Protein participating in the activation of nitrile hydratase...

Details Protein participating in the activation of nitrile hydratase... Protein participating in the activation of nitrile hydratase...

1999-03-10

2004-05-04

Bugaisky, Gabriele (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S254110, C435S320100, C435S069200, C536S023700, C536S023100

Reexamination Certificate

active

06730508

ABSTRACT:

DETAILED DESCRIPTION OF THE INVENTION
1. Technical Background of the Invention
The present invention relates to a protein participating in activation of nitrile hydratase derived from
Pseudonocardia thermophila
JCM3095 (hereinafter simply referred to as “
Pseudonocardia thermophila
”) and a gene encoding the same. Further, the invention relates to a recombinant plasmid containing the gene, a recombinant plasmid containing the gene and a nitrile hydratase gene, a transformant strain obtained through transformation with the recombinant plasmid, and a process for producing a corresponding amide compound from a nitrile compound using the transformant strain, and a culture solution obtained by incubating the transformant treated products thereof.
2. Prior Art
In recent years, nitrile hydratase which is an enzyme having a nitrile hydration activity of converting a nitrile group of various compounds into an amide group through hydration, and a large number of microbial strains that produce this enzyme have been disclosed.
In order to industrially produce an amide compound from a nitrile compound using nitrile hydratase, it is important to reduce the production cost of the enzyme occupied in the production cost of the amide compound. More specifically, the content of the enzyme based on the unit weight of microbes has to be increased. In this respect, an approach to clone the gene of this enzyme has been studied to express the enzyme in a large amount by the method of the genetic engineering using the gene of the enzyme.
The present inventors discovered
Pseudonocardia thermophila
as a microbe having a nitrile hydratase activity (JP-A-8-56684). The inventors further isolated nitrile hydratase from the same strain, and identified that this enzyme composed of an &agr;-subunit and a &bgr;-subunit. Still further, they isolated the nitrile hydratase gene from the same strain, clarified the amino acid sequence and the gene sequence thereof, and successfully produced a recombinant plasmid capable of expressing the gene
E. coli
(
Escherichia coli
) in a large amount and a transformant
E. coli
strain obtained through transformation with the same plasmid (European Patent Application Laid-Open No. 079310). By the way, with respect to
Pseudonocardia thermophila
, this strain is deposited as JCM3095 in International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan, and freely allotted to any person on demand.
Problems that the Invention is to Solve
The invention is to provide a protein participating in activation of nitrile hydratase derived from
Pseudonocardia thermophila
described in European Patent Application Laid-Open No. 0790310 which was clarified by the detailed analysis of a recombinant plasmid (pPT-DB1) capable of expressing this enzyme in a large amount in
E.coli
, a gene encoding the same, a recombinant plasmid containing the gene, a recombinant plasmid containing the gene and a nitrile hydratase gene, a transformant strain obtained through transformation with the recombinant plasmid, and a process for producing a corresponding amide compound from a nitrile compound using the transformant strain, and a culture solution obtained through incubation of the transformant strain, or treated products thereof.
Means for Solving the Problems
The inventors have analyzed in detail the base sequence of the DNA fragment derived from
Pseudonocardia thermophila
in recombinant plasmid pPT-DB1 introduced in MT-10822 strain described in JP-A-9-275978. Consequently, they have acquired the following five findings. First, it has been found that the third open reading frame (hereinafter called “ORF3”) different from nitrile hydratase structural genes (&agr;- and &bgr;subunit structural genes) is present in the DNA fragment in pPT-DB1 and ORF3 encodes a protein having a molecular weight of approximately 15,900. Second, they have produced plasmid pPT-D1 in which only the open reading frame of the &agr;-subunit (hereinafter called “ORF2”), the open reading frame of the &bgr;-subunit (hereinafter called “ORF1”) and ORF3 in pPT-DB1 are cloned, and have identified that transformant
E.coli
obtained through transformation with this plasmid has the nitrile hydratase activity. Third, they have constructed recombinant plasmid pPT-F1 having only ORF1 and ORF2 from pPT-DB1. As a result of measuring the nitrile hydratase activity of transformant
E.coli
obtained through transformation with this plasmid, the nitrile hydratase activity has not been detected from this
E.coli
. Meanwhile, the presence of polypeptide chains corresponding to the &agr;- and &bgr;-subunits has been observed in the cell. Fourth, plasmid pPT-G1 in which only the ORF3 region is cloned has been produced from pPT-DB1, and it has been identified that the nitrile hydratase activity is not observed in transformant
E.coli
obtained through transformation with this plasmid. Fifth, plasmid pPT-H1 in which the region having lacZ promoter, ORF1 and ORF2 is amplified through PCR and the resulting amplified DNA fragment is re-cloned downstream of the 3′-terminus of ORF3 of pPT-G1 has been produced from pPT-F1. The nitrile hydratase activity of transformant
E.coli
obtained through transformation with this plasmid has been measured, and the nitrile hydratase activity has consequently been detected in this
E.coli.
From these findings, the inventors have concluded that in order for
E.coli
, in which nitrile hydratase derived from
Pseudonocardia thermophila
has been introduced to exhibit the same activity, the presence of the ORF3 region is inevitable, and further that the presence of the translation product of ORF3 is inevitable in consideration of the second, third and fifth findings. Further, from the facts that the same enzyme is composed of the &agr;-subunit and the &bgr;-subunit (European Patent Application Laid-Open No. 0790310) and that even when the minimum DNA fragment having the three open reading frames ORF1 to ORF3 is introduced in
E.coli
, recombinant
E.coli
exhibits the nitrile hydratase activity (second finding), it has been concluded that the translation product of ORF3 participates in activation of nitrile hydratase. That is, it has been concluded that ORF3 is a gene locus encoding a protein that participates in activation of nitrile hydratase derived from
Pseudonocardia thermophila
. Consequently, the invention has been completed.
That is, the invention is to provide a protein participating in activation of nitrile hydratase derived from
Pseudonocardia thermophila
and a gene encoding the same. Further, the invention is to provide a recombinant plasmid containing the gene, a recombinant plasmid containing the gene and a nitrile hydratase gene, a transformant strain obtained through transformation with the recombinant plasmid, and a process for producing a corresponding amide compound from a nitrile compound using the transformant strain, and a culture solution obtained through incubation of the transformant strain or treated products thereof.


REFERENCES:
patent: 5910432 (1999-06-01), Ito et al.
patent: 0 790 310 (1997-08-01), None
patent: 8-56684 (1996-03-01), None
patent: WO 97/12964 (1997-04-01), None
Kobayashi et al. “Metalloenzyme nitrile hydratase: structure, regulation, and application to biotechnology.” Nature Biotechnology 16 (8):733-736, Aug. 1998.*
Hashimoto, Yoshihiro et al., “Nitrile Hydratase Gene from Rhodococcus sp. N-774 Requirement for Its Downstream Region for Efficient Expression.”Bioscience Biotechnology Biochemistry, JP, Japan Soc. for Bioscience, Biotechnology and Agrochem, Tokyo, vol. 58, No. 10, Oct. 1994, pp. 1859-1865.

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