KR20030062884A - Cellulases and xylanase-producing Aspergillus niger KK2, produced enzymes and solid materials thereof - Google Patents

Abstract

PURPOSE: Cellulase and xylanase-producing Aspergillus niger KK2, and a process for preparing enzymes and solid culture products using the same microorganism are provided, thereby cheaply producing cellulase and xylanase in higher yield. CONSTITUTION: Cellulase and xylanase-producing Aspergillus niger KK2(KFCC-11285) is provided. A process for preparing enzymes comprises the steps of: seed culturing the cellulase and xylanase-producing Aspergillus niger KK2(KFCC-11285); inoculating the seed cultured medium into a broth or solid medium;culturing the broth or solid medium; and recovering the cultured medium, wherein the enzyme is selected from FPase(filter paper decomposing enzyme), CMCase(carboxymethylcellulose F decomposing enzyme), beta-glucosidase and beta-xylanase; the broth medium contains the combination of rice-straw and wheat bran, and the solid medium contains single or combination of rice-straw and wheat bran; and the solid medium has the water content of 50 to 80%.

Description

Aspergillus niger producing cellulase and xylanase (ｘｙｌａｎａｓｅ) 균주 2 strains and enzymes and solid cultures produced by it }

The present invention relates to a novel strain producing high concentrations of cellulase and xylanase, and to a method for producing enzymes and solid cultures produced by the strain. More specifically, the present invention provides a novel Aspergillus Niger which produces high concentrations of cellulase and xylanase with cellulose and xylan resolution.

Aspergillus niger The present invention relates to a method for producing solid cultures produced by screening KK2 strains and enzymes and solid cultures through liquid and solid cultures thereof.

Cellulose and xylan are the most abundant organic matter on earth, and are forever renewable resources without worrying about depletion like oil or coal. On the other hand, these resources are also a major source of environmental pollution in the present day when agricultural waste is flooded. Therefore, the development of an efficient saccharification process for these polysaccharides can be a great help in solving food, fuel and environmental problems.

Therefore, there have been many studies for glycosylating cellulose and xylan for a long time, and a lot of research has been focused on the development of glycosylase among them. As a result, the use of these glycosylation enzymes has been developed in various ways and commercialized in various fields, and new applied researches are being actively conducted.

Cellulase is widely used in the textile industry, the paper industry, the detergent industry, the feed industry, etc. In addition to the food industry, it is applied to various uses such as the production of low-calorie foods and the fermentation of food waste. On the other hand, xylanase (xylanase) has been applied to a lot of paper industry, food industry, feed industry.

In plants, cell walls are cellulose (insoluble β-1,4-glucan fiber), hemicellulose (hemicellulose, galactan; galactan, mannan; mannan, xylan, including xylan It is composed of polymers such as noncellulosic polysaccharides, lignin, and polyphenolic structure, and the most cellulose is present among the components, followed by xylan. Its main component, hemicellulose, is abundant and these two components make up more than 50% of the total plant biomass.

Cellulose is a homopolymer in which glucose units are linked by β-1,4 bonds. In order to completely decompose cellulose, cellulose may be endo-β-1,4-glucanase [ EC 3. 2. 1. 4; endo-glucanase, CMCase (CMC; carboxymethylcellulose)], exo-β-1,4-glucanase [EC 3. 2. 1. 91; Three kinds of enzymes are required: exo-glucanase, cellobiohydrolase, FPase (FP; filter paper)] and beta-glucosidase (EC 3. 2. 1. 21; cellobiase). Endo-glucanse randomly cleaves β-1,4 glucose bonds from the inside and exo-glucanase cleaves it with cellobiose, a glucose disaccharide at the non-reducing sugar end. I'm going. Cellobiose is finally broken down into glucose by beta-glucosidase.

Xylan is a heteropolymer in which acetate, arabinose or glucuronic acid are branched on xylose backbones connected by β-1,4-glycosidic bonds. (heteropolymer). In general, in order to hydrolyze xylan, endo-β-1,4-xylanase (EC 3. 2. 1. 8; endo-xylanase), beta-xylosidase ( Two enzymes, β-xylosidase (EC 3. 2. 1. 37), are required. Endo-xylanase breaks down xylan to produce xylose, xylobiose, and xylo-oligosaccharides, which contain undecomposed xylobiose and Xylo-oligosaccharides are converted to xylose by beta-xylosidase.

In the prior art, the production of these enzymes has been generally used a mold (fungi), in particular the enzyme production in the industrial aspect has been mainly used for Aspergillus (Aspergillus) Wat Rico Delmas (Trichoderma). Cellulase-producing strains were intensively studied as Trichoderma reesei , and xylanase was mainly studied in Bacillus and Aspergillus genus. However, there was a problem that the concentration and activity of enzymes were not sufficient to meet the industrialization, and the production cost was due to the use of expensive purified medium containing expensive substances such as lactose as substrates for the production of these enzymes. There was a high issue. For example, the process of producing ethanol from biomass has many advantages such as regeneration of raw materials and environmental friendliness of fuel produced, but ethanol produced from lignocellulosic materials is produced compared to gasoline. The unit price is so high that it becomes an obstacle to practical use. The largest part of the cost of this ethanol production process is about 60% of the total cost of enzyme production.

Therefore, an object of the present invention is to provide an enzyme produced economically through the selection of a new strain with high productivity of the enzyme and the development of a medium with low production cost.

An object of the present invention is to select an improved Aspergillus niger KK2 strain that produces high concentrations of cellulase and xylanase using mutations by UV irradiation, and includes rice straw, bran, A low cost medium containing other nutrient fortifications has been developed and achieved.

Hereinafter, the specific configuration of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Figure 1 shows the change in the activity of xylanase (xylanase) with the incubation time when inoculated with Aspergillus niger KK2 of the present invention in solid culture using rice straw.

Figure 2 in solid culture with rice straw, when inoculated with Aspergillus niger KK2 of the present invention according to the culture time according to the CMCase (beta)-beta-glucosidase (β-glucosidase), beta- It shows a change in the activity of xyloxysidase (β-xylosidase).

Figure 3 shows the change in activity of xylanase when inoculated with Aspergillus niger KK2 of the present invention in a solid culture in which rice straw and bran are mixed in a constant ratio.

Figure 4 in a solid culture mixed with rice straw and bran in a constant ratio, when inoculated with the Aspergillus niger (K) aspergillus niger (CMCase), beta-glucosidase (β-glucosidase), beta -Changes in activity of xylosidase (β-xylosidase).

The present invention relates to an Aspergillus niger KK2 (KFCC-11285) strain that produces cellulase and xylanase.

In addition, the present invention comprises the steps of spawn culture of the strain;

Provided is a method for producing an enzyme, characterized in that consisting of the step of recovering the seed culture in the incubator containing the culture medium.

Hereinafter, Aspergillus niger KK2 screening of the present invention

It will be described in detail with respect to the preparation method of the cellulase (cellulase), xylanase (xylanase) using the same.

First, the Aspergillus niger KK2 screening process is described in detail. Aspergillus niger KKS parent isolate isolated from soil was inoculated in a PDA (potato dextrose agar) slope medium, incubated at 28 ° C for 7 days, and then suspended in spores by adding 10 mL of 0.1% Tween 80 solution. good. Aspergillus niger KKS spore suspension is transferred to a sterile petri dish and irradiated for 10 to 40 seconds with a 30W UV lamp at a height of 50 cm. Preferably it is good to irradiate for 25 seconds. Aspergillus niger UV-irradiated Aspergillus niger KKS spore suspension was plated in a selective medium adjusted to pH 7 and incubated in a 28 ° C. incubator for 6 days, resulting in the degradation of colony size and birch xylan. It is recommended to select strains with a ratio of clear zone size of 0.7 or less.

Aspergillus niger KK1 is selected by repeated experiments in the same manner. Aspergillus Niger KK1 strain was prepared by spore suspension in the same manner as above, and injected into a cap tube. The gamma ray (γ-ray) emitted from a radiation mutagen [ ⁶⁰ Co] was 0 to 2.5 KGy. It is good to investigate. Preferably, the irradiation is at 1.25 KGy. Aspergillus niger KK1 spore suspension irradiated with gamma rays is preferably plated on the screening medium. After six days of incubation in a plated sorting medium, strains with a ratio of colony size and clear zone size resulting from degradation of birch xylan are recommended to be screened at 0.5 or less. Aspergillus niger ) KK2 was selected and deposited in Korea Microorganism Conservation Center (Accession No .: KFCC-11285) on November 14, 2001.

The selected strains by the above method has a productivity of the enzyme significantly excellent bacteriological properties as compared to the tricot Delmas (Trichoderma) and in Aspergillus (Aspergillus) in the isolates collected from the natural world.

Second, the method for producing cellulase, xylanase using selected Aspergillus niger KK2 will be described in detail. The cellulase and xylanase can be prepared by two culture methods of liquid culture and solid culture, which will be described in detail below in the case of liquid culture and solid culture. Particularly in the case of solid culture The medium will be described in detail, each divided into a form containing the rice straw and bran alone or combined.

Detailed description of the production of enzymes by liquid culture is as follows.

Aspergillus Niger KK2 strains were inoculated on a PDA (potato dextrose agar) slope medium and incubated at 28 ° C. for 7 days, and then 0.1% Tween 80 solution was added to suspend spores. 5-15 mL of the spore suspension having 2-5 5 10 ⁷ spores per 1.0 mL was inoculated into a 250 mL Erlenmeyer flask containing 80-100 mL of malt extract, followed by 200 ALPM in a 28 ° C shake incubator. It is recommended to seed culture for 48 hours at rpm. Preferably, a spore suspension containing 3.5 × 10 ⁷ spores is inoculated into a seedling medium containing 90 mL of malt extract.

250 mL Erlenmeyer flask containing 45-50 mL of this culture medium was sterilized for 20 minutes at 121 ° C and 1.5 atm, and then inoculated with 2.0-3.0 mL of the spawn culture solution and incubated at 200 Alm (rpm) for 5 days in a 28 ° C shaker. Good to do. Preferably, 47.5 mL of the main culture medium and 2.5 mL of the seed culture medium are used. The composition of this culture medium is shown in Table 1.

ingredient Furtherance (%) Rice straw (size less than 0.3 mm) 2-3 Bran (wheat bran, 0.4 mm or less) 1 to 1.5 Corn Steep Liquor 3 to 6 Industrial yeast extract 0.1 to 0.15 Monobasic Potassium Phosphate 0.5 to 0.8 Copper sulfate pentahydrate 0.05 0.01 Adjust pH = 7.0 with 5 N sodium hydroxide

In the composition of the culture medium, preferably, 2.5% rice straw, 1.25% bran, 4.5% corn concentrate, 0.125% industrial yeast extract, and 0.65% monobasic potassium phosphate may be used.

After centrifugation of the main culture (main culture) for 10 minutes at 3000 rpm (rpm) it is good to take the supernatant to recover the enzyme.

Detailed description of the solid culture of Aspergillus niger KK2 using rice straw is as follows.

Aspergillus niger ( Aspergillus niger ) KK2 strains were inoculated on a PDA (potato dextrose agar) slope medium and incubated at 28 ° C. for 7 days. Measure the spore number of Aspergillus niger KK2 spore suspension under a microscope using a hemocytometer to measure 5 × 10 ⁵ spores per gram of rice straw. It is good to inoculate. The medium is prepared by adding nutrient medium for solid culture to 5.0 grams of rice straw and adding water to adjust the initial moisture content to 50 to 80%. Preferably, the initial moisture content is adjusted to 65%. Sterilizing the medium for 20 minutes at 21 ℃, 1.5 atm.

Nutritional medium for solid culture ingredient Composition for 100g rice straw Corn Steep Liquor 40 to 60 mL Industrial yeast extract 0.4 to 0.6 g Monobasic Potassium Phosphate 4.0-6.0 g Copper sulfate pentahydrate 0.4 to 0.6 g Cobalt Sulfate Heptahydrate 0.05-0.15 g Adjust pH = 6.5-7.5 with 5 N sodium hydroxide

Among the media components, preferably, 50 mL of corn concentrate, 0.5 g of industrial yeast extract, 5.0 g of potassium phosphate monobasic, 0.5 g of copper sulfate pentahydrate, and 0.1 g of cobalt sulfate heptahydrate are used. The pH is preferably adjusted to 7.0.

Inoculate spores evenly distributed in the medium, and then well mixed and incubated for 7 days at 28 ℃ incubator. 0.1% Tween 80 solution was added to the culture complete solid culture, homogenized with 12,000 Alp (rpm) for 5 minutes by homogenizer, and centrifuged at 3,000 Alp (rpm) for 10 minutes to collect supernatant to recover enzyme. It is good.

Detailed description of the solid culture of Aspergillus niger KK2 using bran is as follows.

It is preferable to adjust the initial moisture content to 50-80% by adding the nutrient medium for solid culture of Table 2 to 5.0 g of wheat bran and adding water. Preferably, the initial moisture content is 65%. The medium is sterilized at 121 ° C. and 1.5 atm for 20 minutes. Since the solid culture is the same as the solid culture using the rice straw, fermentation is good to proceed for 5 days.

Detailed description of the solid culture of Aspergillus niger KK2 strain using a mixture of rice straw and bran is as follows.

The nutritional medium for the solid culture of Table 2 is preferably added to the rice straw and wheat bran mixed medium of the ratio of rice straw and wheat bran 4 to 1, 3 to 2, 2 to 3, 1 to 4. It is advisable to adjust the initial moisture content to 50 to 80% by adding water. Preferably, the initial moisture content is 65%. The medium is sterilized at 121 ° C. and 1.5 atm for 20 minutes. Since the solid culture is the same as the solid culture using the rice straw, fermentation is good to proceed for 5 days.

Hereinafter, the specific configuration and operation of the present invention will be described in detail by way of examples.

Example 1: Aspergillus Niger ( Aspergillus niger ) KK2 Screening

Aspergillus niger KKS parent strain isolated from the soil was inoculated in a PDA (potato dextrose agar) slope medium, incubated at 28 ° C. for 7 days, and 10 mL of 0.1% Tween 80 solution was added to suspend the spores. Aspergillus niger KKS spore suspension was transferred to a sterile petri dish and irradiated with 30W ultraviolet light at 50 cm for 25 seconds. UV-irradiated Aspergillus niger KKS spore suspension was adjusted to pH 7 [1.0% birch wood xylan, 0.5% proteose peptone, 0.05% Yeast extract, 0.5% potassium monobasic phosphate, 0.05% copper sulfate pentahydrate, 0.01% cobalt sulfate heptahydrate, 0.1% triton X-100, 1.8% agar] After smearing on, incubation for 6 days in a 28 ℃ incubator was selected strains with a ratio of less than 0.7 of the size of the colony and the clear zone (clear zone) resulting from the decomposition of birch xylan.

Aspergillus niger KK1 strains were selected by repeated experiments in the same manner. Aspergillus niger KK1 strain was prepared in the above manner, spore suspension was injected into a cap tube with a lid, and gamma rays (γ-ray) emitted from a radiation mutation [ ⁶⁰ Co] were injected. Irradiated with 1.25 KGy. Gamma-irradiated Aspergillus niger KK1 spore suspension was plated on the selection medium. After 6 days of incubation at 28 ° C, strains with a ratio of colony size and clear zone size resulting from degradation of birch xylan were below 0.5 were selected, and Aspergillus niger KK2 was finally selected. It was deposited on November 14, 2001 at the Korea Microorganism Conservation Center (Accession No .: KFCC-11285).

Example 2: Aspergillus Niger ( Aspergillus niger ) Liquid culture of KK2 bacteria

Aspergillus Niger KK2 strain was inoculated on a PDA (potato dextrose agar) slope medium and incubated at 28 ° C. for 7 days, and then spores were suspended by adding 0.1% Tween 80 solution. 10 mL of the spore suspension (3.5 × 10 ⁷ spores per 1.0 mL) was inoculated into a 250 mL Erlenmeyer flask containing 90 mL of malt extract, followed by 48 hours at 200 Alp (rpm) in a shaker at 28 ° C. Seed culture was performed. The 250 mL Erlenmeyer flask containing 47.5 mL of the culture medium was sterilized for 20 minutes at 121 ° C. and 1.5 atm. The culture medium contains 2.5% rice straw (0.3 mm or less), 1.25% wheat bran (0.4 mm or less), 4.5% corn steep liquor, 0.125% industrial yeast extract (industrial yeast extract), 0.65% monobasic potassium phosphate, 0.05% copper sulfate pentahydrate, 0.01% cobalt sulfate heptahydrate and pH was adjusted to 7.0 with 5N sodium hydroxide.

After the main culture (main culture) was centrifuged for 10 minutes at 3000 rpm (rpm), the supernatant was taken to measure the enzyme activity. The spawn and main culture showed rheological behavior different according to the morphological characteristics of Aspergillus niger KK2, which influenced enzyme production.

In several replicates, the average range of beta-glucosidase, beta-xylosidase, and xylanase activities ranged from 8.5 to 10.8 IU per 1.0 mL of main culture, 14.1 It was -15.8 IU and 380-613 IU. The maximum activities of beta-glucosidase, beta-xylosidase and xylanase were 11.8 IU, 16.5 IU and 665 IU per 1.0 mL of the culture, respectively.

Example 3: Aspergillus niger using rice straw Aspergillus niger ) KK2 solid culture

Aspergillus NigerAspergillus niger) KK2 strains were inoculated on a PDA (potato dextrose agar) slope medium and incubated at 28 ° C. for 7 days, and then spores were suspended by adding 0.1% Tween 80 solution. 5 × 10 per gram of rice straw by measuring the spore number of Aspergillus Niger KK2 spore suspension under a microscope using a hemocytometer.⁵The medium was inoculated to spores. The medium was composed by adding a medium culture medium to 5.0 grams of rice straw and sterilized for 20 minutes at 121 ℃, 1.5 atm after adding water to adjust the initial moisture content to 65%. Solid culture nutrition medium contains 50 mL of corn steep liquor, 0.5 g of industrial yeast extract, 5.0 g of monobasic potassium phosphate, 0.5 g of copper sulfate pentahydrate, and 0.1 g of rice straw per 100 g of rice straw. Cobalt sulfate heptahydrate was added, and the pH was adjusted to 7.0 with 5N sodium hydroxide. The inoculated spores were mixed well so as to be uniformly dispersed in rice straw, and then cultured in an incubator at 28 ° C. for 7 days.

0.1% Tween 80 solution was added to the complete culture and homogenized with a homogenizer of 12,000 Alp (rpm) for 5 minutes, followed by centrifugation at 3,000 Alp (rpm) for 10 minutes to obtain a supernatant. Enzyme titer was determined by appropriate dilution of the supernatant. The maximum activity of xylanase was 5,071 IU per 1.0 g of solid culture after solid culture for 5 days, and the productivity at this time was 14,790 IU / l · h. (FIG. 1) Exo-β-1 The maximum activity of FPase, which is, 4-glucanase (exo-β-1,4-glucanase), was 19.5 IU per 1.0 g of solid culture on day 4 of solid culture. In addition, endo-β-1,4-glucanase (CMCase), beta-glucosidase and beta-xylosidase (β-xylosidase) Maximal activity was 130 IU, 101 IU and 193 IU per 1.0 g of solid culture in solid culture for 6 days (FIG. 2).

Example 4: Aspergillus niger using bran Aspergillus niger ) KK2 solid culture

50 g of corn steep liquor per 100 g of rice straw, 0.5 g of industrial yeast extract, 5.0 g of potassium phosphate monobasic, 0.5 g of copper sulfate pentahydrate, and 0.1 g of cobalt sulfate heptahydrate After adding them in the ratio, 5N sodium hydroxide was added, and the medium for nutrient culture medium adjusted to pH 7.0 was added to 5.0 g of wheat bran, and water was added to adjust the initial moisture content to 65%. Sterilized for 20 minutes at 1.5 atm. Since the solid culture method is the same as in Example 4 above, but the culture period was 5 days. Exo-β-1,4-glucanase (FPase), endo-β-1,4-glucanase (endo-β-1,4-glucanase) Phosphorus CMCase, β-glucosidase, xylanase and beta-xylosidase activity were 8.8 IU and 118 IU per 1.0 g of solid culture, respectively. , 96 IU, 1,470 IU, 199 IU.

Example 5: Aspergillus niger using rice straw and bran Aspergillus niger ) KK2 solid culture

50 g of corn steep liquor per 100 g of rice straw, 0.5 g of industrial yeast extract, 5.0 g of potassium phosphate monobasic, 0.5 g of copper sulfate pentahydrate, and 0.1 g of cobalt sulfate heptahydrate After adding these by ratio, 5.0 g of rice straw with a ratio of rice straw and wheat bran in a ratio of 4 to 1, 3 to 2, 2 to 3, and 1 to 4, respectively, was added to 5N sodium hydroxide and adjusted to pH 7.0 After adding to the bran mixed medium and water to adjust the initial moisture content to 65% and sterilized for 20 minutes at 121 ℃, 1.5 atm. Since the solid culture method is the same as Example 4 above, but the fermentation period was for 5 days.

3 and 4, FPase, endo-β-1, which is exo-β-1,4-glucanase when mixed with rice straw and wheat bran in a constant ratio and cultured solid CMCase, beta-glucosidase, xylanase and beta-xylosidase, endo-β-1,4-glucanase ) Activity ranges were 10.4-11.9 IU, 118-130 IU, 95-107 IU, 1,884-4,940 IU, and 211-239 IU per 1.0 g of solid culture, respectively.

Rice straw and wheat bran ratio showed the maximum activity of the enzyme, respectively. Exo-β-1,4-glucanase (FPase) and endo-β-1,4-glucanase (endo-β-1,4-glucanase) The maximum activity of phosphorus CMCase was 11.9 IU per 1.0 g of solid culture with rice straw and bran ratio of 4 to 1, and 130 IU per 1.0 g of solid culture with straw of wheat straw and bran ratio of 3 to 2. Glucosidase (β-glucosidase) showed similar enzyme activity of 95-107 IU per 1.0g of solid culture, even if the ratio of rice straw and bran was changed. On the other hand, the maximum activity of xylanase and beta-xylosidase was 4,940 IU per 1.0g of solid culture and rice straw and bran ratio of 1 to 4 when the ratio of rice straw and bran was 4 to 1, respectively. When 239 IU per 1.0 g of solid culture.

As described above, the present invention provides a novel method for producing enzymes having high concentration and high activity in a low-cost medium in the production of cellulase and xylanase. Aspergillus niger KK2 has been screened and cultured in a cheap medium with rice straw and wheat bran alone or in combination with enzymes having high concentration and high activity in low cost medium. Since it is effective to provide the solid culture is a very useful invention in the microbial industry and enzyme manufacturing industry.

Claims (11)

Aspergillus niger KK2 (KFCC-11285), which produces cellulase and xylanase Spawning the strain of claim 1; Enzyme production method characterized in that for producing the enzyme by the step of recovering the culture medium after the liquid or solid culture in the incubator containing the culture medium containing the seed culture medium. The method of claim 2, wherein the enzyme selects any one of Ffaase, CMCase, beta-glucosidase, beta-xylosidase, and beta-xylanase. The method of claim 2, wherein the medium of the liquid culture comprises rice straw and wheat bran combined, and the solid culture medium comprises rice straw and wheat bran alone or in combination. The method of claim 2, wherein the culture medium comprises sorghum concentrate, industrial yeast extract, monobasic potassium phosphate, copper sulfate pentahydrate, and cobalt sulfate heptahydrate. The method for producing an enzyme according to claim 2, wherein the medium of the solid culture has a water content of 50 to 80%. The method according to claim 4, wherein when the rice straw and wheat bran are included in the solid culture medium, the mixing ratio of rice straw and bran is selected from four to one, three to two, two to three, and one to four. Method for producing an enzyme, characterized in that Method for preparing a solid culture obtained by solid culture of the strain of claim 1 (KFCC-11285) The method of claim 8, wherein the solid culture medium is a method for producing a solid culture, characterized in that it comprises rice straw and wheat bran alone or combined. 9. The method of claim 8, wherein the medium of the solid culture comprises a corn concentrate, industrial yeast extract, monobasic potassium phosphate, copper sulfate pentahydrate, cobalt sulfate heptahydrate. 9. The method of claim 8, wherein the medium of the solid culture has a water content of 50 to 80%.