Enzyme

This application is a divisional of U.S. patent application Ser. No. 10/237,386 filed Sep. 9, 2002, which is a continuation-in-part of PCT/IB01/00426, filed Mar. 8, 2001, designating the U.S., published Sep. 13, 2001 as WO 01/66711 and claiming priority from GB 0005585.5 filed Mar. 8, 2000 and GB 0015751.1 filed Jun. 27, 2000. All of the above-mentioned applications, as well as all documents cited herein and documents referenced or cited in documents cited herein, are hereby incorporated by reference.

The present invention relates to mutant xylanase enzymes having an altered sensitivity to xylanase inhibitors. The present invention also relates to the use of these mutant enzymes in processing plant materials.

For many years, endo-β-1,4-xylanases (EC 3.2.1.8) (referred to herein as xylanases) have been used for the modification of complex carbohydrates derived from plant cell wall material. It is well known in the art that the functionality of different xylanases (derived from different micro organisms or plants) differs enormously.

Comprehensive studies characterising the functionality of xylanases have been done on well characterised and pure substrates (Kormelink et al., 1992). These studies show that different xylanases have different specific requirements with respect to substitution of the xylose backbone of the arabinoxylan (AX). Some xylanases require three un-substituted xylose residues to hydrolyse the xylose backbone; others require only one or two. The reasons for these differences in specificity is thought to be due to the three dimensional structure within the catalytic domains, which in turn is dependent on the primary structure of the xylanase, i.e. the amino acid sequence. However, the translation of these differences in the amino acid sequences into differences in the functionality of the xylanases, has up until now not been documented when the xylanase acts in a complex environment, such as plant material.

The xylanase substrates found in wheat (wheat flour), have traditionally been divided into two fractions: The water un-extractable AX (WU-AX) and the water extractable AX (WE-AX). The WU-AX:WE-AX ratio is approx. 70:30 in wheat flour. There have been numerous explanations as to why there are two different fractions of AX. The older literature (D\'Appolonia and MacArthur (1976) and Montgomery and Smith (1955)) describes quite high differences in the substitution degree between WE-AX and WU-AX. The highest degree of substitution was found in WE-AX. This was used to explain why some of the AX was extractable. The high degree of substitution made the polymer soluble, compared to a lower substitution degree, which would cause hydrogen bonding between polymers and consequently precipitation.

The difference between the functionality of different xylanases has been thought to be due to differences in xylanase specificity and thereby their preference for the WU-AX or the WE-AX substrates.

In some applications (e.g. bakery) it is desirable to produce high molecular weight (HMW) soluble polymers from the WU-AX fraction. Such polymers have been correlated to a volume increase in bread making (Rouau, 1993; Rouau et al., 1994 and Courtin et al., 1999).

In other applications it is desirable to modify the HMW WU-AX, making the molecular weight lower, reducing their hydrocolloid effect and hence water-binding in the product (crackers, flour separation, etc.)

These different applications require different functionalities of the xylanases used to do the job. As mentioned above, the difference in functionality has been explained by the different substrate specificities of the xylanases.

By contrast to earlier studies, we have now shown that other factors are more important in determining xylanase functionality than the substrate specificity of the xylanases determined on pure well-characterised substrates. The data presented herein show that endogenous xylanase inhibitors dictate the functionality of the xylanases currently used in, for example, wheat flour systems. This means that a xylanase that normally modifies the WU-AX, giving increased dough liquid viscosity in a wheat flour system, has a different functionality if the endogenous xylanase inhibitor is absent in the wheat flour. Thus, our findings indicate that the design and application of uninhibited xylanases, for example, using site-directed mutagenesis could be a way to mimic the absence of xylanase inhibitors in various plant materials, giving new xylanases with completely new functionality. Such xylanases would be very effective in applications where a reduction in viscosity is required. The uninhibited xylanase would act rapidly on the AX, and be primarily influenced by its specific activity, rather than by endogenous inhibitors. From our studies, we consider that the inhibitory effects are likely to be far more important than the specific activity. Indeed our results show for the first time that there are 10 to 50 fold differences in inhibition levels between the family 11 xylanases.

Furthermore, we have gone on to design and test a series of xylanases modified by site-directed mutagenesis to demonstrate that xylanases can be produced that have reduced sensitivity to xylanase inhibitors present in plant materials. In particular, we have identified a number of residues in family 11 xylanases which influence the degree of inhibition of the xylanase.

Thus, it will be possible to produce variant xylanases having reduced sensitivity to xylanase inhibitors and hence altered functionality. This will, for example, allow a reduction in the amount of xylanase required in a number of applications such as animal feed, starch production, bakery, flour separation (wetmilling) and, paper and pulp production.

Accordingly, the present invention provides a variant xylanase polypeptide, or fragment thereof having xylanase activity, comprising one or more amino acid modifications such that the polypeptide or fragment thereof has an altered sensitivity to a xylanase inhibitor as compared with the parent enzyme.

Here, the “parent enzyme” is the xylanase enzyme from which the variant xylanase enzyme is derived or derivable. With respect to the term “derivable”, the variant need not necessarily be derived from the parent enzyme. Instead, the variant could be prepared, for example, by use of recombinant DNA techniques that utilise nucleotide sequence(s) encoding said variant xylanase sequence—i.e. here the nucleotide sequence(s) are similar to mutated nucleotide sequence(s) but they are not prepared by mutation of the parent nucleotide sequence(s). The variant may even be prepared by chemically modifying a parent enzyme.

For some embodiments the parent enzyme is the wild type enzyme. The term “wild type” is a term of art understood by skilled persons and includes a phenotype that is characteristic of most of the members of a species occurring naturally and contrasting with the phenotype of a mutant. Thus, in the present context, the wild type enzyme may be a form of the enzyme naturally found in most members of the relevant species. Generally, the relevant wild type enzyme in relation to the variant polypeptides of the invention is the most closely related corresponding wild type enzyme in terms of sequence homology. For example, for the particular mutant xylanases described in the examples, the corresponding wild type enzyme is the wild type B. subtilis xylanase A, more specifically the wild type B. subtilis xylanase A published by Paice et al., 1986 and shown as SEQ I.D. 1. However, where a particular wild type sequence has been used as the basis for producing a variant polypeptide of the invention, this will be the corresponding wild type sequence regardless of the existence of another wild type sequence that is more closely related in terms of amino acid sequence homology.

For some embodiments, preferably the variant polypeptide is derived from a family 11 xylanase.

One of our surprising findings is that in our studies so far a mutation in the xylanase active site has no measurable effect on inhibition against the xylanase inhibitor. This is in direct contrast to the mutation(s) that are made outside of the active site—which mutations are discussed in more detail below.