Bacterial galactanases and use thereof

Details Bacterial galactanases and use thereof Bacterial galactanases and use thereof

2000-02-11

2001-12-18

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S069100, C435S183000, C435S200000, C435S252300, C435S320100, C536S023200

Reexamination Certificate

active

06331426

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to bacterial galactanase enzymes for use in different industrial applications, such as in the textile, detergent and cellulose fiber processing industries, and in particular to methods for modifying animal feed using such enzymes.
2. Description of the Related Art
Galactans and arabinogalactans are present in most plants as components of pectic hairy regions and can be found in high quantities e.g. in soy plant seed and in potatoes. Another good source for highly purified galactans and arabinogalactans is the water-soluble polysaccharide extracted with alkali from lupin fibre. This substrate can be treated with arabinofuranosidase (EC 3.2.1.55) resulting in a galactan with a very high content of galactose (more than 91%); such a substrate can be obtained from Megazyme, Australia.
Galactans and arabinogalactans are usually attached to O-4 of rhamnose residues in the rhamnogalacturonan backbone of the hairy region. The distribution and composition of the side chains vary considerably between different cell types and physiological states, but in general about half of the rhamnosyl units in the rhamnogalacturonan regions have side chains attached. The galactan side chains 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 the name 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 water-binding 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).
Beta-1,4-galactanases (EC 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).
WO 92/13945 describes cloning and DNA sequencing of a fungal beta-1,4-galactanase from
Aspergillus aculeatus.
WO 97/32014 describes cloning and DNA sequencing of fungal beta-1,4-galactanase from Humicola insolens and
Myceliophthora thermophilum.
WO 97/32013 describes cloning and DNA sequencing of fungal beta-1,4-galactanase from
Meripilus giganteus.
Braithwaite et al., BIOCHEMISTRY Vol. 36 , No. 49 pp. 15489-15500 (1997) disclose a galactanase from Pseudomonas fluorescens ssp. cellulose which is a retaining family
53
glycosyl hydrolase in which e161 and e270 are the catalytic residues.
WO 91/18521 describes a feed composition comprising, as a source of carbohydrates, a mannan-containing hemicellulose selected from soybeans, corn and alfalfa, as well as a mannanase that catalyzes the degradation of the mannan-containing hemicellulose.
Nakano et al., Eur. J. Biochem. 193(1): 61-67 (1990) describes the purification and characterization of an exo-1,4-&bgr;-galactanase from a strain of
Bacillus subtilis.
The database entries from the publicly available databases EMBL and Swissprot listed below refer to sequences with homology to the galactanases described herein:
Species
Description
wissprot/TREMBL
EMBL Entry
Bacillus
Hypothetical
007013, 032260
Z94043, Z99121
subtilis
protein
Bacillus
Hypothetical
P48843
L03425
circulans
protein
The galactanases in the list above and the galactanases of the invention belong to family
53
of glycosyl hydrolases (Henrissat B., A classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 280: 309-316 (1991)).
In spite of the state of the art e.g. as disclosed above, there remains a need for galactanase enzymes with improved activity for a number of different purposes. The object of the present invention is to provide galactanase enzymes with a high galactanase activity for use in industrial applications, such as the textile, detergent and cellulose fiber processing industries, and in particular for the modification of animal feed.
SUMMARY OF THE INVENTION
The inventors have now found that certain bacterial galactanases, in particular derived from a number of Bacillus species, have advantageous properties that make them suitable for use in the modification of animal feed and in other industrial applications.
In one aspect, the present invention relates to a method for modifying animal feed, the method comprising adding to the animal feed at least one galactanase enzyme comprising at least one consensus amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO 1-6:
(SEQ ID NO.1)
Y-x-x-T-x-E-x-x-D-G

(SEQ ID NO.2)
N-x-x-(M/L)-F-D-F-x-G-x-x-L-x-S

(SEQ ID NO.3)
S-Y-Y-P-x-W-H-G

(SEQ ID NO.4)
YD(S/A)NGNGYGG

(SEQ ID NO.5)
VGP(K/A) (T/H) (Q/R) (I/L)EKNK(V/A)LWETYGS-
GWA(S/T) SYAAEYDPEDAGKW(Y/F)GGSAV

(SEQ ID NO.6)
GG(F/L)AGETD
where x represents any amino acid.
Further aspects of the invention relate to methods for modifying animal feed using other galactanase enzymes as defined below, as well as a method for obtaining a DNA sequence s encoding a galactanase enzyme or a portion thereof, and isolated polynucleotide molecules encoding polypeptides having galactanase activity.
The inventors found novel enzymes having substantial galactanase activity, i.e. an enzyme exhibiting galactanase activity which may be obtained from a bacterial strain of the genus Bacillus, more specifically of the strain
Bacillus licheniformis
ATCC 14580 or
Bacillus agaradhaerens
AC13 (DSM 8721), and have succeeded in identifying DNA sequences encoding such enzymes. The DNA sequences and the deduced amino acid is sequences are listed in the sequence listing as SEQ ID NO. 7 and 8, as well as SEQ ID NO. 11 and 12, respectively.
In a further aspect of the invention there is provided an expression vector comprising a polynucleotide sequence as defined in the previous aspects.
Within yet another aspect of the present invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses the polypeptide encoded by the DNA segment.
A further aspect of the present invention provides an isolated polypeptide having galactanase activity selected from the group consisting of (a) polypeptide molecules comprising an amino acid sequence as shown in SEQ ID NO.8 from residue 1 to residue 399; and (b) polypeptide molecules that are ar least 80% identical to the amino acids of SEQ ID NO.8 from amino acid residue 1 to amino acid residue 399.
One other aspect of the present invention provides an isolated polypeptide having galactanase activity selected from the group consisting of (a) polypeptide molecules comprising an amino acid sequence as shown in SEQ ID NO.12 from residue 1 to residue 245; and (b) polypeptide molecules that are ar least 80% identical to the amino acids of SEQ ID NO.12 from amino acid residue 1 to amino acid residue 245.
Within another aspect of the present invention there are provided methods for producing a polypeptide according to the invention comprising culturing a cell into which has been introduced an expression vector as disclosed above, whereby said cell expresses a polypeptide encoded by the DNA segment and recovering the polypeptide.
Within another aspect of the present invention there is provided an enzyme preparation comprising a

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