Nucleic acid fragment and vector comprising a halogenase,...

Details Nucleic acid fragment and vector comprising a halogenase,... Nucleic acid fragment and vector comprising a halogenase,...

1999-10-29

2003-05-20

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S131000, C435S252300, C435S320100, C435S440000, C536S023200

Reexamination Certificate

active

06566110

ABSTRACT:

The invention relates to a process for the enzymatic halogenation of chemical compounds. The invention further relates to a nucleic acid fragment, a vector and organisms comprising a halogenase or a halogenase gene.
Halogenation reactions have long been known in chemical synthesis. They are used to prepare a large number of halogenated organic compounds. A disadvantage of these synthetic reactions is that special plants are required for the synthesis. These plants must be specially protected against corrosion because the reaction products are frequently very corrosive. There is frequently formation during the synthesis not only of the required product but also of byproducts leading to contamination of the product. If these byproducts cannot be tolerated in the final product, they must undergo elaborate removal. Many byproducts are moreover toxic. Dioxin formation may occur in a number of reactions.
One alternative to chemical halogenation is enzymatic synthesis. It is generally very selective, i.e. generally no byproducts are formed.
The literature discloses enzymes, called haloperoxidases, which halogenate organic compounds in the presence of a halogen ion and hydrogen peroxide. Examples of such enzymes are the haloperoxidase from
Streptomyces aureofaciens
(Kren et al., Liebigs Ann./Red., 1997, 11: 2379-83), from
Rhodococcus erythropolis
(Schrijver et al., Appl. Environ. Microbiol., 1997, 63, 5: 1911-1916), from
Amycolatopsis orientalis
(van Wageningen et al., Chem. Biol., 1998, 5: 155-162), from
Caldariomyces fumago
(Hohaus et al., Angew. Chem. Int. Ed. Engl., 1997, 36, No. 18: 2012-2013), from
Streptomyces lividans
or
Serratia marcescens
(von Pée, K. H., Ann. Rev. Microbiol., 1996, 50: 375-99).
It is not absolutely clear whether the halogenation reaction is the actual enzymatic reaction of these haloperoxidases or whether it is only a side reaction. The enzymes very often show low substrate and cosubstrate affinity and a specificity which is low for enzymes.
Besides the haloperoxidases, other halogenating enzymes are disclosed in the literature. Thus, Kirner et al. (J. Bacteriol., 1998, Vol. 180, No. 7, p. 1939-1943) and Hohaus et al. (Angew. Chem. Int. Ed. Engl., 1997, 36, No. 18, 2012-2013) describe a halogenase which introduces a chlorine atom into position 7 of tryptophan.
Further enzymes are described in Hammer et al. (Appl. Environ. Microbiol., 1997, Vol. 63, No. 6, 2147-2154), de Schrijver et al. (Appl. Environ. Microbiol., 1997, Vol. 63, No. 5, 1911-1916), Nowak-Thompson et al. (J. Bacteriol., 1999, 181: 2166-2174), Solenberg et al. (Chem. Biol., 4, 1997: 195-202) and Dairi et al. (Biosci., Biotechnol. Biochem, 59, 1995, 1099-1106).
A 9.9 kb-long
Amycolatopsis mediterranei
DNA fragment is to be found in GenBank (Y 16952). Pelzer et al. describe two functions for this DNA in the GenBank entry. It codes for oxygenases and glycosyltransferases. No other functions are mentioned.
None of the enzymes has previously been used for the industrial preparation of halogenated organic compounds. It therefore was an object of the present invention to provide an enzyme for the industrial synthesis of halogenated organic compounds.
We have found that this object is achieved by the halogenation process according to the invention, which comprises halogenating a chemical compound in the presence of a halogenase, where the halogenase is
(a) encoded by the sequence specified in SEQ ID NO: 1 or a sequence derived therefrom on the basis of the degeneracy of the genetic code, or is
(b) encoded by a nucleic acid sequence which codes for a functional fragment on (a) or
(c) by a sequence which hybridizes with (a) or (b) under standard conditions, or is
(d) encoded by a sequence which has more than 30% identity or more than 60% similarity with the sequence specified under (a).
The halogenase used in the process according to the invention can be isolated from organisms in a manner known to the skilled worker. It can preferably be isolated from organisms which synthesize halogenated compounds, for example glycopeptide antibiotics. Examples of such organisms are to be found among the bacteria and eukaryota such as algae such as Ascophyllum or Synechocystis, lichens, fungi such as Caldariomyces, yeasts and bacteria such as Gram-positive bacteria such as the Actinomycetales, the Bacillales or Gram-negative bacteria such as Pseudomonas. The halogenase can also be isolated advantageously from nocardioform Actinomycetes or Streptomycetes.
It is possible and particularly preferred for the halogenase or halogenases (singular and plural are to be regarded as synonymous hereinafter and for the application) to be isolated from glycopeptide antibiotic-producing members of the family of Pseudonocardiaceae and related bacteria, such as from the genera Pseudonocardia, Saccharomonospora, Saccharopolyspora. Amycolatopsis, Thermocrispum, Pseudoamycolata, Kibdelosporangium, Amycolata, Actinopolyspora, Actinokineospora or Actinobispora, examples which may be mentioned being the genera and species
Nocardia mediterranei, Amycolatopsis mediterranei, Streptomyces mediterranei
, Nocardia spec., Amycolatopsis spec., Streptomyces spec.,
Nocardia orientalis, Amycolatopsis orientalis, Streptomyces orientalis, Streptomyces toyocaensis
or
Streptomyces viridochromogenes
. Mention may very particularly preferably be made of
Amycolatopsis orientalis
C329.4, A82846 and ATCC19795 and
Amycolatopsis mediterranei
DSM 5908. The enzyme can also be isolated advantageously from organisms of the genus Streptomyces, specifically the genus and species
Streptomyces mediterranei.
The halogenase or the nucleic acid coding for the halogenase can moreover be isolated from the genera Rhodococcus, Thermomonospora, Bacillus, Serratia, Actinosporangium, Actinomadura, Actinoplanes or Micromonospora.
The gene for the halogenase can be isolated from a gene bank from these organisms by various techniques known to the skilled worker. One of these techniques is, for example, the “fishing” for the gene from the gene bank via hybridization with the sequence specified in SEQ ID NO: 1 or parts of this sequence.
An appropriate experimental protocol is to be found, for example, in the textbooks by Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual, Cold Spring, Harbor Laboratory Press, by F. M. Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, or by D. M. Glover et al., DNA Cloning, Vol. 1 (1995), IRL Press (ISBN 019-963476-9). A further method which may be mentioned is the PCR cloning technique (see examples).
Pelzer et al. (J. Biotechnol., 56 [1997], 115-128) describe in section 2.4 the preparation of a gene (DNA) library for
Amycolatopsis mediterranei
. Further methods for other organisms are known to the skilled worker and can be found in textbooks such as Sambrook et al. (1989).
It is possible in principle to use for the process according to the invention all organisms comprising at least one copy of the halogenase gene.
These may be organisms which naturally contain the gene, or which contain the gene in cloned form. It is moreover possible for the gene to have been introduced into the natural host organism or else into another organism.
Host organisms preferably used are those having an advantageous satisfactory or high tolerance of organic solvents, of the substances to be reacted, of elevated temperatures and of altered pressures. Organisms advantageously used are those into which at least one halogenase gene has been introduced.
Hosts which meet these and other advantageous conditions can be found in virtually all regions of the animal, plant and bacterial kingdoms.
Advantageous microorganisms which can act as host organisms can be found among the fungi, yeasts and bacteria.
Prokaryotic host organisms suitable for the process according to the invention are in principle all Gram-negative or Gram-positive bacteria. Examples of Gram-negative bacteria which may be mentioned are the Enterobacteriaceae such as the genera Escherichia, Aerobacter, Enterobacter, Citrobacter, Sh

Affiliated with

Also associated with

Rating

Nucleic acid fragment and vector comprising a halogenase,... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Nucleic acid fragment and vector comprising a halogenase,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid fragment and vector comprising a halogenase,... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3082628