NL8403600A - PROCESS FOR THE BIODEGRADATION OF PENTOSANES. - Google Patents

Description

* u - 1 - * L ·.

Method for the biodegradation of pentosans.

The invention relates to a new method for the biodegradation of pentosans, to an enzyme to be used therefor and to a method for obtaining a composition containing it.

Pentosans are a class of polymers of one or more naturally occurring pentoses. They occur in a number of natural sources, such as grains, for example wheat and oats, plants, for example tea and coffee, seaweed, fruits, vegetables and millet, and their presence is undesirable in a number of applications using the natural source. For example, pentosans occur in wheat flour, in which they bind water and they contribute to the stiffness and aging of bread after baking. Reducing the pentosan content of the flower reduces the chances of becoming stiff in this situation. Another example is the appearance of pentosans in coffee and in soluble gums, which are extracted with the coffee in the preparation of instant coffee granules or powder. Reduction of the pentosan-20 content of such materials makes it possible to obtain higher solids contents in the extract with consequent savings in the manufacturing process.

British Pat. No. 1,421,127 discloses a process for the preparation of enzymes that degrade barley-derived β-glucans and related β-3-1,4 // 3-1,3 glucans.

The main enzyme obtained is α-3-1.4 // 5-1.3 glucanase, which is of particular importance in the treatment of barley, although other β-glucanase, laminarinase, hydroxyethyl cellulase, cellulase and o-amylase activities, is also said to be present. Microorganisms of the species Penicillium emersonii are used as the enzyme source, in particular the strain deposited at Commonwealth Mycological Institute, Kew, England under number IMI 116815.

It has now been found that one can ferment 'Talarómyces emerso-35 nii (formerly known as Penicillium emèrsónii) 3403600 ji

J

7 ..... Λ - 2 - producing another enzyme, a pentosanase, which catalyzes the degradation of pentosans, especially wheat pentosans. The class of such enzymes is well known and various individual enzymes have been used, inter alia, in baking, starch preparation, agriculture, brewery, extraction of vegetable tissue and preparation of vegetable dyes. However, many of these applications usually use high temperatures and there is a need for a thermostable pentosanase which retains good activity over a wide range of high temperatures.

The invention now relates to an enzyme, which can be obtained by fermentation from a strain of the species Talaromyees emersonii, in particular from TalarömyCes emersonii Stolk, syn. PeniciIlium "emèrsónii Stolk IMI 116815, or a mutant thereof, which can catalyze the degradation of pentosans.

It has been found that the enzyme is produced in good yield and at a very satisfactory concentration for commercial use.

The invention particularly relates to an enzyme which can be obtained by fermentation from a strain of the species T. emèrsónii, in particular T. emèrsónii IMI 116815, or a mutant thereof, which can catalyze the degradation of xylan. Xylan is a polymer of the pentose xylose.

The new enzyme has been found to be thermostable, ie it retains good activity at elevated temperatures and is therefore of particular significance in the degradation of pentosans at high temperatures. For example, in stability measurements using the enzyme described in the method of Example I below, the enzyme still retained 50% of its initial activity. Heat at 95 ° C for 6 minutes at pH 5.0 in the absence of substrate. The enzyme also exhibits an advantageous temperature activity relationship with an optimum activity using oat bran xylan as substrate at 87 ± 2 ° C and retention of 45% optimum activity at 95 ° C. This is uncommon with an enzyme 8403500-3 from fungus and is, for example, in contrast to the yd-glucanase described above, which has a poorly defined optimum at 60-70 ° C and only 40% of this peak activity at 80 ° C retains.

The enzyme of the invention has the following properties, measured using a crude enzyme preparation, obtained according to the method of Example I described below: (a) the pH for the optimum activity of the new enzyme is 5, 0, measured at 50 ° C and using oat bran xylan as a pentosah substrate, (b) the temperature for the optimum activity of the new enzyme is 87 ± 2 ° C, measured at pH 5.0 using oat bran xylan as a pentosan substrate, 15 (c ) the presence of pre-added xylose, glucose or maltose has no effect on the enzyme activity when it is subsequently tested at pH 5.0 and 50 ° C using oat bran xylan subs.

The enzyme of the invention can generally be prepared by fermenting an inoculum of T. emèrsónii, for example T. emersonii ME 116815, or a mutant thereof in an appropriate nutrient medium. However, it has further been found that one can increase the amount of pentosanase produced naturally to the amount of 25 y-glucanase produced by appropriate modification of the fermentation broth. An enzyme preparation obtained directly by fermentation of Talarómyces émèrsónii, most preferably T. emersonii ML 116815, in which the pentosanase activity level relative to any yS-1,4 / β-, 1,3-glucanase activity. as a result of such a modification being higher than can normally be achieved, it is a preferred aspect of the invention, as is a method for its preparation.

Fermentation can be stirred by methods well known in the fermentation industry. Thus, the strain of T. émersonii can be grown under aerobic conditions, preferably in submerged culture, under agitation or agitation with air or oxygen. The fermentation medium used should be an assimilable carbon source, a digestible nitrogen source, and, if desired, growth promoters, as well as inorganic salts.

Suitable carbon sources are substances rich in pentosans, for example grains, such as barley and wheat, solubles from the distillery, or other by-products of malt or grain distillation, cellulose, for example Solka Floc, or xylan.

Suitable nitrogen sources are, for example, barley, distillery solubles or other malt or grain distillation by-products, soybean meal, nitrates, or ammonium salts, such as ammonium dihydrogen phosphate.

If it is desired to boost the level of pentosanase-15 activity relative to any β-glucanase activity, then as carbon or nitrogen source, good use can be made of Scotagran (a mixture of solutes and dried barley grains), ammonium nitrate, pot beer syrup, corn pieces (dried corn kernels, mixed with soluble substances), Curne-20 syrup or corn steep liquor.

Inorganic salts which can be used in the fermentation medium are, for example, potassium, magnesium and sodium sulfate and chloride.

Growth promoters which can be used are, for example, trace elements such as manganese, iron, zinc, copper or phosphorus.

The fermentation medium can ferment well in a concentration of 0.2 to 3.5% w / v, solubles from distilleries or other malt or grain distillation by-products in a concentration of 0.4 to 5.5% w / v v and 0.2 to 3.0% w / v of cellulose.

Culture conditions, such as temperature, pH and fermentation time, are selected such that the strain used can accumulate a maximum amount of desired enzyme. For example, the fermentation can be done well at a temperature of 35-60 ° C, 8403600 *. .5 - 5 - preferably from 50-54 ° C, at a pH of 3.5-4.5 and for 1-20 days, preferably 7-10 days.

The crude broth can be used directly for enzymatic action, but if desired, the entire broth can be dried and the resulting powder used. If desired, some purification of the pentosanase can be carried out, for example by chromatographic methods, to obtain an enzyme preparation with increased pentosanase activity relative to any β - 1,4 / 1,3-glucanase activity and a method for carrying out such purification and the product obtained therewith constitute further aspects of the invention.

Optionally, the enzyme, which is exocellular, can be extracted from the fermentation product by, for example, conventional methods. Thus, for example, a first stage 15 is normally filtration of the mycelium formed, preferably with a filter coated with a filter aid. The resulting filtrate can be used directly, but it can also be well concentrated, preferably in a vacuum, to obtain the enzyme in the form of a liquid concentrate. The liquid concentrate itself can be used as an enzyme source. If desired, substances such as sodium chloride, sodium benzoate or sodium metabisulfite can be added to such liquid concentrates which confer storage stability of the enzyme, thermostability of the enzyme and / or bacteriological stability. Optionally, the liquid concentrate can also 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 wet mass to an active enzyme product. If desired, the liquid concentrate ointment can be dried, for example by spray-drying, freeze-drying or roller-drying, to obtain a dry enzyme preparation.

Unless the extraction is rigorous, which is usually avoided for commercial purposes for economic reasons and is not necessary given the range of applications for which a rougher preparation can be used, the preparation usually contains 8403000-6 other enzyme activities.

If a solid enzyme product is desired, it can be obtained from the filtrate or a liquid concentrate thereof in conventional ways, such as precipitation by addition of, for example, an excess of water-miscible organic solvent, such as an alcohol, for example ethanol, or a ketone, for example acetone. Either direct precipitation or precipitation on a support can be used, and good supports are in particular, for example, starch, methyl cellulose or sodium carboxy-methyl cellulose.

For example, the seed of T. emersonii used in the fermentation can be obtained by scaling up from a surface culture of the organism. Upscaling to productive fermentation can be accomplished well by allowing laboratory scale vegetative growth followed by one or more sowing stages in stirred fermenters.

Thus, in true graft preparation, the organism is streaked onto a solid nutrient medium, e.g., an agar medium, containing peptone (e.g., 0.5% w / v), sodium 20 chloride (e.g., 0.4% w / v), glycerol ( e.g. 0.75% w / v), molasses (e.g. 0.8% w / v), potassium dihydrogen phosphate (e.g. 0.006% w / v), and magnesium sulfate (e.g. 0.005% MgSO ^ 7 ^ 0 w / v) and has been pre-sterilized, for example, by heating it in an autoclave at 120 ° C for 15 minutes and then allowing it to cool. Incubation is preferably carried out at 37QG for 10 days, after which the spores produced are used to inoculate a liquid medium for vegetative growth sterilized by, for example, heating in an autoclave at 120 ° C for 15 minutes and malt extraction (e.g. 3, 3% w / v), 30 yeast extract (e.g. 2.0% w / v) and ammonium dihydrogen phosphate (e.g. 0.6% w / v). The culture is preferably carried out until good mycelial growth is present, for example, when using 500 ml medium, at 45 ° C for 72 hours.

The resulting vegetative culture is then used to inoculate a first stirred sowing stage. The 8403600-7 medium for this stage preferably contains similar components to those described above for the auxiliary fermentation medium and is preferably prepared by sterilizing all components together in the seed stage vessel by, for example, steam injection. A 5 good medium for the sowing stage is' Medium. A ', shown below in Table A. Using 40 L of medium, the culture is preferably conducted at 50 ° C for 76 hours with stirring, for example, at 420 rpm and aeration, for example, 50 L of air per minute.

The sowing culture thus obtained can then be used for inoculating a production stage as described above, or if more sowing material is needed, can be used for further sowing stages, which are identical in nature to the first.

Mutants of T. êmèrsónii for use in the above-mentioned fermentation processes can be obtained by the usual methods of strain improvement, for example by using ionizing radiation (for example X-rays and Y-radiation, W-light, or W-light in the presence of a light sensitizing agent, such as 8-methoxypsoralen), chemicals (eg nitric oxide, hydroxylamine, pyrimidine base analogs, such as 5-bromo uracil, acridines, alkylating agents, such as ethyl methanesulfonate or mustard gas, hydrogen peroxide, phenols or formaldehyde), heating or genetic methods (e.g., recombination , transduction, transformation, lyogenization, lyogenic conversion and selective methods for spontaneous mutation).

The desired enzyme activity in the broth or at any point in an isolation procedure can be determined 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, one of the enzyme preparation to be measured is suitably diluted and then incubated with an excess of the pentose xylan for 10 minutes at 50 ° C in a pH 5.0 acetate buffer. The released striker is determined as a maltose equivalent by adding alkaline 3,5-dinitrosalicylic acid reagent 35, heating in a boiling water bath for 5 minutes, in 8403600.

a- s, - 8 -

quench ice water, measure the absorbance of the solution at 540 nm and convert it to a maltose equivalent using a standard calibration graph obtained by preparing standard maltose solutions of known moisture content 5 and those described below conditions with the dinitro salicylic acid reagent. As used herein, one unit of enzyme gives under the test conditions at 50 ° C and pH

5.0 t mg maltose equivalent per minute.

The enzyme of the invention can be used for a number of purposes, for which other pentosanases are used. For example, it can be used in the bakery to reduce the natural pentosan content of wheat flour in order to improve the shelf-life properties of bread. It can be used in starch preparation and hydrolysis to reduce the pentosan content of grains and to reduce the viscosity of starch slurries, which would otherwise adversely affect the efficiency and cost of starch recovery and subsequent hydrolysis. It can be used in agriculture to increase the quality of ensiled harvests and animal feed. It can be used in the brewery for better production and extraction of fermentable sugars, if the batter contains, for example, wheat or millet and to prevent or treat certain types of cloudiness. It can be used for the extraction of vegetable tissue, for example in the extraction of tea, coffee and fruits and for the extraction of alginates from seaweed and in the preparation of natural vegetable dyes. In some of these applications, the thermal stability of the enzyme is particularly advantageous, given the temperatures at which the processes are performed.

The invention therefore relates to a method for reducing the pentosan content of a pentosan-containing substance, whereby this substance is contacted with an enzyme of the invention.

Good examples of pentosans that can be degraded with the enzyme of the invention are the pentos 8403600-9 sanes found in grains such as wheat and oats, plants such as tea and coffee and in extracts thereof, fruits , vegetables, seaweed and millet. In an example, the pentosan may be xylan, for example an oat bran xylan. In a preferred embodiment, the pentosan-degradable material is derived from wheat.

The invention further relates to a method for reducing the xylan content of a xylan-containing substance, wherein this substance is contacted with an enzyme of the invention. The enzyme or composition of the invention can be used using methods commonly found in enzyme technology. For example, the enzyme can be contacted with an aqueous medium containing the substrate either in suspension or in mixture, or in solution. The reaction temperature varies with the proper nature of the reaction, but is generally 35-95 ° C, with the highest range of the range being preferred, therefore, in some applications, 90 ° C and above. The pH of the reaction mixture may be, for example, 4.0-6.5, preferably 4.5-5.5 and most preferably 5.0. If desired, the reaction mixture may contain other added enzymes, for example, or -amylase.

The Talaromyces emersonii strain, which was used, is a subculture of the strain, deposited with Commonwealth Mycological Institute, Kew, England under number 25 DU 116815 in 1972, before the Budapest Convention on the International Recognition of the Deposit of microorganisms for patent procedures (Budapest 1977) were signed. This strain was re-deposited on November 19, 1984 under carpenter CME CC No. 290604 at the same collection but under the terms of the Budapest Treaty 30 and it is believed that the two strains are identical and that either strain can be used.

The following examples illustrate the invention. All temperatures are in eC. All percentages are in w / v.

35 8403 600 9 <- I -ΙΟ Ι

Example I

Talaromyces emersonii IMI 116815 was grown for 10 days at 37 ° on an agar medium autoclaved and 0.5% peptone, 0.4% NaCl, 0.75% glycerol, 0.8% molasses. se, 0.006% K ^ PO ^ and 0.005% MgSO ^ ^ I ^ O.

The spores produced were used to inoculate flasks from a liquid medium which had been sterilized in an autoclave and contained 3.3% malt extract, 2.0% yeast extract and 0.6% ammonium dihydric acid phosphate. The flasks were placed on a rotary shaker at 210 ° for 3 days (210 rpm / 4.9 cm stroke).

The resulting vegetative culture (400 ml) was used to seed 40 L of medium A (Table A) sterilized by steam injection in a 50 L capacity stainless steel fermenter. The culture was stirred at 50 ° C at 420 rpm for 75 hours and 50 L / min aeration.

The resulting culture (4.5 L) was used to seed 40 L of medium A (Table A) sterilized by steam injection in a 50 L capacity stainless steel fermenter. The culture was stirred for 75 hours at 50 ° at 420 rpm and aeration of 50 l / minute.

The resulting culture (4.5 L) was used to inoculate 40 L of medium A, which had been steam sterilized in a 50 L capacity stainless steel fermenter. The culture was stirred for 36 hours at 50 ° at 420 rpm and aeration of 50 1 / minute.

The resulting culture (16 L) was used to inoculate 32 L of medium B (Table A) sterilized by steam injection in a 50 L stainless steel fermenter.

The culture was aerated at 50 1 / minute and stirred at 50 ° at 420 rpm / 35 minute for 10 days with interruptions. Sterile water was added with an average amount of 1.5 X per day.

Exocellular fluid testing yielded a concentration of the enzyme of the invention of 36 units / gram.

5

Table A

Component Medium A (%) Medium B (%)

Ground barley 1.0 3.38 10 Solubles from 2.25 5.06 distillery

Solfca Floc 2.25 2.53 (NH4) 2 H2K> 4 0.34 0.77 K2SG4 0.12 0.27 15 MgS04.7H20 0.01 0.11

NaCl 0.11 0.11

MnS04.4H20 0.0005 0.001

FeSO 4 .7H 2 O 0.004 0.008

ZnS04.7H20 0.004 0.008 20 CuS04.5H20 0.0005 0.001 H3F04 0.5 1.02

Water up to 100%

Example II 25

Demonstration of the variation in pentosanase: glucanase ratio upon fermentation; a) Spores as in Example 1 were produced and used to inoculate a sowing stage into 250 ml flasks containing 40 ml of medium A sterilized in an autoclave. The flasks were incubated for 72 hours at 45 ° with shaking at 200 rpm. The resulting culture (4 ml) was used to seed 40 ml of medium B sterilized in an autoclave into 250 ml flasks. After 12 8403600 _ __j

^ V

incubate at 45 ° for 12 days with shaking at 200 rpm, the exocellular culture fluid was tested for pentosane (P) and barley / glucanase (G) activity and the P: G ratio was calculated. . B) Using the same system as in a) above, omission of Solka Floc and inclusion of oat grain xylan (2.2%) gave a 71% higher P: G ratio than under a).

Example III

10

In order to demonstrate the pentosanase activity in the enzyme produced in Example 1, a 1% pentosan solution was incubated at 50 ° in a 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 bran.

Samples were withdrawn from the test system after 10 minutes, 30 minutes and 22.5 hours, which were quenched to avoid any further reaction and then stained on 20 cm 2 thin layer chromatography plates covered with 0.25 mm silica gel. Stains from selected standard sugar solutions and from the control system were also applied.

The mobile phase was n-butanol: pyridine: ethanol: water (20; 15: 25; 10). Detection was done by spraying the dried plates with 5% ammonium molybdate in 5% sulfuric acid, followed by heating at 105 ° for 15 minutes.

All test samples showed, among other things, a spot of identical Rf value as that given by the standard solution of the sugar xylose.

Since the control sample gave only a single stain at the origin, this test stain could not have come from either xylose contamination of the substrate or from chemical hydrolysis of xylose and thus must have been caused by enzymatic degradation of the pentosan.

35 8403600 * K siff * - 13 -

Example IV

The following comparative test was designed to demonstrate the potential utility of the enzyme of the invention in an enzymatic wheat starch digestion process:

Enzyme (2 ml), prepared according to Example I, lower quality wheat flour (155 g), sodium chloride (2 g), calcium chloride (0.17 g), bacterial alpha amylase, "Termamyl" (Novo Industri A / S ) (1 ml) and water (250 ml), were shaken thoroughly in a glass flask. The pH was adjusted to 6.5 with sodium hydroxide. The neck of the container was covered to minimize evaporation and the flask was kept at 90 ° for 5 hours. Starch hydrolysis (negative result of the iodine test) occurred and the viscosity of the digestion mixture was 43 cps.

In a control test omitting the enzyme of the invention, the final viscosity of the digestion mixture was 127 cps.

The following examples illustrate experiments performed to characterize the enzyme of the invention. In each example, the enzyme used was prepared according to the procedure of Example I, except that small amounts of sodium henzoate and sodium metahisulfite were added to the exocullary liquid as bacteriological stabilizers.

Enzyme activity in each example was determined using an excess of 1% wheat bran xylan in water as pentosan substrate and by measuring the increase in reducing activity after 10 minutes of incubation against a xylose calibration curve. pH and temperature of each breeding are given in the examples.

30

Example Y.

The following relationship between pH and activity was obtained at 50 °: 35 8403600 - 14 - pH 4.0 4.5 5.0 5.5 6.0 6.5% maximum activity 62 85 100 84 70 47 5 Er significant hydrolysis occurred throughout the range shown.

Example VI

Thermal stability was investigated by storing samples at 60, 70, 80, 90 and 95 ° in the absence of substrate and by withdrawing amounts at certain intervals to give a family of residual activity time curves. The residual activity was measured at pH 5.0 and 50 °: at 60 ° there was no detectable loss of activity after 80 minutes, at 70 ° there was still 70% of the initial activity after 1 hour, at 80 ° 70% of the initial activity was still present after 30 minutes, at 90 ° 50% of the initial activity was still present after 8 minutes, at 95 ° 50% of the initial activity was still present after 6 minutes.

25

Example Vil

The relationship between temperature and activity was obtained at a pH of 5. Inspection of the activity temperature-30 curve showed that peak activity occurred at 87 ± 2 °.

The activities at the various temperatures examined, expressed as percentages of this maximum, were as follows:% maximum activity 13 24 41 57 86 / 100_7 90 45 35 Temperature ° C 40 50 60 70 80 / “87 ± 2J 90 95 8403 600 <Φ__ £ - 15 -

The results confirm the inherent thermal stability of the new enzyme as shown in Example V. The enzyme can clearly be used at temperatures above 90 °.

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

Example VIII

10

In a preliminary study to determine whether the activity of the enzymes was affected by reaction products and / or other sugars present in a real digestion mixture of an industrial process, three simple sugars (xylose) were added , glucose and maltose) separately to the enzyme, which was then tested at 50 ° and pH 5.0. Each sugar was added in an amount equivalent to the amount of xylose released from the substrate under the test conditions.

There was no inhibition or promotion of enzyme activity and thus apparently no interaction with the enzyme activity occurred due to the collection of the simple sugars that occurs in many industrial applications.

25 8403300

Claims (14)

1. Enzyme, which can be obtained by fermentation from a strain t of the species Talaromyces emersonii or a mutant thereof, characterized in that it can catalyze the degradation of pentosans. Enzyme according to claim 1, characterized in that it has an optimum activity at pH 5.0 measured at 50 ° C and an optimum activity at 87 ± 2 ° C measured at pH 5.0 and about 50% of retains its initial activity after heating at 95 ° C at pH 5.0 for 6 minutes, all measured using wheat bran xylan as pentosan substrate. Enzyme preparation, characterized in that it contains an enzyme according to claim 1 or 2, mixed with a β ~ \, 4 / β- \, 3-glucanase and the level of its pentosan activity relative to the level of its / 3 -1.4 // 3-1,3-glucanase activity has been boosted by modification of the fermentation medium in which each is produced. Enzyme or preparation according to any one of claims 1 to 3, characterized in that the strain Talaromyces emersonii IMI 116815, GMI CC no. 290604, b £ is a mutant thereof. Process for preparing an enzyme preparation with pentosanase activity, characterized in that a microorganism strain of the species Talaromyces emersonii is fermented in a suitable nutrient medium, whereby a liquid containing this enzyme is produced. 6. Process according to claim 5, characterized in that the level of the pentosanase activity relative to the level of the 3-1,4 / 1,3-glucanase activity is increased by modification of the nutrient medium. Process according to claims 5 to 6, 30, characterized in that the level of pentosanase activity relative to the level of the 1,4-4 / 3-1,3-glucanase activity is increased by a purification treatment after the fermentation . Process according to any one of claims 1 to 7, 8403600 * - 17 - a: characterized in that the strain is Talaromyces emersonii IMI 116815, CMI CC No. 290604 or a mutant thereof. 9. Process for reducing the pentosan content of a pentosan-containing substance, characterized in that the substance is contacted with an enzyme or preparation according to any one of claims 1 to 4. 10. A method according to claim 9, characterized in that the pentosan-containing substance is wheat or oats, a tea or coffee plant, or an extract thereof, a fruit or a vegetable. A method according to claim 9 or 10, characterized in that the pentosan-containing substance is starch. 12. A method according to claim 9 or 10, characterized in that the pentosan-containing substance comes from wheat or from wheat. 13. Process according to any one of claims 9 to 12, characterized in that the enzyme is contacted with the pentosan-containing substance in an aqueous medium at 35-95 ° C and a pH of 4.0 to 6.5. 20 Use of a preparation containing a pentosanase enzyme derived from a strain of the species Talaromyces emersonii in decreasing the pentosan content of wheat or oats, a tea or coffee plant, or an extract thereof, a fruit or a vegetable. 25 8403600 »