Enzyme with galactanase activity

Details Enzyme with galactanase activity Enzyme with galactanase activity

1998-08-21

2001-06-05

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S069100, C435S183000, C435S200000, C435S252300, C435S254100, C435S320100

Reexamination Certificate

active

06242237

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 0-4 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 0-3 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).
&bgr;-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).
The pH optimum of present known fungal galactanases are in the low pH range. Thus, Araujo et al. (J. Industrial Microbiology (1990) 6:171-178) describe a fungal galactanase (
Thielavia terrestris
) with a pH optimum of 5.8; and Hirofumi et al. (Kagaku to Kogyo (science) (science and Industry), (1990) vol. 64, no. 9, pp. 440-445) describe a fungal galactanase from
Aspergillus niger
with a pH optimum around 4.0.
Even though a number of &bgr;-1,4-galactanases have been purified, only one has been cloned and DNA sequenced. Thus WO 92/13945 describe cloning and DNA sequencing of a fungal &bgr;-1,4-galactanase (
Aspergillus aculeatus
)
The object of the present invention is to provide novel galactanases with a pH optimum in the neutral or alkaline range.
SUMMARY OF THE INVENTION
The present invention is based on the cloning and characterization of two DNA sequence obtained from fungal strains within the order of Sordariales, which both encode fungal enzymes exhibiting galactanase activity and have a pH optimum of at least 5.9.
The galactanases of the invention are the first known and purified fungal galactanases with a pH optimum above 5.8. This is presently believed to be advantageous for a number of industrial applications, such as in the animal feed industry (see e.g. a working example disclosed herein (vide infra)).
Accordingly, in a first aspect the invention relates to a fungal galactanase which has a pH optimum above 5.9.
Further the present inventors have identified two amino acid motifs in the amino acid sequences of the two galactanases obtained from Sordariales. It is presently believed that these motifs are characteristic for galactanases from Sordariales. Degenerated PCR DNA primers have been made based on above mentioned two motifs, and it is presently believed that it is possible to clone other galactanase from Sordariales exhibiting similar characteristic as the two described above. Especially the high pH optimum profile which is advantageous for a number of industrial applications (vide infra).
Accordingly in a further aspect the invention relates to a DNA construct obtained from a fungal strain of the order of Sordariales, encoding an enzyme exhibiting galactanase activity, which DNA sequence hybridizes under low stringency conditions with a probe which is a product of a PCR reaction with DNA isolated from
Humicola insolens
(DSM 1800) and/or with DNA isolated from
Myceliophthora thermophila
(CBS 117.65) and the following pairs of PCR primers:
“5′-CTA TTC GGA TCC AG(C/T) GA(C/T) AC(A/C) TGG GC(G/C) GA(C/T) CC(G/T) GC(G/T) C-3′” [SEQID NO 5] as the sense primer, and
“5′-CTA ATG TCT AGA (A/G)AT CCA (A/G/C/T)GC (A/G/C/T)GG (C/T)TC CCA (A/G)TA AAA-3′” [SEQID NO 6] as the anti-sense primer.
In a further aspect the invention relates to a DNA construct comprising a DNA sequence encoding a galactanase enzyme of the invention.
In a further aspect the invention provides a recombinant expression vector, which enables recombinant production of an enzyme of the invention. Thereby it is possible to make a mono-component galactanase composition, which is highly advantageous for a number of industrial applications.
In a further aspect the invention relates to an isolated enzyme exhibiting galactanase activity which comprises the partial amino acid sequence
a) Ser(S)-Asp(D)-Thr(T)-Trp(W)-Ala(A)-Asp(D)-Pro(P)-Ala(A)-His(H) and/or Phe(F)-Tyr(Y)-Trp(W)-Glu(E)-Pro(P)-Ala(A)-Trp(W)-Ile(I).
Finally the invention relates to an isolated substantially pure biological culture of the
Saccharomyces cerevisiae
strain DSM No. 9983 harbouring a galactanase encoding DNA sequence (shown in SEQ ID No 1) (the galactanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in
Saccharomyces cerevisiae
DSM 9983) derived from a strain of the filamentous fungus
Myceliophthora thermophila,
or any mutant of said
Saccharomyces cerevisiae
strain having retained the galactanase encoding capability; and
the invention relates to an isolated substantially pure biological culture of the
Saccharomyces cerevisiae
strain DSM No. 9976 harbouring a galactanase encoding DNA sequence (shown in SEQ ID No 3) (the galactanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in
Saccharomyces cerevisiae
DSM 9976) derived from a strain of the filamentous fungus
Humicola insolens
or any mutant of said
Saccharomyces cerevisiae
strain having retained the galactanase encoding capability.
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 preparartion which is at least about 20% pure, preferably at least about 40% pure, more preferably

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