GB2150933A - Pentosan-degrading enzyme - Google Patents

Abstract

A pentosan-degrading enzyme and a composition containing it which has pentosanase activity, particularly at higher temperatures e.g. around 90 DEG C. The enzyme and composition may be prepared by fermentation of Talaromyces emersonii, particularly strain IMI 116815 and means for enriching the fermentation broth with pentosanase activity relative to other enzyme activity which may also be present is also disclosed.

Description

SPECIFICATION Biochemical process and composition This invention relates to a novel process for the biodegradation of pentosans, to an enzyme useful therefor and a process for obtaining a composition containing it.

Pentosans are a class of polymers of one or more naturally-occurring pentoses. They occur in a variety of natural sources, such as cereals, e.g. wheat and oats, plants e.g. tea and coffee, seaweed, fruits, vegetables and sorghum, and their presence is undesirable in a number of applications to which the natural sources may be put. Thus, for example, pentosans occur in cereal flour, in which they bind water, and they contribute to stiffening or staling of bread after baking A reduction in the pentosan content of the flour reduces the liability to stiffening in this situation. In another example, pentosans occur in coffee and occur in soluble gums co-extracted with the coffee in the manufacture of instant coffee granules or powder.Reducing the pentosan content of such materials enables higher solid levels to be achieved in the extraction with consequent economies in the manufacturing process.

In our British Patent Specification No. 1421127, we describe a method for the production of enzymes which will degrade -glucans derived from barley and related -1,4/ss-1,3 glucans. The principal enzyme obtained is a 1 41(3-1 3-glucanase, of particular value in the treatment of barley, although other (3-glucanase, laminarinase, hydroxyethyl cellulase, cellulase and a-amylase activities are said also to be able to be present. Microorganisms of the species Penicillium emersonii are employed as the enzyme source, and in particular the strain which was deposited with the Commonwealth Mycological Institute, Kew, England underthe number IMI 116815.

We have now been able to ferment Talaromyces emersonii (formerly known as Penicillium emersonii) in order to produce another enzyme, a pentosanase, which will catalyse the degradation of pentosans, particularly pentosans derived from wheat. The class of such enzymes is generally known and various individual enzymes have been used in, interalia, baking, starch manufacture, farming, brewing, the extraction of vegetable tissue and the manufacture of vegetable colouring materials. However, in many of these applications, high temperatures are routinely employed and there is a need for a thermostable pentosanase which will retain good activity over a wide range of elevated temperatures.

Thus in one aspect of the present invention we provide an enzyme obtainable by fermentation from a strain of the species Talaromyces emersonfi, in particular from Talaromyces emerson ii Stolk, syn.

Penicillium emersonii Stolk IMI 116815, or a mutant thereof, which is capable of catalysing the degradation of pentosans. The new enzyme has been found to be produced in good yield and at a concentration highly satisfactory for commercial use.

In a particular aspect of the invention we provide an enzyme obtainable by fermentation from a strain of the species T. emersonii, in particular T. emersonii IMI 116815, or a mutant thereof, which is capable of catalysing the degradation of xylan. Xylan is a polymer of the pentose xylose.

The new enzyme has been found to be thermostable, i.e. it retains good activity at elevated temperatures, and hence is of particular value in the high temperature degradation of pentosans. For example, in stability measurements, using enzyme prepared according to the procedure of Example 1 described hereinafter, the enzyme still retained 50% of its initial activity after heating at 95"C for six minutes at pH 5.0 in the absence of substrate. The enzyme also exhibits an advantageous temperature-activity relationship, with optimum activity using oat hull xylan as substrate at 87 t 2"C and retention of 45% of optimum activity at 950C.This is unusual in an enzyme of fungal origin, and contrasts for example, with the -glucanase described above which has a poorly defined optimum at 60-700C and only retains 40% of this peak activity at 80 C.

The enzyme according to the invention has the following characteristics, measured using a crude enzyme preparation obtained by the procedure of Example 1 described hereinafter: (a) the pH for the optimum activity of the new enzyme is 5.0, measured at 50"C and using oat hull xylan as the pentosan substrate; (b) the temperature for the optimum activity of the new enzyme is 87 + 2"C, measured at pH 5.0 using oat hull xylan as the pentosan substrate; (c) the presence of previously added xylose, glucose or maltose has no effect on enzyme activity when subsequently assayed at pH 5.0 and 50"C using oat hull xylan substrate.

The enzyme according to the invention may generally be prepared by fermenting an inoculum of T.

em erson ii, for example T. emersonii IMI 116815 or a mutant thereof in a nutrient medium therefore.

However, we have further found that the amount of pentosanase naturally produced may be increased relative to the amount of ss-glucanase produced by suitable modification of the fermentation medium. An enzyme preparation obtained directly by the fermentation of Talaromyces emersonii, desirably T. emersonii IMI 116815 in which the level of pentosanase activity relative to any P-1,4/P-1 ,3-glucanase activity is higher as a result of such modification than that which may conventionally be obtained is a preferred feature of the invention, as is a process for its preparation.

The fermentation may be carried out by methods well-known in the fermentation industry. Thus the strain of T. emerson ii may be cultured under aerobic conditions, preferably in submerged culture, with agitation or stirring with air or oxygen. The fermentation medium employed should contain an assimilable source of carbon, a digestible source of nitrogen and, if desired, growth-promoting substances as well as inorganic salts.

Suitable carbon sources include materials rich in pentosans, for example, cereals such as barley and wheat, distillers solubles or other malt-or grain-distillation by-products, cellulose e.g. Solka Floc, orxylan.

Suitable nitrogen sources include, for example, barley, distillers soiubles or other malt- or grain-distillation by-products, soya meal, nitrates or ammonium salts such as ammonium dihydrogen phosphate.

Where it is desired to increase the level of pentosanase activity relative to any ss-glucanase activity, a carbon or nitrogen source selected from Scotagran (a mix of solubles with dried barley grains), ammonium nitrate, pot ale syrup, maize pellets (dried maize grains mixed with solubles), Curne syrup and corn steep liquor will desirably be used.

Inorganic salts which may be used in the fermentation medium may be, for example, sulphates or chlorides of potassium, magnesium or sodium.

Growth promoting substances which may be used include trace elements such as manganese, iron, zinc, copper or phosphorus.

Advantageously, the fermentation medium contains barley in a concentration in the range 0.2 to 3.5% w/v, distillers solubles or other malt or grain distillation by-products in a concentration in the range 0.4 to 5.5% w/v, and 0.2 to 3.0% w/v cellulose.

Culturing conditions such as temperature, pH and fermentation time are selected such that the strain employed may accumulate a maximum amount of the desired enzyme. For example, the fermentation is advantageously carried out at a temperature ranging from 35-60 C, preferably 50-54"C, at a pH from 3.5-4.5 and for from 1-20 days, preferably 7-10 days.

The crude culture liquid can be used directly for its enzymatic action, or if desired the whole culture broth may be dried and the resulting powder used. If desired, some purification of the pentosanase may be carried out, e.g. by chromatographic techniques, to provide an enzyme preparation having increased pentosanase activity relative to any ss-1,4/1,3-glucanase activity and a method for effecting such purification and the product obtained comprise further aspects of the invention.

Alternatively, the enzyme, which is exocellular, may be extracted from the fermentation product by, for example, conventional methods. Thus for example a first stage is normally to filter off the mycelium formed, preferably by means of precoat filtration, i.e. using a filter which has been coated with a filter aid. The resulting filtrate may be used directly, or, conveniently, may be concentrated, preferably in vacuo to yield the enzyme in a liquid concentrate form. The liquid concentrate may itself be employed as the enzyme source.

Materials such as sodium chloride, sodium benzoate or sodium metabisulphite which confer enzyme storage stability, enzyme thermostability and/or bacteriological stability may if desired be added to such liquid concentrates. Alternatively, the liquid concentrate may be absorbed in a suitable solid, for example ground wheat or barley, optionally in the presence of a carrier such as sodium carboxymethyl cellulose, and the resulting damp mass dried to yield an active enzyme product. If desired, the liquid concentrate itself may be dried, e.g. by spray drying, freeze drying or roller drying to yield a dry enzyme composition.

Unless the extraction is rigorous, which for commercial purposes is usually avoided for economic reasons and is not necessary given the range of uses to which a cruder composition can be put, the composition will usually contain other enzyme activities.

Where a solid enzyme product is desired, this may be obtained from the filtrate or a liquid concentrate thereof by conventional methods, such as precipitation by addition for example of an excess of a water-miscible organic solvent such as an alcohol e.g. ethanol or a ketone e.g. acetone. Either direct precipitation or precipitation onto a carrier may be used, typical carriers including for example starch, methyl cellulose or sodium carboxymethyl cellulose.

The inoculum of T. emersonii used in the fermentation may be obtained by, for example, scaling up from a surface culture of the organism. Scaling up to productive fermentation may conveniently be effected by carrying out a laboratory stage of vegetative growth, followed by one or more seed stages in stirred fermentation vessels.

Thus, in a typical inoculum preparation, the organism is streaked onto a solid nutrient medium, e.g. an agar medium containing peptone (e.g. 0.5% w/v), sodium chloride (e.g. 0.4% w/v), glycerol (e.g. 0.75% w/v), molasses (e.g. 0.8w/v), potassium dihydrogen phosphate (e.g. 0.006% w/v) and magnesium sulphate (e.g.

0.005% MgSO4.7H2O w/v) which has previously been sterilised, e.g. by autoclaving at about 120"C for 15 minutes, and allowed to cool. Incubation is preferably carried out at about 37"C for about 10 days, after which the spores produced are used to inoculate a liquid medium for vegetative growth, sterilised by e.g.

autoclaving, at about 120 C for 15 minutes, and containing malt extract (e.g. 3.3% w/v), yeast extract (e.g.

2.0% w/v) and ammonium dihydrogen phosphate (e.g. 0.6% w/v). The culture is preferably effected until good mycelial growth is present, for example, when using about 500ml of medium, for about 72 hours at 45"C.

The resulting vegetative culture is then used to inoculate a first stirred seed stage. The medium for this stage preferably contains similar components to those described previously for the main fermentation medium, and is preferably prepared by sterilising all the ingredients together in the seed stage vessel e.g. by steam injection. A typical medium for the seed stage is 'Medium A' shown hereinafter in Table 1. Using about 40 litres of medium, culturing is preferably effected at about 50"C for about 76 hours with stirring, e.g.

at 420 r.p.m., and aeration, e.g. 50 litres/minute of air.

The seed culture thus obtained may then be used to inoculate a producing stage as described above or, if more seed is required, may be used in further seed stages, identical in nature to be first.

Mutants of T. emersonllfor use in the above fermentation processes may be obtained by conventional methods of strain improvements, e.g. by the use of ionising radiation (for example X and ty-rays; uv light; or uv light in the presence of a photo-sensitising agent such as 8-methoxypsoralen), chemicals (e.g. nitrous oxide; hydroxylamine; pyrimidine base analogues such as 5-bromouracil; acridines; alkylating agents such as ethyl methanesulphonate or mustard gas; hydrogen peroxide; phenols or formaldehyde), heat or genetic techniques (e.g. recombination, transduction, transformation, lysogenisation, lysogenic conversion and selective techniques for spontaneous mutants).

The desired enzyme activity may be determined in the culture liquid or at any point in an isolation procedure by a simple test designed to determine the reducing sugars released by the action of the enzyme on a pentosan substrate. Thus, in one test, a sample of the enzyme preparation to be measured is suitably diluted and is then incubated for 10 minutes with an excess of the pentosan xylan, at 50"C in pH 5.0 acetate buffer.The sugar released is determined as a maltose equivalent by adding alkaline 3,5-dinitrosalicylic acid reagent, heating for 5 minutes on a boiling water bath, quenching in ice water, measuring the absorption of the solution at 540nm and converting this to a maltrose equivalent by reference to a standard calibration graph obtained by preparing standard dilutions of maltose of known moisture content and reacting these with the dinitrosalicylic reagent under the conditions just described. As used herein, one unit of enzyme releases 1 mg of maltose equivalent per minute under the test conditions at 50"C and pH 5.0.

The enzyme according to the invention is useful for a variety of purposes for which other pentosanases are used. Thus, for example, it may be used in baking, to reduce the natural pentosan content of wheat flour and so improve the keeping qualities of bread. It may be used in starch manufacture and hydrolysis, to lower the pentosan content of cereals and reduce the viscosity of starch slurries which otherwise adversely affect the efficiency and cost of starch recovery and subsequent hydrolysis. It may be used in farming, to upgrade the quality of mixed silage and animal feeds. It may be used in brewing, for the improved production and extraction of fermentable sugars when the mash includes e.g. wheat or sorghum, and for the prevention or treatment of certain types of haze.It may be used for the extraction of vegetable tissue, for example in the extraction of tea, coffee and fruits; and to extract alginatesfrom seaweed; and in the manufacture of natural vegetable colouring materials. In some of these applications, the thermostability of the enzyme is particularly advantageous in view of the temperatures at which the processes are conducted.

In a further aspect of the invention, therefore, we provide a method of reducing the pentosan content of a pentosan-containing material which comprises contacting the said material with an enzyme of the invention.

Typical pentosans which may be degraded with the enzyme of the invention include those found in cereals such as wheat and oats; plants such as tea and coffee and in extracts thereof; fruits; vegetables; seaweed and sorghum. In one example, the pentosan may be xylan, for example an oat hull xylan. In a preferred embodiment, the pentosan containing material which is to be degraded is derived from wheat.

In a further embodiment of the invention we provide a method of reducing the xylan content of an xylan-containing material wherein the material is contacted with an enzyme according to the invention.

The enzyme or composition according to the invention may be used employing methods customarily found in enzyme technology. Thus for example the enzyme may be contacted with an aqueous medium containing the substrate either in suspension, admix or in a solution. The reaction temperature will vary according to the exact nature of the reaction but will in general be in the range 35-95"C, advantageously at the higher end of the range, in some applications at 900C and above. The pH of the reaction mixture may be for example in the range 4.0-6.5, preferably 4.5-5.5 and in particular at pH 5.0. If desired, the reaction mixture may contain other added enzymes, for example or-amylase.

The strain of Talaromyces emersoniiwhich we have used is a sub-culture of that which was deposited at the Commonwealth Mycological Institute, Kew, England under Number IMI 116815 in 1972 before the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure (Budapest 1977) was signed. We have recently redeposited this strain (on 19th November 1984) under Number CMI CC No. 290604 at the same depository but under the conditions of the Treaty of Budapest and it is our belief that the two strains are identical and that either strain may be used.

The following Examples illustrate the invention. All temperatures are in "C. All percentages are in w/v.

Example 1 Talaromyces emersonii IMI 116815 was cultured at 37" for 10 days on an agar medium sterilised in an autoclave and containing 0.5% peptone, 0.4% NaCI,0.75% glycerol, 0.8% molasses, 0.006% KH2PO4 and 0.005% MgSO4.7H20).

The spores produced were used to inoculate flasks of a liquid medium sterilised by autoclaving and containing 3.3% malt extract,2.0% yeast extract and 0.6% ammonium dihydrogen phosphate. The flasks were placed on a rotary shaker (210 rev/min; 4.9 cm throw) for 3 days at45 C.

The resulting vegetative culture (400 ml) was used to inoculate 40 litres of Medium A) Table 1 sterilised by steam injection in a 50 litre capacity stainless steel fermenter. The culture was stirred at 420 rev/min and aerated at 50 litres/min for 75 hours at 50"C.

The resulting culture (4.5 L) was used to inoculate 40 litres of Medium A (Table 1) sterilised by steam injection in a 50 litre capacity stainless steel fermenter. The culture was stirred at 420 rev/min and aerated at 50 litres/min for 75 hours at 50 .

The resulting culture (4.5 L) was used to inoculate 40 litres of Medium A sterilised by steam injection in a 50 litre capacity stainless steel fermenter. The culture was stirred at 420 rev/min and aerated at 50 litres/min for 36 hours at 50'.

The resulting culture (16 L) was used to inoculate 32 litres of Medium B (Table 1) sterilised by steam injection in a 50 litre capacity stainless steel fermenter.

The culture was aerated at 50 litres/min. and stirred intermittently at 420 rev/min. at 50" for 10 days. Sterile water was added at an average rate of 1.5 litres per day.

Assay of the exocellular fluid yielded a concentration of the enzyme according to the invention of 36 u/g.

TABLE 1 Ingredient Medium A /O/o/ Medium B { /O) Ground Barley 1.0 3.38 Distillers Solubles 2.25 5.06 Solka Floc 2.25 2.53 (NH4)2H2PO4 0.34 0.77 K2SO4 0.12 0.27 MgSO4.7H2O 0.01 0.11 NaCI 0.11 0.11 MnSO4.4H2O 0.0005 0.001 FeSO4.7H2O 0.004 0.008 ZnSO4.7H2O 0.004 0.008 CuSO45H2O 0.0005 0.001 H3PO4 0.5 1.02 Water to 100% Example 2 Demonstration of variation in pentosanase: glucanase ratio on fermentation a) Spores were produced as in Example 1 and used to inoculate a seed stage in 250 ml flasks containing 40 ml of Medium A sterilised by autoclaving. The flasks were incubated at 45 for 72 hours, shaking at 200 revimin. The resulting culture (4 ml) was used to inoculate 40 ml of Medium B sterilised by autoclaving in 250 ml flasks.After incubating at 45" for 12 days, shaking at 200 rev/min, the exocellular culture fluid was assayed for pentosanase (P) and barley ss-glucanase (G) activities and the P: G ratio was calculated.

b) Using essentially the same system as in a) above.

Omitting the Solka Floc and including oat spelts xylan (2.2%) gave a P: G ratio 71% higher than that obtained in a).

Example 3 In order to demonstrate pentosanase activity in the enzyme produced in Example 1, a 1% pentosan solution was incubated at 50 in buffer at pH 5.0 in the presence of added enzyme (test system) or with the addition of an equivalent amount of water (control system). The pentosan substrate used was xylan, obtained from oat hulls.

Samples of the test system were withdrawn after 10 minutes, 30 minutes and 22.5 hours, quenched to prevent any further reaction and then spotted on to 20 cm square thin layer chromatography plates covered with 0.25 mm kieselgel. Spots of selected standard sugar solutions and of the control system were also applied.

The mobile phase was n-butanol :pyridine:ethanol water (20:15:25:10). Detection was by spraying the dried plates with 5% ammonium molybdate in 5% sulphuric acid followed by heating at 108 for 15 minutes.

All the test samples showed interalia spot of identical Rf value to that given by the standard sugar solution xylose.

Since the control sample gave only a single spot on the origin, this test spot can not have derived either from xylose contamination of the substrate or from its chemical hydrolysis: it must have arisen by enzymic degradation ofthe pentosan.

Example 4 The following comparative test was designed to illustrate the potential utility of the enzyme according to the invention in an enzymatic wheat starch digestion process: Enzyme (2 ml) prepared according to Example 1, low grade wheat flour (155 g), sodium chloride (2 g), calcium chloride (0.17 g). 'Termamyl' bacterial alpha amylase (Novo Industri A/S) (1 ml) and water (250 ml) were shaken thoroughly in a glass flask. The pH was adjusted to 6.5 using caustic soda. The neck of the container was covered to minimise evaporation and the flask was held for 5 hours at a temperature of 90 .

Starch hydrolysis occurred (negative iodine test result) and the digest viscosity was 43 cps.

In a control experiment omitting the enzyme of the invention the final viscosity of the digest was 127 cps.

The following Examples illustrate experiments performed to characterise the enzyme according to the invention. In each Example, the enzyme used was prepared according to the method of Example 1 except that small amounts of sodium benzoate and sodium metabisulphite were added to the exocellular fluid as bacteriological stabilisers. Enzyme activity was determined in each Example using an excess of 1% aqueous oat hull xylan as the pentosan substrate and measuring the increase in reducing activity after a 10 min incubation, against a xylose calibration curve. The pH and temperature of each incubation are stated in the Examples.

Example 5 The pH-activity relationship was obtained at a temperature of 50 pH 4.0 4.5 5.0 5.5 6.0 6.5 % Maximum Activity 62 85 100 84 70 47 Significant hydrolysis occurs throughout the range illustrated.

Example 6 Thermal stability was studied by holding samples at temperatures of 60, 70, 80, 90 and 95 in the absence of substrate and withdrawing amounts at timed intervals to obtained a family of residual activity-time curves. The activity remaining was measured at pH 5.0 and 50 : - at 60 , there was no detectable loss in activity after 80 mins, - at 70 , 70% of the initial activity was still present after 1 h, - at 80 , 70% of the initial activity was still present after 30 mins, - at 90 , 50% of the initial activity was still present after 8 mins, - at 95 , 50% of the initial activity was still present after 6 mins.

Example 7 The temperature - activity relationship was obtained at a pH of 5.0. Inspection of the activity temperature curve showed that peak activity occurred at 87 Activities at the individual temperatures studied, expressed as percentages of this maximum, were as follows: % Maximum Activity 13 24 41 57 86 [100] 90 45 Temperature' 40 50 60 70 80 [87+2] 90 95 The results confirm the inherent thermal stability of the new enzyme, as shown in Example 5. The enzyme can clearly be used at temperatures in excess of 90 .

This contrasts with a poorly defined optimum of 60-70" for the barley -glucanase of British Specification 1,421,127. At 80 , only 40% of peak activity is recorded.

Example 8 In a preliminary study to determine whether activity of the enzymes was affected by products of the reaction and/or other sugars present in a typical digest in an industrial process, three simple sugars (xylose, glucose and maltose) were separately added to the enzyme, which was then assayed at 50 and pH 5.0. Each sugar was added in an amount equivalent to the amount of xylose liberated from the substrate under the assay conditions.

There was no inhibition or promotion of enzyme activity, and thus, on this evidence, there could be no interference with enzyme activity from the accumulation of simple sugars that will occur in many industrial applications.

Claims (21)

1. An enzyme obtainable by fermentation from a strain of the species Talaromyces emersonfi or a mutantthereof which is capable of catalysing the degradation of pentosans.

2. An enzyme as claimed in claim 1 which has a pH for optimum activity of about 5.0 measured at 50"C, and a temperature for optimum activity at 87+2"C measured at pH 5.0 and which retains about 50% of its initial activity after heating at 95"C for six minutes at pH 5.0, all measured using oat hull xylan as pentosan substrate.

3. An enzyme composition comprising an enzyme as claimed in claim 1 or claim 2 in a mixture with a (3-1,41(3-1 ,3-glucanase and wherein the level of pentosanase activity has been increased relative to the level of p-1.4/P-1 ,3-glucanase activity by modification of the fermentation medium in which each is produced.

4. An enzyme or composition as claimed in any of claims 1 to 3 wherein the strain employed is Talaromyces emersonll IMI 116815, CMI CC No. 290604, or a mutant thereof.

5. A process for the preparation of an enzyme composition having pentosanase activity which comprises fermenting a microorganism strain of the species Talaromyces emersonii in a nutrient medium thereof whereby a broth containing said enzyme is produced.

6. A process as claimed in claim 5 wherein the level of pentosanase activity relative to the level of (3-1 ,4/(3-1 3-glucanase activity is increased by modification of the nutrient medium.

7. A process as claimed in claim 5 or claim 6 wherein the level of pentosanase activity relative to the level of p-1,4-/P-1,3-g I uca n ase activity is increased by a purification step subsequent to the fermentation.

8. A process as claimed in any of claims 5 to 7 wherein the strain is Talaromyces emersonii IMI 116815, CMI CC No.290604, our a mutant thereof.

9. A method of reducing the pentosan content of a pentosan-containing material which comprises contacting the said material with an enzyme or composition as claimed in any of claims 1 to 4.

10. A method as claimed in claim 9 wherein the pentosan-containing material is a cereal selected from wheat and oats, a plant selected from tea and coffee or an extract thereof, a fruit or a vegetable.

11. A method as claimed in claim 9 or claim 10 wherein the pentosan-containing material is starch.

12. A method as claimed in claim 9 or claim 10 wherein the pentosan-containing material is wheat or is derived from wheat.

13. A method as claimed in any of claims 9 to 12 wherein the enzyme is contacted with the pentosan-containing material in an aqueous medium at a temperature of from 35-95"C and at a pH of from 4.0 to 6.5.

14. The use of composition containing a pentosanase enzyme derived from a strain of the species Talaromyces emersonii in reducing the pentosan content of a cereal selected from wheat and oats, a plant selected from tea and coffee or an extract thereof, a fruit or a vegetable.

15. An enzyme or enzyme composition as claimed in claim 1 or claim 3 substantially as hereinbefore described.

16. A pentosanase enzyme substantially as hereinbefore described and with reference to any of Examples 1 to 8.

17. An enzyme composition substantially as hereinbefore described and with reference to Example 2.

18. A process as claimed in any of claims 5 to 8 substantially as hereinbefore described.

19. A process for the preparation of a pentosanase substantially as hereinbefore described and with reference to Examples 1 and 2.

20. A method as claimed in any of claims 9 to 13 substantially as hereinbefore described.

21. A method of reducing the pentosan content of a pentosan-containing material substantially as hereinbefore described and with reference to any of Examples 3 to 8.