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,...

2002-04-05

2004-09-21

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S135000, C435S252300, C435S320100, C435S440000, C435S006120, C536S023200

Reexamination Certificate

active

06794170

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 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).


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