Enzyme with galactanase activity

Details Enzyme with galactanase activity Enzyme with galactanase activity

1998-08-04

2001-12-11

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S069100, C435S183000, C435S200000, C435S202000, C435S206000, C435S209000

Reexamination Certificate

active

06329185

ABSTRACT:

FIELD OF INVENTION
The present invention relates to an enzyme with galactanase activity, a DNA construct encoding the enzyme with galactanase activity, a method of producing the enzyme, an enzyme composition comprising said enzyme with galactanase activity, and the use of said enzyme and enzyme composition for a number of industrial applications.
BACKGROUND OF THE INVENTION
Galactans and arabinogalactans are present in most plants as components of pectic hairy regions. They are usually attached to O-4 of rhamnose residues in the rhamnogalacturonan backbone of the hairy region. The distribution and composition of the sidechains vary considerably between different cell types and physiological states, but in general about half of the rhamnosyl units in the rhamnogalacturonan regions have sidechains attached. The galactan sidechains are in most plants type 1 galactans, which are composed of &bgr;-1,4 linked galactopyranose with some branching points and a length of up to 60 saccharide units (DP60). Arabinofuranose residues or short arabinan oligomers can be attached to the galactan chain at the O-3 of the galactosyl unit, thus named arabinogalactan. Galactans (or arabinogalactans) have an important function in the primary cell wall, where they interact with other structural components of the cell wall such as xyloglucans or arabinoxylans. Thus they possibly serve to anchor the pectic matrix in the cell wall. Furthermore, they increase the hydration and waterbinding capacity and decrease inter-chain association between pectin polymers which is thought to be of importance for modulation of porosity and passive diffusion. (Carpita & Gibeaut, 1993, Plant J.,3, 1-30; O'Neill et al.,1990, Methods in Plant Biochemistry, 415-441; Selvendran, 1983, The Chemistry of Plant Cell Walls. Dietary Fibers; Hwang et al., Food Hydrocolloids, 7, 39-53; Fry, 1988, The growing Plant Cell Wall: Chemical and Metabolic Analysis).
b-1,4-galactanases (E.C.3.2.1.89) degrade galactans (and arabinogalactans) and have been purified from a variety of microbial sources (Nakano et al., 1985, Agric. Biol. Chem.,49, 3445-3454; Emi & Yamamoto, 1972, Agric. Biol. Chem., 36, 1945-1954; Araujo & Ward, 1990, J. Ind. Microbiol., 6, 171-178; Van De Vis et al., 1991, Carbohydr. Polym., 16, 167-187).
Even though a number of b-1,4-galactanases have been purified, only one has been cloned and DNA sequenced.
WO 92/13945 decribe cloning and DNA sequencing of a fungal b-1,4-galactanase (
Aspergillus aculeatus
)
SUMMARY OF THE INVENTION
According to the present invention, the inventors have now for the first time succeeded in isolating and characterizing a DNA sequence, from a Basidiomycota fungus, which encodes an enzyme exhibiting galactanase activity, thereby making it possible to prepare a mono-component galactanase composition.
Accordingly, in a first aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting galactanase activity, which DNA sequence comprises
(a) the galactanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in
Escherichia coli
DSM 10355;
(b) the DNA sequence shown in positions 1-1026 in SEQ ID NO 1 or more preferably 55-1026 or its complementary strand;
(c) an analogue of the DNA sequence defined in (a) or (b) which is at least 70% homologous with said DNA sequence;
(d) a DNA sequence which hybridizes with the DNA sequence shown in positions 1-1026 in SEQ ID NO 1 at low stringency;
(e) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with the sequences of (b) or (d), but which codes for a polypeptide having the same amino acid sequence as the polypeptide encoded by any of these DNA sequences; or
(f) a DNA sequence which is a allelic form or fragment of the DNA sequences specified in (a), (b), (c), (d), or (e).
The full length DNA sequence encoding a galactanase has been derived from a strain of the filamentous fungus
Meripilus giganteus
and has been cloned into plasmid pYES 2.0 present in the
Escherichia coli
strain DSM No. 10355.
Said galactanase encoding DNA sequence harboured in
Escherichia coli
DSM 10355 is believed to have the same sequence as that presented in SEQ ID NO 1. Accordingly, whenever reference is made to the galactanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in DSM 10355 such reference is also intended to include the galactanase encoding part of the DNA sequence presented in SEQ ID NO 1.
Accordingly, the terms “the galactanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in DSM 10355” and “the galactanase encoding part of the DNA sequence presented in SEQ ID NO 1” may be used interchangeably.
In further aspects the invention provides an expression vector harbouring the DNA construct of the invention, a cell comprising said DNA construct or said expression vector and a method of producing an enzyme exhibiting galactanase activity, which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
In a still further aspect the invention provides an isolated enzyme exhibiting galactanase activity selected from the group consisting of:
(a) a polypeptide encoded by the galactanase enzyme encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in
Escherichia coli
DSM 10355;
(b) a polypeptide comprising an amino acid sequence as shown in positions 19-342 of SEQ ID NO 2;
(c) an analogue of the polypeptide defined in (a) or (b) which is at least 70% homologous with said polypeptide; and
(d) an allelic form or fragment of (a), (b) or (c).
In a still further aspect, the present invention relates to the use of an enzyme or an enzyme composition of the invention for various industrial applications.
Finally the invention relates to an isolated substantially pure biological culture of the
Escherichia coli
strain DSM No. 10355 harbouring a galactanase encoding DNA sequence (the galactanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in
Escherichia coli
DSM 10355) derived from a strain of the filamentous fungus
Meripilus giganteus
, or any mutant of said
E. coli
strain having retained the galactanase encoding capability; and to an isolated substantially pure biological culture of the filamentous fungus
Meripilus giganteus
CBS No. 521.95, from which the DNA sequence presented as SEQ ID No. 1 has been derived.
DEFINITIONS
Prior to discussing this invention in further detail, the following terms will first be defined.
“A DNA construct”: The term “A DNA construct”, refers to a DNA sequence cloned in accordance with standard cloning procedures used in genetic engineering to relocate a segment of DNA from its natural location to a different site where it will be reproduced. The cloning process involves excision and isolation of the desired DNA segment, insertion of the piece of DNA into the vector molecule and incorporation of the recombinant vector into a cell where multiple copies or clones of the DNA segment will be replicated.
The “DNA construct” of the invention may alternatively be termed “cloned DNA sequence” or “isolated DNA sequence”.
“Obtained from”: For the purpose of the present invention the term “obtained from” as used herein in connection with a specific microbial source, means that the enzyme is produced by the specific source, or by a cell in which a gene from the source have been inserted.
“An isolated polypeptide”: As defined herein the term, “an isolated polypeptide” or “isolated galactanase”, as used about the galactanase of the invention, is a galactanase or galactanase part which is at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE. The term “isolated polypeptide” may alternatively be termed “purified polypeptide”.
“Homologous impurities”: As used herein the term “homologous impurities” means any impurity (e.g.

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