DNA fragment encoding the C. testosteroni 5-MGAM-4D nitrile hydratase α- and β-subunits plus 0.9 kb of downstream DNA under the control of the trc promoter. The 0.9 kb of downstream DNA encodes an accessory protein (“P7K”) useful in recombinant expression of nitrile hydratase. E. coli strain SW137 harbors plasmid pSW137 and has ATCC accession number PTA-5074.
“Open reading frame” is abbreviated ORF.
“Polymerase chain reaction” is abbreviated PCR.
As used herein, an “isolated nucleic acid fragment” or “isolated polynucleotide” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.
The term “accessory nucleic acid” refers to the 0.9 kb sequence (SEQ ID NO:10), located downstream of the nitrile hydratase α (alpha) and β (beta) subunit genes, which contains an open reading frame (SEQ ID NO:13) encoding a polypeptide (SEQ ID NO:14) useful in increased recombinant expression of the nitrile hydratase.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the CLUSTAL method of alignment (Higgins and Sharp CABIOS. 5:151–153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters typically used for pairwise alignments using the CLUSTAL method are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the instant microbial polypeptides as set forth in SEQ ID NOs:4, 6, 14, and 17. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
“Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. “Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. In the present invention, the host cell's genome includes chromosomal and extrachromosomal (e.g., plasmid) genes. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The term “carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention, and particularly refers to carbon sources selected from, but not limited to, the group consisting of aliphatic carboxylic acids or dicarboxylic acids, monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.
Nitriles particularly pertinent to the invention are nitriles of

R—C≡N  Formula 1

or

N≡C—R—C≡N  Formula 2

wherein N is Nitrogen, C is Carbon, and R is selected from the group consisting of: a) C1–C9 alkyl that is linear, branched or cyclic, optionally substituted with a hydroxyl group, an amino group, a carboxylic acid group, a carboxamide group, a halogen atom, or an oxo group; b) C1–C9 alkenyl, linear, branched or cyclic, optionally substituted with a hydroxyl group, an amino group, a carboxylic acid group, a carboxamide group, a halogen atom, or an oxo group; and c) C6–C9 aryl, optionally substituted with a hydroxyl group, an amino group, a carboxylic acid group, a carboxamide group, or a halogen atom. Particularly useful nitriles in the invention include, but are not limited to, acrylonitrile, methacrylonitrile, 3-hydroxypropionitrile, 3-hydroxybutyronitrile, 3-hydroxyvaleronitrile, butyronitrile, adiponitrile, benzonitrile, and glycolonitrile.

Additional nitrites particularly pertinent to the invention are nitriles of Formula 3:

R2—C≡N  Formula 3

wherein N is Nitrogen, C is Carbon, and R2 is selected from the group consisting of the general formulae 4, 5, 6, 7, and 8:





wherein Formulae 4, 5, and 6 X is N, in Formula 7 X is NH, O, or S, and in Formula 8 X is NH and Y is N, O, or S, and where R3 and R4 are, independently, selected from the group consisting of: a) a hydrogen atom, b) a halogen atom, c) a C1–C9 alkyl group that is linear, branched, or cyclic and optionally substituted with a hydroxyl group, an amino group, a carboxylic acid group, a carboxamide group, a halogen atom, or an oxo group, d) a C1–C9 alkenyl group that is linear, branched, or cyclic and optionally substituted with a hydroxyl group, an amino group, a carboxylic acid group, a carboxamide group, a halogen atom, or an oxo group, and e) a C6–C9 aryl, optionally substituted with a hydroxyl group, an amino group, a carboxylic acid group, a carboxamide group, or a halogen atom. Halogen atoms may be independently F, Cl, Br, or I. Particularly useful heterocyclic nitriles in the invention include, but are not limited to, 3-cyanopyridine, 4-cyanopyridine, pyrazinecarbonitrile, 2-furancarbonitrile, 2-thiophenecarbonitrile, and 4-thiazolecarbonitrile.
The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
The term “altered biological activity” will refer to an activity, associated with a polypeptide (protein) encoded by a microbial nucleotide sequence which can be measured by an assay method, where that activity is either greater than or less than the activity associated with the native microbial sequence. “Enhanced biological activity” refers to an altered activity that is greater than that associated with the native sequence. “Diminished biological activity” is an altered activity that is less than that associated with the native sequence.
The terms “suitable aqueous reaction mixture” or “suitable reaction mixture” refer to the materials and water in which the nitrile and/or amide substrate and enzyme catalyst come into contact. Components of suitable aqueous reaction mixtures are referred to herein and those skilled in the art appreciate the range of component variations suitable for this process.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403–410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA), and the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111–20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.). The term “MEME” refers to a software program used to identify conserved diagnostic motifs based on a hidden Markov model (Timothy L. Bailey and Charles Elkan, Fitting a mixture model by expectation maximization to discover motifs in biopolymers, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28–36, AAAI Press, Menlo Park, Calif. (1994)). “MAST” (Timothy L. Bailey and Michael Gribskov, “Combining evidence using p-values: application to sequence homology searches” Bioinformatics, Vol. 14, pp. 48–54 (1998)) is a program that takes the output from the MEME program and searches the identified motifs against the protein databases such as EMBL and SwissProt. Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters which originally load with the software when first initialized.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984) (hereinafter “Silhavy”); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987) (hereinafter “Ausubel”).
Sequence Identification
Comparison of the present amino acid sequences for the nitrile hydratase a and β-subunits and the amidase from Comamonas testosteroni 5-MGAM-4D to public databases revealed that the most similar-known sequences were all from Pseudomonas putida 5B (NRRL-18668; see: U.S. Pat. No. 5,811,286, and Wu et al., supra). The nitrile hydratase α-subunit was about 97.1% identical to the corresponding nitrile hydratase α-subunit from P. putida 5B (Table 1). The nitrile hydratase β-subunit was about 82.0% identical to the corresponding nitrile hydratase β-subunit from P. putida 5B. Lastly, the amidase was about 92.3% identical to the corresponding amidase from P. putida 5B.







TABLE 1







Sequence Analysis Results












Gene

%
%




Name
Similarity Identified
Identitya
Similarityb
E-valuec
Citation





Amidase
Gi|6225048|sp|O69768|
92.3
92.5
0
Wu et al., DNA Cell



AMID_PSEPU



Biol. 17 (10), 915–920







(1998)


nitrile
Gi|2499193|sp|P97051
97.1
97.1
10e−108
Payne et al.,


hydratase
|NHAA_PSEPU



Biochemistry 36


α-subunit




(18), 5447–5454







(1997)


nitrile
Gi|2499195|sp|P97052|
82.0
82.5
6e−92
Payne et al.,


hydratase
NHAB_PSEPU



Biochemistry 36


β-subunit




(18), 5447–5454







(1997)





aIdentity is defined as percentage of amino acids that are identical between the two proteins.


bSimilarity is defined as percentage of amino acids that are identical or conserved between the two proteins.


cExpect value. The Expect value estimates the statistical significance of the match, specifying the number of matches, with a given score, that are expected in a search of a database of this size absolutely by chance.






Despite the sequence similarities between the nitrile hydratase and amidase enzymes of Comamonas testosteroni 5-MGAM-4D to those of Pseudomonas putida 5B (NRRL-18668), the accompanying Examples demonstrate that both the thermal stability, and stability under reaction conditions, of the Comamonas testosteroni 5-MGAM-4D nitrile hydratase enzyme (expressed in E. coli transformant SW132) are both different and markedly superior to the Pseudomonas putida 5B nitrile hydratase (expressed in E. coli transformant SW30, Wu et al., supra).
Identification of Homologs
The instant sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes of the present invention are typically single stranded nucleic acid sequences which are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions which will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration the shorter the hybridization incubation time needed. Optionally a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res., 19:5143–5151, (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture, typically 30–50% (v/v).
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30–50% v/v formamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6–9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between 0.5–20 mM EDTA, FICOLL (about 300–500 kilodaltons), polyvinylpyrrolidone (about 250–500 kdal), and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA, e.g., calf thymus or salmon sperm DNA, or yeast RNA, and optionally from about 0.5 to 2% wt./vol. glycine. Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as polyethylene glycol, anionic polymers such as polyacrylate or polymethylacrylate, and anionic saccharidic polymers, such as dextran sulfate.
Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
Microbial Recombinant Expression
The genes and gene products of the instant sequences may be produced in heterologous host cells, particularly in the cells of microbial hosts. Expression in recombinant microbial hosts may be useful for the expression of various pathway intermediates; for the modulation of pathways already existing in the host, or for the synthesis of new products heretofore not possible using the host.
Preferred heterologous host cells for expression of the instant genes and nucleic acid fragments are microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances. For example, it is contemplated that any of bacteria, yeast, and filamentous fungi will be suitable hosts for expression of the present nucleic acid fragments. Because of transcription, translation and the protein biosynthetic apparatus is the same irrespective of the cellular feedstock, functional genes are expressed irrespective of carbon feedstock used to generate cellular biomass. Large-scale microbial growth and functional gene expression may utilize a wide range of simple or complex carbohydrates, organic acids and alcohols, saturated hydrocarbons such as methane or carbon dioxide in the case of photosynthetic or chemoautotrophic hosts. However, the functional genes may be regulated, repressed or depressed by specific growth conditions, which may include the form and amount of nitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrient including small inorganic ions. In addition, the regulation of functional genes may be achieved by the presence or absence of specific regulatory molecules that are added to the culture and are not typically considered nutrient or energy sources.
Examples of host strains include but are not limited to bacterial, fungal, or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Methylobacterium, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, Myxococcus, and Staphylococcus. In another embodiment, suitable host strains are selected from the group consisting of Aspergillus, Saccharomyces, Pichia, Candida, Hansuela, Bacillus, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Escherichia, Pseudomonas, Methylomonas, Synechocystis, and Klebsiella. In a further embodiment, suitable host strains are selected from the group consisting of Bacillus, Rhodococcus, Escherichia, Pseudomonas, Klebsiella, and Methylomonas. 
Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for expression of the present nitrile hydratase, amidase, and PK7. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high-level expression of the enzymes
Accordingly it is expected, for example, that introduction of chimeric genes encoding the instant bacterial enzyme under the control of the appropriate promoter, will demonstrate increased nitrile to amide and/or carboxylic acid conversion. It is contemplated that it will be useful to express the instant genes both in natural host cells as well as in a heterologous host. Introduction of the present genes into native hosts will result in altered levels of existing nitrile hydratase and amidase activity. Additionally, the instant genes may also be introduced into non-native host bacteria where an existing nitrile-amide-carboxylic acid pathway may be manipulated.
Vectors or cassettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive expression of the instant ORF in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus. Additionally, the deoxy-xylulose phosphate synthase or methanol dehydrogenase operon promoter (Springer et al., FEMS Microbiol Lett 160:119–124 (1998)), the promoter for polyhydroxyalkanoic acid synthesis (Foeliner et al., Appl. Microbiol. Biotechnol. 40:284–291 (1993)), promoters identified from native plasmids in methylotrophs (EP 296484), promoters identified from methanotrophs (PCT/US03/33698), and promoters associated with antibiotic resistance [e.g., kanamycin (Springer et al., supra; Ueda et al., Appl. Environ. Microbiol. 57:924–926 (1991)) or tetracycline (U.S. Pat. No. 4,824,786)] are suitable for expression of the present coding sequences, especially in C1 metabolizers.
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.
Methods of manipulating genetic pathways are common and well known in the art. Selected genes in a particularly pathway may be up-regulated or down-regulated by variety of methods. Additionally, competing pathways may be eliminated or sublimated by gene disruption and similar techniques.
Once a key genetic pathway has been identified and sequenced, specific genes may be up-regulated to increase the output of the pathway. For example, additional copies of the targeted genes may be introduced into the host cell on multicopy plasmids such as pBR322. Alternatively the target genes may be modified so as to be under the control of non-native promoters. Where it is desired that a pathway operate at a particular point in a cell cycle or during a fermentation run, regulated or inducible promoters may used to replace the native promoter of the target gene. Similarly, in some cases the native or endogenous promoter may be modified to increase gene expression. For example, endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868).
Alternatively, it may be necessary to reduce or eliminate the expression of certain genes in a pathway or in competing pathways that may serve as competing sinks for energy or carbon. Methods of down-regulating genes for this purpose have been explored. Where sequence of the gene to be disrupted is known, one of the most effective methods of gene down regulation is targeted gene disruption where foreign DNA is inserted into a structural gene so as to disrupt transcription. This can be effected by the creation of genetic cassettes comprising the DNA to be inserted (often a genetic marker) flanked by sequence having a high degree of homology to a portion of the gene to be disrupted. Introduction of the cassette into the host cell results in insertion of the foreign DNA into the structural gene via the native DNA replication mechanisms of the cell (Hamilton et al., J. Bacteriol. 171:4617–4622 (1989); Balbas et al., Gene 136:211–213 (1993); Gueldener et al., Nucleic Acids Res. 24:2519–2524 (1996); and Smith et al., Methods Mol. Cell. Biol. 5:270–277(1996)).
Antisense technology is another method of down regulating genes where the sequence of the target gene is known. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. This construct is then introduced into the host cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the protein of interest. The person skilled in the art will know that special considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
Although targeted gene disruption and antisense technology offer effective means of down regulating genes where the sequence is known, other less specific methodologies have been developed that are not sequence based. For example, cells may be exposed to a UV radiation and then screened for the desired phenotype. Mutagenesis with chemical agents is also effective for generating mutants and commonly used substances include chemicals that affect non-replicating DNA such as HNO2 and NH2OH, as well as agents that affect replicating DNA such as acridine dyes, notable for causing frameshift mutations. Specific methods for creating mutants using radiation or chemical agents are well documented in the art. (See for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass. (hereinafter “Brock”), or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992) (hereinafter “Deshpande”)).
Another non-specific method of gene disruption is the use of transposable elements or transposons. Transposons are genetic elements that insert randomly in DNA but can be latter retrieved on the basis of sequence to determine where the insertion has occurred. Both in vivo and in vitro transposition methods are known. Both methods involve the use of a transposable element in combination with a transposase enzyme. When the transposable element or transposon, is contacted with a nucleic acid fragment in the presence of the transposase, the transposable element will randomly insert into the nucleic acid fragment. The technique is useful for random mutagenesis and for gene isolation, since the disrupted gene may be identified on the basis of the sequence of the transposable element. Kits for in vitro transposition are commercially available (see for example The Primer Island Transposition Kit, available from Perkin Elmer Applied Biosystems, Branchburg, N.J., based upon the yeast Ty1 element; The Genome Priming System, available from New England Biolabs, Beverly, Mass.; based upon the bacterial transposon Tn7; and the EZ::TN Transposon Insertion Systems, available from Epicentre Technologies, Madison, Wis., based upon the Tn5 bacterial transposable element).
Industrial Production of Biocatalyst
Commercial production of biocatalyst for preparing amides using transformants harboring the nitrile hydratase catalyst disclosed herein (encoded by genes for the α- and β-subunits, and optionally, accessory protein P7K) and for preparing carboxylic acids from amides using the amidase catalyst disclosed herein may be conducted using a variety of culture methodologies. Large-scale production of a specific gene product may be produced by both batch and continuous culture methodologies.
A classical batch culturing method is a closed system where the composition of the media is set at the beginning of the culture and not subject to artificial alterations during the culturing process. Thus, at the beginning of the culturing process the media is inoculated with the desired organism or organisms and growth or metabolic activity is permitted to occur adding nothing to the system. Typically, however, a “batch” culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase are often responsible for the bulk of production of end product or intermediate in some systems. Stationary or post-exponential phase production can be obtained in other systems.
A variation on the standard batch system is the Fed-Batch system. Fed-Batch culture processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and Fed-Batch culturing methods are common and well known in the art and examples may be found in Brock (supra) and Deshpande (supra).
Commercial production of biocatalysts may also be accomplished with a continuous culture. Continuous cultures are an open system where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high-liquid-phase density where cells are primarily in log phase growth.
Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to aliphatic carboxylic acid and dicarboxylic acids (such as lactic acid or succinic acid), glycerol, monosaccharides (such as glucose and fructose), disaccharides (such as lactose or sucrose), oligosaccharides (such as soluble starch), polysaccharides (such as starch or cellulose or mixtures thereof, and unpurified mixtures from renewable feedstocks (such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt). Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, methane, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon-containing compounds such as methylamine, glucosamine, and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1-Compd., [Int. Symp.], 7th (1993), 415–32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485489 (1990)). Therefore, it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing substrates and will only be limited by the microorganism employed.
Biocatalytic Conversion of Nitriles to Amides or Carboxylic Acids
An aqueous reaction mixture containing the aliphatic, aromatic, or heterocyclic aromatic nitrile is prepared by mixing the nitrile with an aqueous suspension of the appropriate enzyme catalyst. Intact microbial cells can be used as catalyst without any pretreatment, such as permeabilization or heating. Alternatively, the cells can be immobilized in a polymer matrix (e.g., alginate, carrageenan, polyvinyl alcohol, or polyacrylamide gel (PAG)) or on a soluble or insoluble support (e.g., celite, silica) to facilitate recovery and reuse of the catalyst. Methods to immobilize cells in a polymer matrix or on a soluble or insoluble support have been widely reported and are well known to those skilled in the art. The enzyme can also be isolated from the microbial cells and used directly as catalyst, or the enzyme can be immobilized in a polymer matrix or on a soluble or insoluble support. These methods have also been widely reported and are well known to those skilled in the art (Methods in Biotechnology, Vol. 1: Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, N.J., USA; 1997).
Intact microbial cells, either immobilized or unimmobilized, containing genes that encode a polypeptide having nitrile hydratase activity or amidase activity, or containing genes that encode a combination of polypeptides separately having nitrile hydratase and amidase activities, can be used as catalyst without any pretreatment, such as permeabilization, freeze thawing or heating. Alternatively, the microbial cells may be permeabilized by methods familiar to those skilled in the art (e.g., treatment with toluene, detergents, or freeze-thawing) to improve the rate of diffusion of materials into and out of the cells. Methods for permeabilization of microbial cells are well-known to those skilled in the art (Felix, H., Anal. Biochem., 120:211–234 (1982)).
Some of the aliphatic or aromatic nitriles used as starting material in the present invention are only moderately water soluble. Their solubility also depends on the temperature of the solution and the salt concentration in the aqueous phase; the optional inclusion of a buffer, or the production of the ammonium salt of a carboxylic acid by hydrolysis of the corresponding amide are two possible sources of salt in a reaction mixture. In the present case, producing a hydrated or hydrolyzed reaction product at a concentration greater than the solubility limit of the starting aliphatic or aromatic nitrile is accomplished using a reaction mixture that is initially composed of two phases: an aqueous phase (containing the enzyme catalyst and dissolved aliphatic or aromatic nitrile) and an organic phase (the undissolved aliphatic or aromatic nitrile, optionally dissolved in an organic solvent not miscible with the aqueous phase). As the reaction progresses, the aliphatic or aromatic nitrile dissolves into the aqueous phase, eventually yielding a product mixture which may be a single phase, depending on the solubility of the products in water, and on the presence or absence of an optional organic solvent not miscible with water.
The aqueous phase of a two-phase reaction mixture can contain, at a minimum, only as much water as is sufficient to result in a) complete conversion of the aliphatic or aromatic nitrile to the corresponding amide or carboxylic acid (dependent on whether only active nitrile hydratase or a combination of active nitrile hydratase and amidase enzyme are present), and b) maintenance of the hydrolytic activity of the enzyme catalyst. The reaction may also be run by adding the aliphatic or aromatic nitrile to the reaction mixture at a rate approximately equal to the enzymatic hydration or hydrolysis reaction rate, thereby maintaining a single-phase aqueous reaction mixture, thereby avoiding the potential problem of substrate inhibition of the enzyme at high starting material concentrations.
The final concentration of aliphatic or aromatic amide or carboxylic acid in solution in the product mixture at complete conversion of the corresponding aliphatic or aromatic nitrile may range from 0.001 M to the solubility limit of the aliphatic or aromatic nitrile in the product mixture. Product may precipitate from the reaction mixture during the course of the reaction, allowing for the production of amide or carboxylic acid in excess of the solubility of said product in the reaction mixture. Typically, the concentration of the aliphatic or aromatic amide or carboxylic acid product in solution in the product mixture ranges from 0.001 M to 7.0 M. The aliphatic or aromatic amide or carboxylic acid may also be isolated from the product mixture (after removal of the catalyst) by optionally adjusting the pH of the reaction mixture to between 2.0 and 2.5 with concentrated HCl when the product of the reaction is a carboxylic acid, saturating the resulting solution with sodium chloride, and extracting the aliphatic or aromatic amide or carboxylic acid with a suitable organic solvent, such as ethyl acetate, ethyl ether, methyl isobutyl ketone or dichloromethane. The organic extracts are then combined, stirred with a suitable drying agent (e.g., magnesium sulfate), filtered, and the solvent removed (e.g., by rotary evaporation) to produce the desired product in high yield and in high purity (typically 98–99% pure). If desired, the product can be further purified by recrystallization or distillation.
The concentration of enzyme catalyst in the reaction mixture depends on the specific catalytic activity of the enzyme catalyst and is chosen to obtain the desired rate of reaction. The wet cell weight of the microbial cells used as catalyst in hydrolysis reactions typically ranges from 0.001 grams to 0.300 grams of wet cells per mL of total reaction volume, preferably from 0.002 grams to 0.050 grams of wet cells per mL; the cells may be optionally immobilized as described above. The specific activity of the microbial cells (IU/gram dry cell weight) is determined by measuring the rate of conversion of a 0.10–0.50 M solution of a nitrile substrate to the desired amide or carboxylic acid product at 25° C., using a known weight of microbial cell catalyst. An IU of enzyme activity is defined as the amount of enzyme activity required to convert one micromole of substrate to product per minute.
The temperature of the hydrolysis reaction is chosen to optimize both the reaction rate and the stability of enzyme catalyst. The temperature of the reaction may range from just above the freezing point of the reaction mixture (ca. 0° C.) to 65° C., with a preferred range of reaction temperature of from 5° C. to 45° C. An enzyme catalyst solution or suspension may be prepared by suspending the unimmobilized or immobilized cells in distilled water, or in an aqueous reaction mixture of a buffer that will maintain the initial pH of the reaction between 5.0 and 10.0, preferably between 6.0 and 8.0, or by suspending the immobilized enzyme catalyst in a similar mixture, or by preparing a solution of a cell extract, partially purified or purified enzyme(s), or a soluble form of the immobilized enzymes in a similar mixture. After the nitrile is added and as the reaction proceeds, the pH of the reaction mixture may change due to the formation of an ammonium salt of the carboxylic acid from the corresponding nitrile functionality of the aliphatic or aromatic nitrile (when using a combination of nitrile hydratase and amidase enzymes). The reaction can be run to completely convert the nitrile with no pH control, or a suitable acid or base can be added over the course of the reaction to maintain the desired pH.
EXAMPLES
The present invention is further defined in the following Examples that indicate preferred embodiments of the invention. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
In the following Examples, the percent recovery of nitrile, and the percent yields of the corresponding amide and carboxylic acid products were based on the initial concentration of nitrile present in the reaction mixture, and were determined by HPLC. Analyses of 3-hydroxyvaleronitrile, adiponitrile, butyronitrile, benzonitrile, and methacrylonitrile were performed by HPLC using a refractive index detector in combination with a Supelco LC-18-DB column (15 cm×4.6 mm diameter) with precolumn at 25° C. and 10 mM acetic acid, 10 mM sodium acetate in 7.5% methanol in water as eluent at 1.5 mL/min. Analyses for glycolonitrile, acrylonitrile, 3-HPN, 3-HBN, and their corresponding reaction products were performed by HPLC using a Bio-Rad HPX-87H organic acid analysis column (30 cm×7.8 mm dia.) with precolumn at 50° C. and 0.010 N H2SO4 as eluent at 1 mL/min. Analyses for 3-cyanopyridine, pyrazinecarbonitrile, 2-furancarbonitrile, 2-thiophenecarbonitrile, 4-thiazolecarbonitrile and their corresponding reaction products, were performed by HPLC using a UV detector at 254 nm in combination with a 10-cm×4-mm ID, 5 μm C8 Discovery column (Supelco) with precolumn, 1.0 mL/min of 5% CH3CN/95% 10 mM NaOAc, 10 mM AcOH as solvent, and N,N-dimethylbenzamide as external standard.
General Methods
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Maniatis (supra) and Ausubel (supra).
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillip Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, D.C. (1994)) or in Brock, supra.
The following abbreviations in the specification correspond to units of measure, techniques, properties, or compounds as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “d” means day(s), “mL” means milliliters, “L” means liters, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “rpm” means revolutions per minute, “sipm” means standard liters per minute, “psig” means pounds per square inch, and “wt” means weight. “HPLC” means high performance liquid chromatography, “ca” means approximately, “O.D.” means optical density at the designated wavelength, “dcw” means dry cell weight, and “IU” means International Units.
Example 1
Identification of a Genomic DNA Fragment Encoding C. testosteroni 5-MGAM-4D Nitrile Hydratase
Comamonas testosteroni 5-MGAM-4D (ATCC 55744) was grown in LB media at 37° C. with shaking. Genomic DNA was prepared using a Puregene DNA Isolation Kit according to the manufacturer (Gentra Systems, Minneapolis, Minn.). A Southern analysis (Southern et al., J. Mol. Biol., 98:503 (1975)) was performed on EcoRI restricted genomic DNA using Psuedomonas putida NRRL-18668 genes (SEQ ID NOs:1 and 2) encoding nitrile hydratase alpha and beta subunits (U.S. Pat. No. 5,811,286) as probes. Probe labeling, hybridization, and detection were done using ECL random prime labeling and detection systems version 11 according to the manufacturer (Amersham International, Buckinghamshire, UK). The alpha (SEQ ID NO:1) and beta (SEQ ID NO:2) probes each showed positive hybridization to the same 5.7 kb EcoRI DNA fragment.
Example 2
Cloning of a Genomic DNA Fragment Encoding C. testosteroni 5-MGAM-4D nitrile hydratase
Genomic DNA from C. testosteroni 5-MGAM-4D was prepared (Example 1), restricted with EcoRI, and subjected to standard agarose gel electrophoresis. DNA fragments in the size range of approximately 5–7 kb were isolated and ligated to EcoRI restricted pUC19 (New England Biolabs, Beverly, Mass.). This plasmid library was plated and screened with the P. putida NRRL-18668 nitrile hydratase α-subunit gene probe (SEQ ID NO:1). Probe labeling, hybridization and detection were done using ECL random prime labeling and detection systems version 11 according to the manufacturer (Amersham International). A positively hybridizing colony was isolated and determined to contain an insert of 5.7 kb (pKP57).
Example 3
Determination of the nucleotide sequence of the genes encoding C. testosteroni 5-MGAM-4D nitrile hydratase
The nucleotide sequence of the pKP57 (EXAMPLE 2) insert was determined using an ABI 377-XL DNA sequencer and BigDye Terminator Cycle Sequencing chemistry. A BlastN analysis (Altschul et al., Nucleic Acids Res., 25:3389–3402 (1997)) of the obtained sequence to the GenBank® database confirmed the presence of complete genes encoding nitrile hydratase a and β-subunits and a partial gene encoding amidase. Nucleotide sequences of the pKP57 insert encoding nitrile hydratase α- and β-subunits are given in SEQ ID NO:3 and SEQ ID NO:5, respectively. Deduced amino acid sequences of the pKP57 insert for the α- and β-subunits are given in SEQ ID NO:4 and SEQ ID NO:6, respectively.
BlastP analysis was conducted using the deduced amino acid sequence for the nitrile hydratase α- and β-subunits. Results are shown in Table 1. The closest match to the instant amino acid sequence of the nitrile hydratase α-subunit was the nitrile hydratase α-subunit from Pseudomonas putida NRRL-18668 (97.1% identity, 97.1% similarity, E value=10e−108). The closest match to the instant amino acid sequence of the nitrile hydratase β-subunit was the nitrile hydratase β-subunit from Pseudomonas putida NRRL-18668 (82.0% identity, 82.5% similarity, E value=6e−92).
Example 4
Production of C. testosteroni 5-MGAM-4D Nitrile Hydratase in E. coli 
A DNA fragment encoding the nitrile hydratase alpha and beta subunits from C. testosteroni 5-MGAM-4D (SEQ ID NO:7) was obtained from pKP57 by standard PCR via a GeneAmp Kit according to the manufacturer (Roche, Branchburg, N.J.) using Primer 1 (SEQ ID NO:8) and Primer 2 (SEQ ID NO:9) and subcloned into pGEM-T (Promega, Madison, Wis.) under control of the T7 promoter to generate pSW131. E. coli BL21(DE3) (Novagen, Madison, Wis.) was transformed with pSW131 using standard procedures. Growth and induction of E. coli BL21 (DE3) harboring pSW131 was carried out essentially as recommended by Novagen. Modifications included the addition of cobalt chloride and sodium citrate at the time of induction to a final concentration of 0.01 mg/ml and 0.1 mg/ml respectively. Induction was carried out at 30 C for 16 hrs. Production of alpha (23 kDa) and beta (24 kDa) proteins was confirmed by standard SDS-PAGE analysis.
Example 5
Conversion of 3-hydroxyvaleronitrile (3-HVN) by Transformed E. coli 
Growth and induction of E. coli BL21 (DE3) cells harboring pSW131 was carried out as described in EXAMPLE 4. Cells were then harvested by centrifugation, washed twice in buffer (0.1 M potassium phosphate pH 7.0) and suspended at 100 mg wet cells/ml in buffer. The nitrilase activity assay mix included cells (50 mg/mL), 3-hydroxyvaleronitrile (0.3 M) and buffer (0.1 M potassium phosphate, pH 7.0) stirred at ambient temperature. HPLC analysis demonstrated 17% conversion of 3-HVN to the corresponding amide (3-hydroxyvaleramide) in 15 min.
Example 6
Production of high-level C. testosteroni 5-MGAM-4D nitrile hydratase activity in E. coli requires downstream sequence
A plasmid (pSW132; ATCC PTA-5073), containing a DNA fragment encoding the C. testosteroni 5-MGAM-4D nitrile hydratase alpha and beta subunits plus 0.9 kb of downstream DNA encoding the accessory protein P7K (SEQ ID NO:11) under control of the T7 promoter was constructed by replacing the smaller BamHI/PstI fragment in pSW131 with the corresponding BamHI/PstI fragment from pKP57. E. coli BL21(DE3) (Novagen) was transformed with pSW132 using standard procedures. Growth and induction of E. coli BL21 (DE3) harboring pSW132 (“E. coli strain SW132”) was carried out as described in EXAMPLE 4, and production of alpha and beta proteins was confirmed by standard SDS-PAGE analysis.
A DNA fragment (SEQ ID NO:11) encoding the C. testosteroni 5-MGAM-4D nitrile hydratase alpha and beta subunits plus 0.9 kb of downstream DNA encoding the accessory protein P7K was also obtained from pKP57 by standard PCR via a GeneAmp Kit according to the manufacturer (Roche) using Primer 1 (SEQ ID NO:8) and Primer 3 (SEQ ID NO:12) and subcloned into pTrcHis2-TOPO under control of the trc promoter to generate pSW137 in E. coli TOP10 according to the manufacturer (Invitrogen). Growth and induction of E. coli TOP10 harboring pSW137 was carried out as described in EXAMPLE 4, and production of alpha and beta proteins was confirmed by standard SDS-PAGE analysis. Production of the alpha and beta proteins in E. coli BL21(DE3) harboring pSW132 and in E. coli TOP10 harboring pSW137 was qualitatively indistinguishable to that obtained from E. coli BL21 (DE3) harboring pSW131 (EXAMPLE 4).
Growth and induction of E. coli BL21 (DE3) harboring pSW132 was carried out as described in EXAMPLE 4. Cells were then harvested by centrifugation, washed twice in buffer (0.1 M potassium phosphate pH 7.0) and suspended at 100 mg wet cells/ml in buffer. The nitrile hydratase activity assay mix included cells (2 mg/mL), 3-hydroxyvalerontrile (0.3 M) and buffer (0.1 potassium phosphate, pH 7.0) stirred at ambient temperature. HPLC analysis demonstrated 100% conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleramide in 5 min. Similarly, nitrile hydratase activity assay of E. coli TOP10 harboring pSW137 demonstrated 100% conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleramide in 5 min. Comparing these results to those obtained from pSW131 (Example 5) demonstrated the importance of the DNA downstream of the nitrile hydratase beta gene, in obtaining maximal nitrile hydratase activity (Table 2). The downstream nucleotide sequence (SEQ ID NO:10) contains a small open reading frame, the sequence of which is given in SEQ ID NO:13. The deduced amino acid sequence (called P7K) is given in SEQ ID NO:14.







TABLE 2







Comparison of Expression Vectors pSW131 and pSW132











% Conversion of




0.35 M 3-HVN to 3-




HVAm


Expression
Expressed Genetic Components from
at Room


Vector
C. testosteroni 5-MGAM-4D
Temperature





PSW131
Nitrile hydratase α- and β-subunits
 17% in 15 min


PSW132
Nitrile hydratase α- and β-subunits
100% in 5 min



plus accessory protein “P7K”









Example 7
Determination of the Nucleotide Sequence of the Gene Encoding C. testosteroni 5-MGAM-4D amidase
Genomic DNA from C. testosteroni 5-MGAM-4D was prepared (EXAMPLE 1), restricted with PstI, and subjected to Southern analysis using a standard PCR product comprising the first 0.6 kb of the pKP57 (EXAMPLE 2) insert as a probe (SEQ ID NO:15). Probe labeling, hybridization and detection were done using ECL random prime labeling and detection systems version II according to the manufacturer (Amersham International). This probe gave hybridized to a 2.4 kb PstI fragment. Genomic DNA digested with PstI was subjected to standard agarose gel electrophoresis. DNA fragments in the size range of approximately 2–4 kb were isolated and ligated into PsfI restricted pUC19. This plasmid library was plated and screened with the same 0.6 kb probe (SEQ ID NO:15). Probe labeling, hybridization and detection were done using ECL random prime labeling and detection systems version 11 according to the manufacturer (Amersham International). A positively hybridizing colony was isolated and determined to contain an insert of 2.4 kb (pKP59).
Nucleotide sequencing confirmed that the insert is a DNA fragment that overlaps the EcoRI DNA fragment previously cloned (pKP57). Thus, by combining the nucleotide sequences from pKP57 and pKP59, the complete nucleotide sequence for the amidase gene was determined (SEQ ID NO:16). The deduced amidase amino acid sequence is given in SEQ ID NO:17. The nucleotide sequence of a 7.4 kb DNA fragment from C. testosteroni 5-MGAM-4D comprising complete coding sequences for amidase and nitrile hydratase is given in SEQ ID NO:18.
BlastP analysis was conducted using the deduced amidase amino acid sequence (SEQ ID NO:17; Table 1). The closest publicly known match was to that of the amidase from Pseudomonas putida NRRL-18668 (92.3% identity, 92.5% similarity, E value=0).
Example 8
Production of C. testosteroni 5-MGAM-4D Amidase in E. coli 
A DNA fragment encoding the amidase from C. testosteroni 5-MGAM-4D was obtained from genomic DNA by standard PCR via a GeneAmp Kit according to the manufacturer (Roche), using Primer 4 (SEQ ID NO:19) and Primer 5 (SEQ ID NO:20) and subcloned into pGEM-T (Promega) under control of the T7 promoter to generate pKP60. E. coli BL21 (DE3) (Novagen) was transformed with pKP60 using standard procedures. Growth and induction of E. coli BL21(DE3) harboring pKP60 was carried out according to Novagen, and production of amidase protein was confirmed by standard SDS-PAGE analysis
A DNA fragment encoding the amidase from C. testosteroni 5-MGAM-4D was also obtained by standard PCR using Primer 6 (SEQ ID NO:21) and Primer 7 (SEQ ID NO:22) and subcloned into pTrcHis2 TOPO under control of the trc promoter to generate pSW133 in E. coli TOP10 according to the manufacturer (Invitrogen). Growth and induction of E. coli TOP10 harboring pSW133 was carried out according to Invitrogen, and production of amidase protein was confirmed by standard SDS-PAGE analysis.
Example 9
Co-production of C. testosteroni 5-MGAM-4D nitrile hydratase and amidase in E. coli 
A DNA fragment encoding the amidase, NHase alpha and beta, and accessory protein P7K from C. testosteroni 5-MGAM-4D (SEQ ID NO:23) was obtained from genomic DNA by standard PCR via a GeneAmp Kit according to the manufacturer (Roche), using Primer 6 (SEQ ID NO:21) and Primer 3 (SEQ ID NO:12), and subcloned into pTrcHis2-TOPO (Invitrogen) under control of the Trc promoter to generate pSW136.
Example 10
Fermentation of Escherichia coli SW132 Cells
The production of nitrile hydratase in a 14 L Braun Biostat C fermentor (B. Braun Biotech International Gmbh, Melsungen, Germany) was made in mineral medium with glucose, ammonia, and yeast extract. E. coli strain SW132 (E. Coli BL21(DE3) harboring plasmid pSW132 as described in Example 6) was grown in a seed culture for 10 h prior to inoculation of the fermentor. IPTG (1 mM) was added to the fermentor at 30–35 ODλ=550 and cells were harvested 5 h after IPTG addition. Fermentation protocol: vessel medium was prepared in an initial batch of 7.5 L containing 32 g KH2PO4, 8.0 g MgSO4.7H2O, 8.0 g (NH4)2SO4, 50 g yeast extract, and 10 mL Mazu DF204 antifoam (BASF Corporation, Mount Olive, N.J.). Following sterilization, 369 g glucose solution (60% w/w), 160 mL trace element solution (Table 3), and 100 mg/L ampicillin were added. NH4OH (40% w/v) and 20% w/v H2SO4 were used for pH control. The set points for agitation, aeration, pH, pressure, dissolved oxygen concentration (DO), and temperature are described in Table 4 below. The dissolved oxygen concentration was controlled at 25% of air saturation with the agitation to rise first with increase oxygen demand and the aeration to follow. The 500 mL seed culture was grown in a 2 L flask at 36° C., 300 rpm for 10 h to an ODλ=550 of >2.0. In the fermentor at culture densities of 20–30 OD additional AMP was added to 100 mg/L. IPTG was added to 1 mM at culture densities of 30–35 OD. Glucose feed was started at <5 g/L and the scheduled rates are described in Table 5. Glucose feed rate was reduced if glucose accumulated above 2 g/L. Five hours after IPTG addition the cells were chilled to 5–10° C. and harvested by centrifugation; 490 g (wet cells) was harvested. The kinetics of growth and nitrile hydratase production are presented in Table 6.







TABLE 3







Trace elements solution:











Concentration



Chemical
g/L














Citric acid
10.0



CaCl2*2H2O
1.50



FeSO4*7H2O
5.00



ZnSO4*7H2O
0.39



CuSO4*5H2O
0.38



CoCl2*6H2O
0.20



MnCl2*4H2O
0.30

















TABLE 4







Fermentation Run Conditions











Initial Set-Point
Minimum
Maximum
















Stirrer (rpm)
400
400
850



Airflow (slpm)
2
2
16



pH
6.8
6.8
6.8



Pressure (psig)
0.5
0.5
0.5



DO
25%
25%
25%



Temp. C.
36
36
36

















TABLE 5







Glucose feed protocol










Time, h
Rate (g/min)














0–2
0.39



2–8
0.78



8–End
0.60

















TABLE 6







The kinetics of growth and nitrile hydratase production















Nitrile Hydratase





Glucose
Production



Time, h
ODλ=550
(g/L)
(U/g dry cell wt.)
















2.9
5.8
26.2




4.9
15.7
20.2



6.3
28
8.5
9157



8.1
44
6
10220



11.2
57.6
0.04
10888










Example 11
Hydration of Nitriles to Corresponding Amides by Unimmobilized E. Coli SW132 Cells
To a 20-mL reaction vessel equipped with magnetic stirring was added 0.04, 0.4, 2.0, or 5.0 mmol of acrylonitrile, methacrylonitrile, 3-hydroxypropionitrile, 3-hydroxybutyronitrile, 3-hydroxyvaleronitrile, butyronitrile, adiponitrile, benzonitrile, or glycolonitrile and distilled, deionized water was added to adjust the final volume of the mixture to 3.0 mL. To the reaction vessel was next added 1.0 mL of an aqueous suspension of 0.44–8.8 mg dry cell weight (dcw)/mL of E. coli SW132 cells (prepared as described in Example 10) in 0.10 M potassium phosphate buffer (pH 7.0, except for glycolonitrile, which was run at pH 6.0), and the mixture was stirred at 25° C. Samples (0.100 mL) of the reaction mixture were mixed with 0.400 mL of water, and then 0.200 mL of the diluted sample was either (a) mixed with 0.200 mL of 0.200 M sodium butyrate (acrylonitrile and 3-hydroxyvaleronitrile HPLC standard), 0.200 M N-ethylacetamide (methacrylonitrile HPLC standard), 0.200 M isobutyric acid (butyronitrile and 3-hydroxypropionitrile HPLC standard), or 0.200 M malonic acid (3-hydroxybutyronitrile HPLC standard) in water, or (b) measured against a calibration curve for product at 100% nitrile conversion (5-cyanovaleramide, adipamide, benzamide, glycolamide). The resulting mixture was centrifuged, and the supernatant analyzed by HPLC.
All reactions produced only the amide as the hydration product at 100% conversion of nitrile, with no hydrolysis of the nitrile to the corresponding carboxylic acid.







TABLE 7







Hydration of Nitriles to Corresp. Amides by E. coli SW132 Cells














mg


Amide



conc.
dcw/
time

yield


Nitrile
(M)
mL
(h)
amide
(%)















Acrylonitrile
0.50
2.2
3
acrylamide
100


Acrylonitrile
1.25
8.8
5
acrylamide
98


Methacrylonitrile
0.50
2.2
20
methacrylamide
100


3-
0.50
2.2
3
3-
100


hydroxy-



hydroxypropionamide


propionitrile


3-
0.50
4.4
4
3-hydroxybutryamide
99


hydroxy-


butyronitrile


3-
0.51
2.2
1
3-hydroxyvaleramide
99


hydroxy-


valeronitrile


Butyronitrile
0.50
2.2
16
butyramide
100


Adiponitrile
0.50
0.44
1.5
5-cyanovaleramide
93






adipamide
8


Adiponitrile
0.50
4.4
1
adipamide
100


Benzonitrile
0.010
2.2
2
benzamide
99


Glycolonitrile
0.10
2.2
2
glycolamide
63









Example 12
Hydration of 3-Hydroxyvaleronitrile using a Partially-Purified Protein Extract of E. coli SW132 Cells
E. coli SW132 cells (0.4874 g) were suspended in 2.0 mL of cold breaking buffer consisting of 1 mM DTT and 0.1 mM PMSF in 0.1 M potassium phosphate buffer (pH 7). The suspension (200 mg wet cell weight/mL) was loaded in a French Pressure Mini Cell, and the cells were ruptured at 16000–17000 psi. Cell debris was removed from the resulting mixture by centrifugation at 38000 RCF for 15 min. Approximately 1.68 mL of extract supernatant was recovered, having a nitrile hydratase activity equivalent to a 199 mg wet cell weight/mL cell suspension.
To a 20-mL reaction vessel equipped with magnetic stirring was added 0.4 mL of the E. coli SW132 extract supernatant, 0.6 mL of deionized water, and 3 mL of 0.667 M 3-hydroxyvaleronitrile in water. The mixture was stirred at 25° C. Samples (0.100 mL) of the reaction mixture were mixed with 0.100 mL of water, and then 0.200 mL of the diluted sample was mixed with 0.200 mL of 0.200 M sodium butyrate (external standard) and analyzed by HPLC. After 120 min, the yield of 3-hydroxyvaleramide was 99% at 100% conversion of 3-hydroxyvaleronitrile.
Example 13
Comparison of Thermal Stability of Nitrile Hydratase from Comamonas testosteroni 5-MGAM-4D and Pseudomonas putida 5B Cells
A 44 mg dry cell weight/mL suspension of either E. coli SW30 wet cells (having active nitrile hydratase from Pseudomonas putida 5B) or E. coli SW132 wet cells (having active nitrile hydratase from Comamonas testosteroni 5-MGAM-4D) in 0.50 M phosphate buffer was heated to 50° C. in a water bath. At predetermined times, aliquots of the 50° C. cell suspensions were rapidly cooled to 25° C. in a water bath, and these suspensions were assayed for remaining nitrile hydratase activity by adding 1.0 mL aliquots of the heated/cooled cell suspensions with stirring to 3.0 mL of 0.667 M 3-hydroxyvaleronitrile in water at 25° C. Samples (0.100 mL) of the reaction mixture were withdrawn at predetermined times and mixed with 0.100 mL of water, then 0.200 mL of the diluted sample was mixed with 0.200 mL of 0.200 M sodium butyrate (external standard), centrifuged, and the supernatant and analyzed by HPLC. The rate of hydration of 3-hydroxyvaleronitrile in each reaction was determined, and remaining nitrile hydratase specific activity of the cells calculated. The nitrile hydratase specific activity and % of enzyme recovered for E. coli SW30 and E. coli SW132, respectively, as a function of time at 50° C. is listed in Table 8, below.







TABLE 8







Comparison of E. coli SW30 and E. coli SW132 Nitrile Hydratase


Thermostability.














SW132



Time
SW30 Nitrile
Nitrile
Nitrile
Nitrile


At 50° C.
Hydratase
Hydratase
Hydratase
Hydratase


(min)
(IU/g dcw)
Recovery (%)
(IU/g dcw)
Recovery (%)














0
103
100
7752
100


30
1.6
1.6
6940
90


60
0
0
6437
83









Example 14
Comparison of Comamonas testosteroni 5-MGAM-4D and Pseudomonas putida 5B Nitrile Hydratase for Hydration of Acrylonitrile to Acrylamide
To a 20-mL reaction vessel (equipped with magnetic stirring) was added 0.270 g (5.1 mmol) of acrylonitrile and a suspension of either 107.6 mg dry cell weight E. coli SW30 wet cells (expressing the active nitrile hydratase from Pseudomonas putida 5B) or 4.41 mg dry cell weight E. coli SW132 wet cells (expressing the active nitrile hydratase from Comamonas testosteroni 5-MGAM-4D) in a total volume of 9.664 mL of 0.10 M potassium phosphate buffer (pH 7.0); the amount of dry cell weight present in each reaction was chosen to provide ca. equivalent nitrile hydratase activities (IU/mL) in the reaction mixtures. The final concentration of acrylonitrile was 0.51 M. The mixture was stirred at 25° C. Samples (0.200 mL) of the reaction mixture were mixed with 0.200 mL of 0.200 M sodium butyrate (HPLC standard) in water, and 0.020 mL of 6 N acetic acid. The resulting sample was centrifuged, and the supernatant analyzed by HPLC. The reaction time, % acrylonitrile conversion, and % yield of acrylamide are listed in Table 9. For reactions using E. coli SW30 as biocatalyst, a loss of nitrile hydratase activity was observed over the course of the reaction, and incomplete conversion of nitrile was obtained at extended reaction times.







TABLE 9







Comparison of E. coli SW30 and E. coli SW132 in Acrylonitrile


Hydration Reactions











E. coli
mg
time
acrylonitrile conv.
acrylamide yield


Construct
dcw/mL
(h)
(%)
(%)














SW30
10.8
1.5
42
38


SW30
10.8
20
80
82


SW30
10.8
45
96
97


SW132
0.44
1.5
100
98









Example 15
Comparison of Comamonas testosteroni 5-MGAM-4D and Pseudomonas putida 5B Nitrile Hydratase for Hydration of 3-Hydroxyvaleronitrile to 3-Hydroxyvaleramide
To a 20-mL reaction vessel (equipped with magnetic stirring) was added 3.0 mL of a solution of 0.204 g (2.0 mmol) of 3-hydroxyvaleronitrile in distilled, deionized water and a 1.0 mL suspension of 44 mg dry cell weight of either E. coli SW30 wet cells (expressing the active nitrile hydratase from Pseudomonas putida 5B) or E. coli SW132 wet cells (expressing the active nitrile hydratase from Comamonas testosteroni 5-MGAM-4D) in 0.10 M potassium phosphate buffer (pH 7.0). The final concentration of 3-hydroxyvaleronitrile was 0.50 M. The mixture was stirred at 25° C. Samples (0.100 mL) of the reaction mixture were mixed with 0.100 mL of water, 0.200 mL of 0.200 M sodium butyrate (HPLC standard) in water, an 0.020 mL of 6 N HCl. The resulting mixture was centrifuged, and the supernatant analyzed by HPLC. The reaction time, % 3-hydroxyvaleronitrile conversion, and % yield of 3-hydroxyvaleramide are listed in Table 10. For reactions using SW30 as biocatalyst, a loss of nitrile hydratase activity was observed over the course of the reaction, and incomplete conversion of nitrile was obtained at extended reaction times.







TABLE 10







Comparison of E. coli SW30 and E. coli SW132 in


3-Hydroxyvaleronitrile Reactions











E. coli






Con-
mg
Time
3-hydroxyvaleronitrile
3-hydroxyvaleramide


struct
dcw/mL
(h)
conv. (%)
yield (%)














SW30
11
1
12
12


SW30
11
7
42
40


SW30
11
24
61
61


SW132
11
0.25
100
100









Example 16
Hydration of 3-Hydroxyvaleronitrile to 3-Hydroxyvaleramide by E. coli SW137
To a 4-mL reaction vessel (equipped with magnetic stirring) was added 0.75 mL of an aqueous solution containing 0.404 M 3-hydroxyvaleronitrile and 0.25 mL of a suspension of 18 mg dry cell weight E. coli SW137 wet cells (prepared as described in Example 6) in 0.10 M potassium phosphate buffer (pH 7.0). The E. coli SW137 cells express the polypeptide having nitrile hydratase activity from Comamonas testosteroni 5-MGAM-4D. The final concentration of 3-hydroxyvaleronitrile was 0.303 M. The mixture was stirred at 25° C. Samples (0.100 mL) of the reaction mixture were mixed with 0.100 mL of water, 0.200 mL of 0.200 M sodium butyrate (HPLC standard) in water, an 0.020 mL of 6 N HCl. The resulting mixture was centrifuged, and the supernatant analyzed by HPLC. After 10 min, the conversion of 3-hydroxyvaleronitrile was 100%, and the yield of 3-hydroxyvaleramide was 100%.
Example 17
Immobilization of Escherichia coli SW132 Cells in Calcium Cross-linked Alginate
Into a 250-mL media bottle (equipped with magnetic stir bar and containing 59.7 g of distilled, deionized water at 50° C.) was slowly added 3.30 g of FMC BioPolymer Protanal® LF 10/60 alginate with rapid stirring. The mixture was heated to 75–80° C. with rapid stirring until the alginate was completely dissolved, and the resulting solution cooled to 25° C. in a water bath. To the alginate suspension was added 40.8 g of Escherichia coli SW132 wet cell paste (22% dry cell weight) and 16.2 mL of distilled water with stirring. The cell/alginate mixture was added dropwise by syringe to 640 mL of 0.20 M calcium acetate buffer (pH 7.0) at 25° C. with stirring. After stirring for 2 h, the buffer was decanted from the resulting beads (82 g), which were resuspended in 200 mL of 0.20 M calcium acetate buffer (pH 7.0) at 25° C. With stirring, 4.10 g of 25 wt % glutaraldehyde (GA) in water was added and the beads mixed for 1.0 h at 25° C. To the suspension was then added 16.4 g of 12.5 wt % polyethylenimine (PEI) (BASF Lupasol® PR971 L, average molecular weight ca. 750,000) in water, and the beads mixed for an additional 1 h at 25° C. The GA/PEI-crosslinked beads were then washed twice with 250 mL of 0.05 M calcium acetate buffer (pH 7.0) at 25° C., and stored in this same buffer at 5° C.
Example 18
Hydration of Nitriles (0.50 M to 3.0 M) to Corresponding Amides by Alginate-Immobilized Escherichia coli SW132 Cells in Consecutive Batch Reactions with Biocatalyst Recycle
Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer (temperature-controlled at 25° C. or 35° C.) with a recirculating temperature bath) was placed 4.0 g of GA/PEI-crosslinked Escherichia coli SW132 cell/alginate beads prepared as described in Example 17. To the reaction vessel was added 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture), 10, 20, 40, or 60 mmol of acrylonitrile, methacrylonitrile, or 3-hydroxy-valeronitrile, and the final volume of the reaction mixture adjusted to 20 mL by the addition of distilled, deionized water. The mixture was stirred at 25° C. or 35° C. Samples (0.100 mL) of the reaction mixture were mixed with 0.400 mL of water, and then 0.200 mL of the diluted sample was mixed with 0.200 mL of 0.200 M sodium butyrate (acrylonitrile and 3-hydroxyvaleronitrile HPLC external standard) or 0.200 M N-ethylacetamide (methacrylonitrile HPLC external standard) in water. The resulting mixture was centrifuged, and the supernatant analyzed by HPLC.
At the completion of the reaction (100% conversion of nitrile), the product mixture was decanted from the biocatalyst beads, and additional distilled, deionized water, 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture) and 10, 20, 40 or 60 mmol of acrylonitrile, methacrylonitrile, or 3-hydroxy-valeronitrile mixed with the reaction heel (immobilized-cell catalyst and remaining product mixture from the first reaction) at 25° C. or 35° C. At the completion of the second reaction, the product mixture was decanted and a third reaction performed as before. The reaction time, product yield for acrylamide, methacrylamide, or 3-hydroxyvaleramide, and the percent recovered biocatalyst activity for each recycle reaction is listed in Table 11 below.







TABLE 11







Hydration of Nitriles (0.50 M to 3.0 M) to Corresponding


Amides by Immobilized E. coli SW132 Cells in Consecutive


Batch Reactions with Biocatalyst Recycle



















Recov-








ered








Bio-







Amide
catalyst



Conc.
Temp.
Rxn
Time
Yield
Activity


Nitrile
(M)
(° C.)
#
(h)
(%)
(%)
















Acrylonitrile
0.53
25
1
0.18
100
100





2
0.18
100
94





3
0.12
100
112


Acrylonitrile
1.02
25
1
0.25
100
100





2
0.25
100
118





3
0.25
100
124


acrylonitrile
2.05
25
1
0.5
100
100





2
0.5
100
95





3
0.5
100
100


acrylonitrile
3.04
25
1
0.75
100
100





2
0.83
100
98





3
0.5
100
106


acrylonitrile
1.06
35
1
0.15
100
100





2
0.25
100
107





3
0.15
100
110


methacrylonitrile
1.00
25
1
1.0
100
100





2
1.0
100
106





3
1.5
99
103


3-hydroxyvaleronitrile
1.05
25
1
0.75
100
100





2
0.75
100
93





3
0.75
100
84


3-hydroxyvaleronitrile
2.05
25
1
1.5
100
100





2
2.0
100
102





3
2.0
100
93









Example 19
Immobilization of E. coli SW132 Cells in Carrageenan
Into a 250 mL media bottle equipped with magnetic stir bar and containing 54.6 g of water at 50° C. is slowly added 2.88 g of kappa-carrageenan (FMC RG300) with rapid stirring. The mixture is heated to 75–80° C. with rapid stirring until the carrageenan is completely dissolved, and the resulting solution cooled to 55–56° C. (ca. 52° C. gelling temperature) in a thermostated water bath. A suspension of 18.6 g of E. coli SW132 wet cell paste (22.0% dry cell wt) in 19.7 g of 0.35 M sodium phosphate buffer (pH 7.3) is heated to 50° C. for 15 min, then added to the carrageenan solution at 55–56° C. with stirring. The cell/carrageenan mixture is immediately added slowly to 383 mL of soybean oil at 50° C. with stirring using an overhead stirrer. After cell/carrageenan droplets of the desired size are produced in the oil by controlling the stirring rate, the temperature of the oil is reduced to 40–42° C. to gel the droplets, and the oil decanted from the resulting beads. The beads are washed with 150 mL of 0.1 M potassium bicarbonate buffer (pH 7.0), then suspended in 182 mL of this same buffer, and 1.9 g of 25 wt % glutaraldehyde in water is added and the beads mixed for 1 h at 25° C. To the mixture is then added 7.6 g of 12.5 wt % polyethylenimine (BASF Lupasol PR971L) average Mw ca. 750,000) in water, and the beads mixed for 1 h at 25° C. The beads are then washed twice with 0.30 M ammonium bicarbonate (pH 7.0), and stored in this same at 5° C.
Example 20
Hydration of Acrylonitrile by Carrageenan-Immobilized E. coli SW132 Cells
Into a 50-mL jacketed reaction vessel equipped with an overhead stirrer (temperature-controlled at 35° C. with a recirculating temperature bath) is placed 4.0 g of GA/PEI-crosslinked E. coli SW132 cell/carrageenan beads prepared as described in Example 19. To the reaction vessel is then added 1.06 g of acrylonitrile (1.0 M final concentration), the final volume of the reaction mixture adjusted to 20 mL by the addition of distilled, deionized water, and the mixture stirred at 35° C. Samples (0.100 mL) of the reaction mixture are mixed with 0.400 mL of water, and then 0.200 mL of the diluted sample is mixed with 0.200 mL of 0.200 M sodium butyrate (HPLC external standard) in water. The resulting mixture is centrifuged, and the supernatant analyzed by HPLC. At complete conversion of acrylonitrile, there is a quantitative yield of acrylamide.
Example 21
Hydration of Acrylonitrile Using Immobilized Nitrile Hydratase from Partially-Purified Protein Extract of E. Coli SW132 Cells
Into a 25-mL Erlenmeyer flask is weighed 1.0 g of oxirane acrylic beads (Sigma). To the flask is then added ca. 7.5 mL of a solution containing potassium phosphate buffer (50 mM, pH 8.0), and the oxirane acrylic beads suspended in the buffer by briefly mixing the contents of the flask. After cessation of mixing, the beads settle to the bottom of the flask, and the fine particles which float to the top of the mixture are removed by pipette, along with as much of the supernatant which can be removed without disturbing the settled beads. This washing procedure is repeated a second time. To the flask is then added 1.0 mL of the E. coli SW132 cell extract supernatant described in Example 12 and the final volume of the mixture adjusted to 10 mL with additional potassium phosphate buffer. The resulting mixture is mixed on a rotary platform shaker for 16 h at 25° C. The mixture is then transferred to a chromatography column equipped with a fritted bed support, and the immobilized nitrile hydratase washed three times with 10 mL of potassium phosphate buffer and stored at 5° C. in this same buffer.
Into a 50-mL jacketed reaction vessel equipped with an overhead stirrer (temperature-controlled at 35° C. with a recirculating temperature bath) is placed 1.0 g of immobilized E. coli SW132 nitrile hydratase prepared as described above. To the reaction vessel is then added 1.06 g of acrylonitrile (1.0 M final concentration), the final volume of the reaction mixture adjusted to 20 mL by the addition of distilled, deionized water, and the mixture stirred at 35° C. Samples (0.100 mL) of the reaction mixture are mixed with 0.400 mL of water, and then 0.200 mL of the diluted sample is mixed with 0.200 mL of 0.200 M sodium butyrate (HPLC external standard) in water. The resulting mixture is centrifuged, and the supernatant analyzed by HPLC. At complete conversion of acrylonitrile, there is a quantitative yield of acrylamide.
Example 22
Hydration of 3-Cyanopyridine (0.5 M) to Nicotinamide by Unimmobilized E. coli SW132 Cells
To a 15-mL polypropylene centrifuge tube was added 3.73 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mg dry cell weight E. coli SW132 wet cells (prepared as described in Example 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2660 g of 3-cyanopyridine. The final concentration of 3-cyanopyridine was 0.501 M. The reaction mixture was mixed on a rotating platform at 23° C. After 15 minutes, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) was added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of 3-cyanopyridine was 100%, and the yields of nicotinamide and nicotinic acid were 100% and 0%, respectively.
Example 23
Hydration of 3-Cyanopyridine (1.0 M) to Nicotinamide by Unimmobilized E. coli SW132 Cells
To a 15-mL polypropylene centrifuge tube was added 3.47 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 55.2 mg dry cell weight E. coli SW132 wet cells (prepared as described in Example 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.5339 g of 3-cyanopyridine. The final concentration of 3-cyanopyridine was 1.00 M. The reaction mixture was mixed on a rotating platform at 23° C. After 30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) was added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of 3-cyanopyridine was 100%, and the yields of nicotinamide and nicotinic acid were 99% and 0%, respectively.
Example 24
Hydration of 3-Cyanopyridine using a Partially-Purified Protein Extract of E. coli SW132 Cells
E. coli SW132 cells (0.5377 g, (prepared as described in Example 10)) were suspended in 5.377 mL of cold breaking buffer consisting of 1 mM DTT and 0.1 mM PMSF in 0.50 mM potassium phosphate buffer (pH 7.0). The suspension (100 mg wet cell weight/mL) was loaded in a French Pressure Mini Cell, and the cells were ruptured at 16000–17000 psi (approximately 110.3–117.2 megapascal (Mpa)). Cell debris was removed from the resulting mixture by centrifugation at 38000 RCF for 15 min. Approximately 4.20 mL of extract supernatant was recovered, having a nitrile hydratase activity equivalent to a 100 mg wet cell weight/mL (22.06 mg dry cell weight/mL) cell suspension.
To a 15-mL polypropylene centrifuge tube was added 3.73 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of an E. coli SW132 cell extract suspension prepared as described above, and 0.2626 g of 3-cyanopyridine. The final concentration of 3-cyanopyridine was 0.494 M. The reaction mixture was mixed on a rotating platform at 23° C. After 15 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) was added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of 3-cyanopyridine was 100%, and the yields of nicotinamide and nicotinic acid were 100% and 0%, respectively.
Example 25
Hydration of Pyrazinecarbonitrile (0.5 M) to Pyrazinamide by Unimmobilized E. coli SW132 Cells
To a 15-mL polypropylene centrifuge tube was added 3.73 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mg dry cell weight E. coli SW132 wet cells (prepared as described in Example 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2694 g of pyrazinecarbonitrile. The final concentration of pyrazinecarbonitrile was 0.512 M. The reaction mixture was mixed on a rotating platform at 23° C. After 15 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) was added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of pyrazinecarbonitrile was 100%, and the yields of pyrazinamide and pyrazinecarboxylic acid were 100% and 0%, respectively.
Example 26
Hydration of Pyrazinecarbonitrile (1.0 M) to Pyrazinamide by Unimmobilized E. coli SW132 Cells
To a 15-mL polypropylene centrifuge tube was added 3.47 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 55.2 mg dry cell weight E. coli SW132 wet cells (prepared as described in Example 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.5330 g of pyrazinecarbonitrile. The final concentration of pyrazinecarbonitrile was 1.00 M. The reaction mixture was mixed on a rotating platform at 23° C. After 30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) was added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of pyrazinecarbonitrile was 100%, and the yields of pyrazinamide and pyrazinecarboxylic acid were 100% and 0%, respectively.
Example 27
Hydration of 2-Furancarbonitrile (0.5 M) to 2-Furancarboxamide by Unimmobilized E. coli SW132 Cells
To a 15-mL polypropylene centrifuge tube was added 3.78 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mg dry cell weight E. coli SW132 wet cells (prepared as described in Example 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2381 g of 2-furancarbonitrile. The final concentration of 2-furancarbonitrile was 0.506 M. The reaction mixture was mixed on a rotating platform at 23° C. After 30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) was added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of 2-furancarbonitrile was 100%, and the yields of 2-furancarboxamide and 2-furancarboxylic acid were 99% and 0%, respectively.
Example 28
Hydration of 2-Thiophenecarbonitrile (0.3 M) to 2-Thiophenecarboxamide by Unimmobilized E. coli SW132 Cells
To a 15-mL polypropylene centrifuge tube was added 3.86 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mg dry cell weight E. coli SW132 wet cells (prepared as described in Example 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.1691 g of 2-thiophenecarbonitrile. The final concentration of 2-thiophencarbonitrile was 0.307 M. The reaction mixture was mixed on a rotating platform at 27° C. After 30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) was added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of 2-thiophenecarbonitrile was 99.5%, and the yields of 2-thiophenecarboxamide and 2-thiophenecarboxylic acid were 98% and 0%, respectively.
Example 29
Hydration of 4-Thiazolecarbonitrile (0.5 M) to 4-Thiazolecarboxamide by Unimmobilized E. coli SW132 Cells
To a 15-mL polypropylene centrifuge tube is added 3.70 mL of 50 mM potassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mg dry cell weight E. coli SW132 wet cells (prepared as described in Example 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2754 g of 4-thiazolecarbonitrile. The final concentration of 4-thiazolecarbonitrile is 0.500 M. The reaction mixture is mixed on a rotating platform at 25° C. After 30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard) is added to the reaction, the resulting mixture centrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC. The conversion of 4-thiazolecarbonitrile is 100%, and the yields of 4-thiazolecarboxamide and 4-thiazolecarboxylic acid are 100% and 0%, respectively.
Example 29
Hydration of 3-Cyanopyridine (1.0 M) to Nicotinamide by Alginate-Immobilized Escherichia coli SW132 Cells in Consecutive Batch Reactions with Biocatalyst Recycle at 25° C.
Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer (temperature-controlled at 25° C. with a recirculating temperature bath) was placed 1.0 g of GA/PEI-crosslinked Escherichia coli SW132 cell/alginate beads prepared as described in Example 17. To the reaction vessel was added 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture), 16.67 mL of distilled, deionized water, and 2.124 g of 3-cyanopyridine. The final concentration of 3-cyanopyridine was 1.00 M in 20 mL of reaction mixture. Samples (0.100 mL) of the reaction mixture were mixed with 0.400 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of the resulting solution mixed with 0.900 mL of acetonitrile and analyzed by HPLC. After 24 h, the conversion of 3-cyanopyridine was 100%, and the yields of nicotinamide and nicotinic acid were 100% and 0%, respectively. The initial reaction rate for production of nicotinamide, measured during the first 30 min of reaction, was 7.65 mM/minute.
At the completion of the reaction (100% conversion of nitrile), the product mixture was decanted from the biocatalyst beads, and 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture), 16.56 mL of distilled, deionized water, and 2.127 g of 3-cyanopyridine was added to the catalyst beads in the reaction vessel. The final concentration of 3-cyanopyridine was 1.06 M in 20 mL of reaction mixture. Samples (0.100 mL) of the reaction mixture were mixed with 0.400 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of the resulting solution mixed with 0.900 mL of acetonitrile and analyzed by HPLC. After 23 h, the conversion of 3-cyanopyridine was 100%, and the yields of nicotinamide and nicotinic acid were 100% and 0%, respectively. The initial reaction rate for production of nicotinamide, measured during the first 30 min of reaction, was 4.60 mM/minute.
Example 30
Hydration of 3-Cyanopyridine (1.0 M) to Nicotinamide by Alginate-Immobilized Escherichia coli SW132 Cells in Consecutive Batch Reactions with Biocatalyst Recycle at 10° C.
Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer (temperature-controlled at 10° C. with a recirculating temperature bath) was placed 4.0 g of GA/PEI-crosslinked Escherichia coli SW132 cell/alginate beads prepared as described in Example 17. To the reaction vessel was added 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture), 13.63 mL of distilled, deionized water, and 2.127 g of 3-cyanopyridine. The final concentration of 3-cyanopyridine was 1.00 M in 20 mL of reaction mixture. Samples (0.100 mL) of the reaction mixture were mixed with 0.400 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of the resulting solution mixed with 0.900 mL of acetonitrile and analyzed by HPLC. After 1 h, the conversion of 3-cyanopyridine was 100%, and the yields of nicotinamide and nicotinic acid were 100% and 0%, respectively. The initial reaction rate for production of nicotinamide, measured during the first 30 min of reaction, was 23.4 mM/minute.
At the completion of the reaction (100% conversion of nitrile), the product mixture was decanted from the biocatalyst beads, and 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture), 13.14 mL of distilled, deionized water, and 2.125 g of 3-cyanopyridine was added to the catalyst beads in the jacketed reaction vessel at 10° C. The final concentration of 3-cyanopyridine was 1.00 M in 20 mL of reaction mixture. Samples (0.100 mL) of the reaction mixture were mixed with 0.400 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of the resulting solution mixed with 0.900 mL of acetonitrile and analyzed by HPLC. After 1 h, the conversion of 3-cyanopyridine was 94%, and the yields of nicotinamide and nicotinic acid were 94% and 0%, respectively. The initial reaction rate for production of nicotinamide, measured during the first 30 min of reaction, was 18.5 mM/minute.
Example 31
Hydration of 3-Cyanopyridine (3.0 M) to Nicotinamide by Alginate-Immobilized Escherichia coli SW132 Cells in Consecutive Batch Reactions with Biocatalyst Recycle at 25° C.
Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer (temperature-controlled at 25° C. with a recirculating temperature bath) was placed 2.0 g of GA/PEI-crosslinked Escherichia coli SW132 cell/alginate beads prepared as described in Example 17. To the reaction vessel was added 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture), 11.42 mL of distilled, deionized water, and 6.3648 g of 3-cyanopyridine. The final concentration of 3-cyanopyridine was 3.00 M in 20 mL of reaction mixture. Samples (0.100 mL) of the reaction mixture were mixed with 1.400 mL of 95:5 acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of the resulting solution mixed with 0.900 mL of acetonitrile and analyzed by HPLC. After 48 h, the conversion of 3-cyanopyridine was 100%, and the yields of nicotinamide and nicotinic acid were 100% and 0%, respectively.

## Claims

1. An isolated polynucleotide encoding a polypeptide comprising the alpha-subunit of a nitrite hydratase enzyme, said polypeptide having the amino acid sequence as represented in SEQ ID NO:4.

2. An isolated polynucleotide encoding a polypeptide having at least 98% identity to SEQ ID NO:4, wherein the polvpentide is an alpha subunit of a nitrile hydratase enzyme.

3. An isolated polynucleotide encoding a polypeptide comprising the alpha-subunit of a nitrite hydratase enzyme, said isolated polynucleotide having the nucleic acid sequence represented in SEQ ID NO: 3.

4. An isolated polynucleotide encoding a polypeptide comprising the beta-subunit of a nitrile hydratase enzyme, said polypeptide having the amino acid sequence as represented in SEQ ID NO:6.

5. An isolated polynucleotide encoding a polypeptide comprising the beta-subunit of a nitrile hydratase enzyme, said isolated polynucleotide having the nucleic acid sequence represented in SEQ ID NO: 5.

6. An isolated polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO:6, wherein the polypeptide is the beta-subunit of a nitrile hydratase enzyme.

7. An isolated polynucleotide encoding the alpha- and beta-subunits of a nitrite hydratase enzyme, said isolated polynucleotide having the nucleic acid sequence as represented in SEQ iD NO:7.

8. An isolated polynucleotide encoding the alpha- and beta-subunits of a nitrite hydratase enzyme and an accessory protein, said isolated polynucleotide having the nucleic acid sequence as represented in SEQ ID NO:11.

9. An isolated polynucleotide encoding a polypeptide comprising an accessory protein, said isolated polynucleotide having the nucleic acid sequence represented in SEQ ID NO: 13.

10. An isolated polynucleotide encoding the alpha- and beta-subunits of a nitnie hydratase enzyme, an accessory protein, and an amidase. said alpha- and beta-subunits of a nitrile hydratase enzyme, the accessory protein, and the amidase encoded by the nucleic acid sequence as represented in SEQ ID NO: 23.

11. An expression vector comprising any one of the nucleic acid sequences of claims 1-3, 4-6, 7 or 10.

12. The expression vector as contained in E. coil SW132 designated ATCC PTA-5073.

13. The expression vector as contained in E. coil SWI37 designated ATCC PTA-5074.

14. A transformed microbial host cell comprising the expression vector of claim 11.

15. A transformed microbial host cell comprising the expression vector of claims 12 or 13.

16. The transformed microbial host cell of claim 15 wherein the microbial host cell is a bacterium, yeast, or filamentous fungi.

17. The transformed microbial host cell of claim 16 wherein the microbial host cell is
a) a bacterium selected from the group consisting of the genera Escherichia, Pseudomonas, Rhodococcus, Acinectobacter, Bacillus, Methylomonas, and Streptomyces; 
b) a yeast selected from the group consisting of the genera Pichia, Hansenula, and Saccharomyces; or
c) a filamentous fungi selected from the group consisting of the genera Aspergillus, Neurospora, and Peniclllium.

18. The transformed microbial host cell of claim 12 wherein the microbial host cell is Escherichia coil.

19. A purified transformed microbial host cell selected from the group consisting of Escherichia coil SW132 designated ATCC PTA-5073 and a purified microbial host cell Escherichia coil SW137 designated ATCC PTA-5074.

20. A method for producing polypeptides comprising
a) culturing, under suitable conditions, a transformed microbial host cell containing the expression vector of claim 11; and
b) recovering the polypeptides produced in step a).