&bgr;-xylosidase, nucleotide sequence encoding it, and use...

Details &bgr;-xylosidase, nucleotide sequence encoding it, and use... &bgr;-xylosidase, nucleotide sequence encoding it, and use...

1997-12-22

2001-10-09

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S320100, C435S325000, C435S069100, C536S023200, C536S063000

Reexamination Certificate

active

06300112

ABSTRACT:

The present invention relates to a novel peptide having &bgr;-xylosidase activity, to a nucleotide sequence encoding such a peptide, and to the use of such a &bgr;-xylosidase-like peptide.
BACKGROUND
Beta-xylosidase (1,4-&bgr;-D-xylan-xylohydrolase; EC 3.2.1.37) is one of the xylanolytic enzymes. Xylans are a major constituent of the cell walls of plants, and are second only to cellulose. They are abundantly found in most land plants, especially in agricultural by-products such as straw, wheat-bran, corn cobs, cotton seed, etc. Xylan is a complex polymer consisting of a &bgr;-1,4-linked xylose polymer with arabino-furanose, glucuronic acid, methylglucuronic acid and acetyl side-groups. Endoxylanase (EC 3.2.1.8) randomly cleaves the &bgr;-1,4-bonds in the xylan backbone to yield oligosaccharides, xylobiose and xylose. Beta-xylosidase cleaves terminal xylose units from the non-reducing end of the xylose oligomers resulting from endoxylanase activity. &agr;-Glucuronidase cleaves glucuronic acid side groups from backbone xylose units, whereas &agr;-L-arabinofuranosidases (EC 3.2.1.55) cleave arabinose units from the xylan backbone and acetylesterases (EC 3.1.1.6) remove the acetyl side-groups.
Beta-xylosidase is also effective in transglycosylation reactions wherein monosaccharide units or alcohols are attached to or cleaved from xylose units. Beta-xylosidase is rate-limiting in xylan hydrolysis (Dekker 1983, Poutanen and Puls 1988).
The hydrolysing and transglycosylating reactions of &bgr;-xylosidases are economically important for the breakdown and utilisation of agricultural waste material e.g. in the production of xylose, xylose oligomers and xylitol, which are useful as sweeteners in foodstuffs, candies and medicaments, especially as a sugar substitute. Also, the enzyme or its products can be used as bread improvers and in the beer brewing industry.
Beta-xylosidases have been isolated from various sources including bacteria and fungi. For example, the purification of &bgr;-xylosidase from
Aspergillus niger
was reported by Rodionova et al. (1983); the molecular weight was reported to be 253,000 on the basis of gel filtration and 122,000 on the basis of SDS electrophoresis, whereas its isoelectric point was at pH 4.9.
Three endoxylanases and one &bgr;-xylosidase were isolated from
Aspergillus awamori
by Kormelink et al. (1993); the &bgr;-xylosidase had a molecular weight of 110,000, a pH optimum of 6.5 and a temperature optimum of 70° C.
Beta-D-xylosidase from rumen fungus
Neocallirnastix frontalis
was reported by Garcia-Campayo and Wood (1993) and had an apparent molecular weight (gel filtration) of 150,000, a pH optimum of 6.4 and a temperature optimum of 37° C. Utt et al (1991) report the sequencing of the xylB of the ruminal bacterium
Butyrivibrio fibrisolvens
encoding both &bgr;-xylosidase and &agr;-arabinofuranosidase activities.
Known &bgr;-xylosidases have activity patterns that do not always correspond to the industrial needs. In particular it is often desirable that the enzyme has a high xylosidase specificity and low specificities for other substrates, such as glucosides and galactosides. Especially fungal &bgr;-xylosidases are highly advantageous for their activity levels and specificity patterns.
In order to be able to provide &bgr;-xylosidase-like enzymes having the desired activity patterns from the desired production organisms, sequence information of the &bgr;-xylosidase gene should be available. Up to now however, no sequence information on fungal &bgr;-xylosidases has been reported.
DESCRIPTION OF THE INVENTION
A novel &bgr;-xylosidase has now been found and its amino acid sequence as well as its encoding nucleotide sequence have been determined. The protein is denoted herein as xylD, whereas the encoding gene is denoted as xlnD. The primary structure of the novel &bgr;-xylosidase appears to be different form known &bgr;-xylosidase-like enzymes. Also, its activity pattern is different form known &bgr;-xylosidase-like enzymes, and its xylosidase activity is about two times higher than that of the &bgr;-xylosidase reported by Rodionova et al (supra).
Accordingly, the invention relates to a nucleotide sequence encoding a peptide having &bgr;-xylosidase activity and exhibiting at least 30% amino acid homology with the amino acid sequence shown in SEQ ID NO. 1 and described in SEQ ID NO. 3 or hybridising under stringent conditions with a nucleotide sequence shown in SEQ ID NO. 1, or a part thereof having at least 15, preferably at least 21, more preferably at least 24 or even at least 30 nucleotides encoding an amino acid sequence shown in SEQ ID NO. 1. By amino acid homology is meant here amino acid identity in the primary structure. Amino acid similarity is usually higher than the figures given for identity.
In this context, heterologous hybridisation conditions are as follows: hybridisation in 6×SSC (20×SSC per 1000 ml: 175.3 g NaCl, 107.1 g sodium citrate.-5H
2
O, pH 7.0), 0.1% SDS, 0.05% sodium pyrophosphate, 5* Denhardt's solution (100×Denhardt's solution per 500 ml: 10 g Ficoll-400, 10 g polyvinylpyrrolidone, 10 g Bovine Serum Albumin (Pentax Fraction V)) and 20 &mgr;g/ml denatured herring sperm DNA at 56° C. for 18-24 hrs followed by two 30 min. washes in 5×SSC, 0.1 % SDS at 56° C. and two 30 min. washes in 2×SSC, 0.1% SDS at 56° C.
The nucleotide sequence of the invention encodes a peptide having substantial &bgr;-xylosidase activity, i.e. it has &bgr;-xylosidase activity as its predominant enzymic activity, and thus may be used for the production of &bgr;-xylosidases or mutants thereof. The coding sequences may contain mutations (insertions, deletions or both) which serve to modify the structure and/or the activity of the expression product. For an active expression product, the minimum identity and/or the hybridisation characteristic as defined above should preferably be maintained. The nucleotide sequence may also correspond to regulating or signal sequences of &bgr;-xylosidases. For these uses, the nucleotide sequence comprises substantially the encoding or regulating sequences of the &bgr;-xylosidase. On the other hand, the nucleotide sequence may be used as a primer or probe in detecting &bgr;-xylosidase encoding sequences. For these uses, the sequence comprises at least 15, up to e.g. 60, consecutive nucleotides of the sequence of SEQ ID NO. 1.
The invention also relates to an isolated peptide having &bgr;-xylosidase activity and exhibiting at least 30% amino acid homology (identity) with the amino acid sequence shown in SEQ ID NO. 1 and described in SEQ ID NO. 3 or a part thereof, said peptide having no &bgr;-glucosidase and/or no &bgr;-galactosidase activity; essentially no &bgr;-glucosidase or &bgr;-galactosidase activity means that these activities are less than 2%, in particular less than 1% of the &bgr;-xylosidase activity. A peptide exhibiting at least 40%, preferably at least 60%, most preferably at least 75% amino acid identity with the amino acid sequence shown in SEQ ID NO. 1 and described in SEQ ID NO. 3 forms a preferred embodiment of the invention. Also part of the invention are peptides comprising a series of at least 8 contiguous amino acids of the amino acid sequence shown in SEQ ID NO.1. These can be produced by translating a nucleotide sequence as described above. Preferably the peptide has a contiguous series of at least 10, most preferably at least 12 amino acids from the sequence set forth in SEQ ID NO. 1and described in SEQ ID NO. 3.
The peptide according to the invention is especially from fungal origin, in particular from filamentous fungi, e.g. strains from the genera Aspergillus (especially
A. niger, A.niger
var
tubigensis, A. niger
var
awarnori, A. niger
var
kawachii, A. oryzae, A. sydowii, A. japonicus, A. aculeatus, A. ochraceus, A. terreus, A. fumigatus, A. versicolor, A. flavus, A. phoenicis, A. nidulans, A. foetidus
and
A. carbonarius
), Trichoderma (especially
T. reesei, T viride, T. longibrachiatum, T. harzianum, T. kongingii, T. pseudokongii
), Penicilliu

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