Cellulose degrading enzymes of aspergillus

Details Cellulose degrading enzymes of aspergillus Cellulose degrading enzymes of aspergillus

2000-04-04

2003-05-06

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S099000, C426S622000, C426S052000

Reexamination Certificate

active

06558937

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to polypeptides which have cellulolytic activity and which can be derived from fungae; and to polynucleotides which encode the polypeptides and vectors which can express the polypeptides in a host cell. The polypeptides can be used to degrade cellulose. In particular they can be used in the production or processing of food, animal feed, wood pulp, paper and textiles.
BACKGROUND OF THE INVENTION
Cellulose is a linear polysaccharide of glucose residues connected by beta-1,4 linkages. In nature, cellulose is usually associated with other compounds, such as hemicelluloses or lignin.
Three different classes of enzymatic activity have been shown to be required for the complete degradation of cellulose into glucose, viz. endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21).
Cellobiohydrolases attack cellulose either from the reducing or the non-reducing ends of the cellulose polymer and yield cellobiose (a glucose dimer) as the major product. Two types of cellobiohydrolase are known, cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II). CBH I attacks cellulose from the reducing end of the cellulose polymer, and CBH II from the non-reducing end.
Cellobiohydrolase I and II from
Trichoderma reesei
have been described in EP-B-0137 280 and U.S. Pat. No. 4,894,338, respectively. CBH I has also been identified in
Agaricus bisporus, Phanerochaete chrysosporium, Trichoderma viride
and
Humicola grisea
. Depending on both the natural origin and the previous treatment cellulose exists in many varieties differing in crystallinity, fibre composition, fibre length, fibre thickness. For fast and complete degradation of crystalline cellulose CBH and endoglucanases work synergistically (Teeri, TT., TIBTECH May 15, 1997, p. 160-167).
SUMMARY OF THE INVENTION
The invention seeks to provide novel polypeptides with CBH activity which can be used to degrade cellulose. Accordingly, the invention provides in a first aspect a polypeptide which comprises the sequence of SEQ ID No:8 (CBH A) or SEQ ID No:10 (CBH B), a sequence substantially homologous to either sequence, or a fragment of any of these sequences.
The first aspect of the invention also provides a polypeptide which is a CBH from Aspergillus.
The first aspect additionally provides a polypeptide which:
(i) has CBH activity;
(ii) has an activity of at least 50% of the maximum activity over the pH range from 3 to 5;
(iii) has an optimum activity at a temperature which is greater than 50° C.; and optionally
(iv) does not have a cellulose binding domain or a linker peptide.
A second aspect of the invention provides a polynucleotide which encodes the polypeptide of the first aspect, and a polynucleotide capable of selectively hybridising to SEQ ID No:5, 9 or a complement thereof.
A third aspect of the invention provides a vector which comprises a nucleotide of the second aspect.
A fourth aspect of the invention provides a host cell which comprises a polynucleotide or vector of the second or third aspects.
A fifth aspect of the invention provides a composition comprising a polypeptide of the invention and, for example, one or more enzyme(s).
A sixth aspect of the invention provides a use of the polypeptide or composition of the first or fifth aspects to degrade cellulose.
DETAILED DESCRIPTION OF THE INVENTION
One of the advantages of the present invention is that cellobiohydrolases may be produced which are free from endoglucanases. Since CBHs, unlike endoglucanases, do not significantly affect fibre strength they are more suitable than crude cellulase complexes for fibre modification in applications were strength is important, such as in textile and paper manufacture and finishing.
Another advantage is that the availability of cloned CBHs and endoglucanases (WO 97/13862) makes it possible to design tailor-made combinations of CBHs and endogucanases for specific applications.
Yet another advantage of the invention is that the polypeptides of the invention have optimum activity at low pH, unlike known cellobiohydrolases. Therefore, the polypeptides of the invention are well suited for use in industrial processes which are performed at low pH.
Polypeptides
The polypeptide of the invention typically has CBH activity. Generally such an activity would be the activity defined by E.C. 3.2.1.91. The term “CBH activity” includes the ability to produce cellobiose from cellulose (as a substrate).
The CBH activity may be CBH I-like activity and/or CBH II-like activity. The cellulose which is acted on by the polypeptide of the invention may be crystalline cellulose. The level of CBH activity of the polypeptide of the invention may be the same as, substantially the same as, or higher or lower than the polypeptide shown in SEQ ID No:8 or 10.
The polypeptide of the invention may or may not comprise a cellulose binding domain. Such a domain is often linked to the catalytic domain via a linker peptide in prior art CBH enzymes. Therefore if the polypeptide of the invention does not comprise a cellulose binding domain then generally it will not comprise a linker peptide either.
The polypeptide of the invention may comprise the sequence of SEQ ID No:8 or 10 or a substantially homologous sequence. The polypeptide may be 65% homologous to SEQ ID No:8 or 10, preferably 80% or 90%, and more preferably at least 95% homologous thereto over the length of SEQ ID No:8 or 10.
The polypeptide of the invention may be derived from a fungus, such as a filamentous fungus. Preferably the (filamentous) fungus is of the species Aspergillus oryzae, Aspergillus sojae, Aspergillus nidulans, species from the
Aspergillus niger
Group (as defined by Raper and Fennell, The Genus
Aspergillus
, The Williams & Wilkins Company, Baltimore, pp 293-344, 1965), specifically including but not limited to
Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus japonicus
and
Aspergillus ficuum.
In the context of the invention the term “derived from” includes polypeptides which naturally occur in or are produced by these fungae and therefore can be obtained from these fungae, or fragments of such polypeptides. The term also includes polypeptides which are substantially homologous (such as at least 65% homologous to SEQ ID No:8 or 10) to these naturally occurring polypeptides of the invention. Thus the invention includes polypeptides which are not naturally occurring.
The polypeptide of the invention may co-purify with an &agr;-arabinofuranosidase (see for e.g. Example 1). The polypeptide of the invention may thus co-elute with an &agr;-arabinofuranosidase. The &agr;-arabinofuranosidase may be detected using the chromogenic substrate 4- methylumbelliferyl-&agr;-L-arabinofuranoside. The &agr;-arabinofuranosidase may be a fungal &agr;-arabinofuranosidase, such as one from an Aspergillus species, e.g. Aspergillus niger. The &agr;-arabinofuranosidase may be one from-any of the genera or species of fungae mentioned above from which the polypeptide of the invention may be derived.
A polypeptide of the invention may comprise:
(a) a polypeptide comprising the sequence of SEQ ID No:8 or 10; or
(b) a polypeptide from Aspergillus which is a CBH or has CBH activity; or
(c) a homologue or an allelic variant of any of the polypeptides of (a) or (b) from a fungus, an Aspergillus species, from Aspergillus niger or from any of the genera or species of fungus mentioned above from which the polypeptide of the invention may be derived; or
(d) a homologue which is at least 65% homologous to (a), (b) or (c).
Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as “hard homology”).
The polypeptide of the invention or fragments thereof may be used to identify cellobiohydrolases in other organisms. For example, they may be used for the production of antibodies. The sequence of the polyp

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