SU1521760A1 - Method of obtaining glucose - Google Patents

Abstract

This invention relates to biotechnology, in particular to methods for producing glucose. The purpose of the invention is to increase the yield of glucose. The enzymatic hydrolysis of waste from the production of viscose or paper was carried out using the GEOTRICHUM CANDIDUM CPM F-220 culture. Culture support on agar mineral medium containing 0.5-3% paper and 0.5-2% wheat bran, and enzymatic hydrolysis is carried out on medium containing 0.5-3% waste production of viscose (fine fiber) or waste paper production (skop) as a carbon source. As a result, the cellulosic raw material is completely converted into glucose.

Description

The invention relates to biotechnology, in particular to a method for producing glucose by enzymatic hydrolysis of paper and rayon production waste.

The purpose of the invention is to increase the yield of glucose.

The invention is as follows.

The method involves the use of viscous production waste - fine fiber or paper production waste - skop as a cellulose-containing raw material. This waste

accumulate in the places of production of pulp, viscose, paper in the amount of about 100 thousand CtoT / g. and pollute the environment. The waste does not need to be pretreated. Organic matter is readily hydrolyzed by the combined action of endoxy-i, 4-6-glucanases and and -glucosidases. Concentration of waste containing up to 80% is also not required.

It has been established that the ratio of these enzymes, which is necessary for a completely pregragtsen substrate of cellulose-containing waste to glucose, is contained in the culture of the fungus Geotri-cvr candidura VKPM 7-220 tested on a nutrient medium containing the same waste in a concentration of 0.5–3% in as a carbon source ,,

When the concentration of the carbon source is below 0.5%, an insufficient amount of enzymes is formed, and the concentration above 3% is economically unfeasible.

The culture must be maintained on an agar medium containing 0.5–3% ground filter paper and 0.5–2% wheat bran. At concentrations of substrates lower than indicated, the producer produces a complex of enzymes of non-optimal composition, i.e., during hydrolysis the waste does not achieve a 100% yield of glucose, and the concentrations of substrates more than the indicated ones are not appropriate.

The remaining nutrient components are known for the cultivation of the fungus Geotrichum candidu VKPM F-220,

 The carbon sources used in the proposed method induce the formation of the fungus Geotrichum candidura VKPM F-220 balanced culture enzyme systems hydrolyzing fine fiber - waste from the production of viscose and scop without full pretreatment to glue. goats, as the only product of substrate hydrolysis.

The method is carried out as follows.

The culture of the fungus Geotrichum candidum VKPM F-220 is maintained for several months on an agar medium containing 0.5–3% shredded paper and 0.5–2% wheat

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bran and mineral salts. The fungus is cultivated for 90–100 h in salt or 60–72 h in a fermenter with aeration on a nutrient medium containing 0.5–3% waste from the production of viscose (fine fiber) or a scop as a carbon source, wheat bran, nitrogen sources , phosphorus and magnesium. The cultivation temperature is 28 ° C. At the end of the cultivation, the biomass is separated, and the supernatant is used to hydrolyze the waste of viscose or osprey production. If the cellulolytic activity of the supernatant is high, it is advisable to dilute it with water in such a way that the cellulolytic activity is 0.1-15 u / mp of enzymes saccharifying cotton fiber. Hydrolysis is carried out at pH 4-5 and 47 ° C. for 8 to 18 hours. As a result, the cellulose-containing substrate is completely hydrolyzed to glucose, the content of which in the final mixture is 3-6%,

Example 1, the Culture of the fungus Geotrichum candidum VKPM F-220 support on agar medium containing 1% ground filter paper, 0.5% wheat bran and mineral salts: (NH) S04 0.15%, NaNO, 0.15%, 0 , 5% and MgS04 x-7 0.05%, for 6 months, after which sterile water is added to the tube with agarized medium and the suspension of conidia with a concentration of 10 conidia in 1 ml is added to the culture medium in an amount of 2% volume. Cultivation is carried out at 28 ° C on a medium containing 0.5% fine fiber, viscose, 2% wheat bran, (NH4.) TS04 0.15%, HaAs 0.15%, KH4PO4 0.15%, MgS04 7 0.05 %, on rocking chairs in 750 ml flasks from 150 ml of medium at a rotation speed of 180 rpm for 4 days

Cellulolytic activity is determined by the ability to break down cotton fiber into reducing sugar into 0.1 M acetate buffer, pH 4.7, for 1 h. The reducing sugars are determined using the Somogyi-Nelson method. enzymes, which atalize the formation of 1 µmol of glucose per 1 minute under the conditions of the experiment. 1 MP culture fluid

contains 1 units of sugar. cotton fiber enzymes.

At the end of the cultivation, 11.4 l of Geotrichum sapidura VKPM F-220 culture liquid separated from the culture, containing 0.2 units of enzymes saccharifying cotton fiber, is added to 3.0 kg of fine fiber — a production waste

goats and incubated at pH 4.5 and 47 ° C for 8 hours. During this time, the substrate is completely hydrolyzed to glucose. Received 5% glucose solution.

Example 2 The culture of the fungus Seo-trichum Candida VKPM F-220 is maintained on an agar medium containing 0.5% paper, 2% wheat bran, and the above mineral components of the medium, Suspension containing 10 conidia in 1 ml, t in the amount of 2% to the medium for submerged cultivation, containing 3% fine fiber - waste from the production of viscose and 2% Lpenichny bran, and the mineral components listed in the indicated concentrations in tap water are added in the amount of 150 ml to 750 ml of liter flasks and 15 liter fermenters by 30 respectively, and cultured for 60 hours, 1 ml lennoy separated from the culture broth contains culture. 0.3 units of enzymes saccharifying cotton fiber, 11.4 liters of cultured liquid diluted 3 times, are added to 3.0 kg of fine fiber, -. waste from the production of viscose, incubated at pH 4.0 and 47 ° C for 18 hours. During this time, the substrate is completely hydrolyzed to glucose as the only hydrolysis product.

Example 3, the culture of the fungus is maintained on an agar medium containing 3% paper and 0.5% wheat bran. Cultivation is carried out on a medium containing 3% of the organic matter of the osprey waste of the pulp and paper industry and 2% of wheat bran, as well as the above listed mineral components of the medium, 0.2% by volume of seed containing 10 ° conidia in 1 ml, Cultivation lead under the conditions described above

1 ml of culture liquid contains 0.3 units of enzymes saccharifying cotton fiber.

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7.5 g of waste pulp and paper industry (Osprey) are incubated with 32 ml of filtrate of culture fluid Geotrichum candidum WYM F-220, diluted 3 times at pH 4.5 and. 48 ° C for 24 hours. During this time, the organic matter of the pulp and paper industry waste (skop) 0 is completely hydrolyzed to glucose, the content of which, as in other cases, was determined by the Somogyi-Nelson method, by the glucosidase method using ka-ferrocyanide) 5 LIA and by thin layer chromatography or chromatography on HPLC paper and HPLC. It has been shown that glucose is the only product of substrate hydrolysis,

20 The resulting glucose solutions were incubated with 0.1% activated carbon for 3 hours at 30 ° C, followed by ultrafiltration through a column with polyamide fibers for 25 separation of glucose and proteins. The protein solution containing endo-, exo-1,4-B-glucanase and cellobiase is again used for enzymatic hydrolysis of the waste after the activity has been adjusted to 30 after adding the culture fluid.

The glucose solution is concentrated to 80% concentration, and after seed is applied (glucose crystals), glucose crystals fall out of the solution for 1 day, which are separated by filtration, dried by washing with ethanol and air-dried.

The resulting glucose can be used as an additive in animal feed, can serve as a carbon source for refining organic acid producers, in particular amino acids, antibiotics, as well as feed yeast 45, after further purification can be used for food purposes, can be turned into fructose, sugar sweeter than sucrose

Thus, the proposed method allows to obtain, by enzymatic hydrolysis of cellulose-containing industrial wastes, one sugar-glucose with full conversion of the substrate.

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Claims (1)

Invention Formula The method of producing glucose, envisioning enzymatic hydrolysis 715217608 cellulose-containing raw material, selected-Geotriehura candidum using culgo from waste production viscose geotrichum candidum VKPM F-220, or pulping of paper prom hash which is supported on agarizanti culture of the fungus Geotrichum candi- mineral medium containing dura at optimum temperature of 0.5–3.0 wt.% ground filter media and medium saccharide paper and 0.5–2.0 wt.% pulping of sponge, and cultivating due to the fact that, in order to maintain the output on a nutrient medium, yes glucose, as a cellulose-10 containing 0.5-3.0 masl of osprey or containing raw materials, use an osprey or fine fiber pulp as fine fiber viscose, from a mushroom source carbon.