KR20130050399A - Cellulase producing armillaria gemina sku0114 and its use for saccharification - Google Patents

Abstract

PURPOSE: Armillaria gemina which produces cellulase and a use thereof for saccharification are provided to ensure excellent saccharification and to be used in various purposes such as production of low calorie foods and fermentation of food waste. CONSTITUTION: A composition for producing cellulase contains Armillaria gemina(deposit number KCCM 11186BP) which produces cellulase or a culture medium thereof as an active ingredient. A method for producing cellulase comprises a step of culturing Armillaria gemina and isolating cellulase. The culture medium of cellulase contains 10-30 g/L of rice straws, 8-10 g/L of corn steep, 2-5 g/L of yeast extract, 3-5 g/L of monobasic potassium phosphate, 3-5 g/L of potassium diphosphate, and 0.005-0.01 g/L of thiamine HCl. Cellulase is produced at 250rpm, at an infiltration rate of 0.8-1.2vvm, in pH 5, and at 30 deg. C.

Description

Cellulase producing Armillaria gemina SKU0114 and its use for saccharification

The present invention relates to a strain for producing cellulase and a saccharification method using the same.

Cellulose is the most abundant organic substance on earth and is a renewable resource that you don't have to worry about running out like oil or coal. However, most of the cellulose resources are now a major source of environmental pollution, inundated by agricultural and forest wastes. Therefore, the development of an efficient saccharification process can be a great help in solving food, fuel and environmental problems. The world's agricultural and forestry waste is more than 3 billion tonnes annually, with more than 800 million tonnes produced in Asia alone. It consists mainly of cellulose and hemicellulose, so the production of monosaccharides, including glucose by their saccharification, is an important raw material production technology of bioenergy resources. Currently, in order to recover monosaccharides from agricultural and forest waste resources, sulfuric acid is added in most cases to pressurize and decompose at high temperature. In this case, expensive production equipment that can withstand acids and high temperatures is required, and the decomposition products are also very diverse due to various side reactions, which makes the separation process difficult, and the cost of waste disposal is high, resulting in high production costs and environment-friendly conditions. have.

Therefore, there have been many studies for glycosylating cellulose with high efficiency for a long time, and a lot of research has been made, especially focusing on the development of glycosylase. As a result, various uses of these glycosylases have been developed 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, the cell wall of the plant is composed of polymers such as cellulose (insoluble β-1,4-glucan fiber), hemicellulose (hemicellulose, non-cellulose polysaccharide), and lignin (lignin, polysaccharide of complex polyphenol structure). Cellulose is the most abundant component, followed by hexylcellulose, the main component of xylan, 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 decompose it into monosaccharides, cellulose [endo-β-1,4-glucanase] [EC 3. 2. 1 4], exo-β-1,4-glucanase [EC 3. 2. 1. 91], beta-glucosidase (EC 3. 2. 1. 21) Three enzymes are required. Endo-glucanase randomly cleaves β-1,4 glucose bonds from the inside and exo-glucanase cleaves into cellobiose, a glucose disaccharide at the non-reducing sugar end. Cellobiose is finally degraded into glucose by beta-glucosidase.

Conventionally, the production of these enzymes mainly used fungi (fungi), particularly in the industrial aspect of the production of enzymes (Aspergillus) and Trichoderma (Trichoderma). As a cellulase producing strain, Tricoderma reese ZU-02 (Trichoderma reesei ATCC 56764) has been intensively studied as a representative strain, but there is a problem that the concentration and activity of the enzyme is not sufficient to meet the industrialization. For example, the process of producing ethanol from biomass has many advantages such as regeneration of raw materials and environmental friendliness of fuel produced. However, ethanol produced from lignocellulosic materials is significantly more expensive than gasoline. It is high and becomes obstacle to practical use. The largest part of the cost of this ethanol production process is about 60% of the total cost of glycosylase production. Therefore, there is an urgent need for the development of highly active strains that produce cellulase.

As a related prior art, Korean Patent Publication No. 1020000049127 relates to 'Crysporium cellulase and a method of use', and relates to novel compositions of neutral and / or alkaline cellulase and to chrysosporium cultures, in particular to chrysospore. It relates to a method for obtaining neutral and / or alkaline cellulase compositions from Leeum Luknowen, and also relates to genes encoding enzymes comprising neutral and / or alkaline cellulase compositions.

In another related prior art, Korean Patent Publication No. 1020100049223 relates to 'Bacillus rickeniformis secreting cellulase and xylanase and its use', and to Bacillus rickeniformis strain secreting cellulase and xylanase, Produced from the strain, it exhibits activity at low temperature, the optimum temperature is 40 ~ 50 ℃, the cellulase having an optimum pH of 5.5 ~ 6.5, feed additive for cellulose degradation containing the cellulase and cellulose and xylan containing the strain It relates to a feed additive for enhancing degradation.

The present invention solves the above problems, and the object of the present invention as devised by the necessity of the above is to provide a strain for producing high activity cellulase.

Another object of the present invention is to provide a method for producing cellulase using the strain.

Still another object of the present invention is to provide a method for saccharifying cellulose using the cellulase.

In order to achieve the above object, the present invention is a brown mulberry mushroom ( Arillaria) producing cellulase gemina ) (deposit number KCCM 11186P).

In addition, the present invention is the brown mulberry mushroom of the present invention ( Armillaria) gemina ) (Accession No. KCCM 11186P) or a cellulase production composition comprising the culture medium as an active ingredient.

In addition, the present invention is the brown mulberry mushroom of the present invention ( Armillaria gemina ) (accession number KCCM 11186P) provides a method of producing cellulase comprising the step of culturing in the production medium to separate the cellulase.

In one embodiment of the present invention, the production medium of the cellulase rice straw 10 ~ 30 g / L, corn steep liquor 8 ~ 10 g / L, yeast extract 2 ~ 5 g / L, potassium monophosphate 3 ~ 5 g / L, potassium diphosphate 3-5 g / L and thiamine hydrochloride 0.005-0.01 g / L, preferably

In another embodiment, the production of the cellulase is preferably performed at a stirring speed of 250 rpm, aeration rate 0.8 ~ 1.2 vvm, pH 5 and the incubation temperature at 30 ℃, but is not limited thereto.

In another aspect, the present invention is a brown mulberry mushroom of the present invention ( Armillaria gemina ) (Accession No. KCCM 11186P) provides a method for glycosylation of biomass comprising the treatment of cellulase produced from the strain.

In one embodiment of the present invention, the biomass is preferably a North American Aspen tree, but is not limited thereto.

In another embodiment, glycosylation of the biomass is preferably performed at a cellulase concentration of 20 to 42.5 FPU / g-substrate, pH 4 to 6, temperature 50 to 70 ° C, but is not limited thereto.

In addition, the present invention is the brown mulberry mushroom of the present invention ( Armillaria gemina ) (Accession No. KCCM 11186P) Provides a composition for biomass glycosylation comprising the cellulase produced from the strain as an active ingredient.

In another aspect, the present invention is characterized by the use of North American Aspen cellulose as a source of cellulose in the saccharification method of cellulose.

Brown mulberry mushroom SKU0114 isolated from the mushroom of the present invention produces a highly active cellulase, can be used for cellulose glycosylation, cellulase produced from the strain of the present invention shows better glycation yield than conventional glycosylase, so production of bioenergy In the textile, paper, detergent, feed, and food industries, it can be applied to a variety of uses, including the production of low-calorie foods and the fermentation of food waste.

1 is a result of analyzing the soft relationship with the similar species of the ITS-5.8S rDNA sequence of the strain of the present invention.
Figure 2 is a graph showing the filter paper degradation activity of the endoclucanase and enzyme of brown mulberry mushroom SKU0114 strain according to the culture time. ●; Endoclucanase activity, (circle): The production | generation rate of glucose which a unit amount of enzyme produces | disassembles and produces.
3 is a brown mulberry mushroom ( Armillaria) gemina ) endo-glucanase optimum activity pH (a) and optimal activity temperature (b) produced by the strain.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: cellulase Production  Selection

To isolate cellulase-producing strains, 10ul of various cultures of mushrooms were suspended in 10 ml of physiological saline, 10 ul (1x10 ⁴ cfu ml ^-1 ) of the suspension was taken, and 2% carboxymethyl cellulose was added. After dextrose agar) was incubated for 3 days at 27 ^° C. After colonies were formed on a solid agar medium, stained with 0.1% Congo red reagent, and decolorized with 1 M sodium chloride to screen for bacteria that produced fibrinolytic rings around the colonies. Explored.

Solid agar to which carboxymethyl cellulose was added as described above using Tricoderma Reese ZU-02, which was used as a conventional cellulase producing strain as the control (C), the first selected strain (S1 to S6) through the above search process. After confirming the fibrinolytic ability in the medium, the S4 strain having the best fibrin resolution was selected.

Example  2: Identification of the strain

In order to identify the S4 strain isolated in Example 1, the ITS-5.8S rDNA sequence was analyzed at the Korea Microorganism Conservation Center. The ITS-5.8S rDNA sequence of the S4 strain is shown in SEQ ID NO: 1.

As a result of analyzing the soft relationship with the similar species of the ITS-5.8S rDNA sequence of the S4 strain, it was identified as brown mulberry mushroom (FIG. 1).

The S4 strain is brown mulberry mushroom ( Armillaria gemina ) was named SKU0114 and was deposited internationally in accordance with the Budapest Treaty under the accession number KCCM 11186P on April 20, 2011 to the Korea Center for Microbiological Conservation.

Example  3: Medium Optimization Experiment for Cellulase Production of Strains

(One) Carbon source  Cellulase Activity Test by Type

Cellulase production experiments of the brown mulberry mushroom SKU0114 strain of the present invention according to rice straw concentration in a 7L fermenter were performed. For measuring cellulase activity, cellulose, bagasse, palm nucleus, cellobiose, carboxymethylcellulose, xylan, rice straw, avicel and the like were used as carbon sources.

Species culture: A single colony of stored brown mulberry mushrooms was inoculated into a 50 mL flask containing 50 mL of preculture medium (Potato starch 4 g / L, Dextrose 20 g / L) for 5 days at 150 rpm and 25 ° C in a shaker. Incubated.

Main culture: 50 mL of seed culture medium was produced (peptone 8 g / L, yeast extract 2 g / L, potassium dihydrogen phosphate 5 g / L, potassium hydrogen phosphate 5 g / L, magnesium sulfate (hexahydrate) 3 g / L, 0.02 g / L hydrochloric acid 0.02 g / L and rice straw 20 g / L, the final pH 5) was inoculated into a 50 ml flask was carried out for 7 days at a stirring speed of 150 rpm, incubation temperature 25 ℃, pH 5 for 7 days. Experimental results of different types of carbon source were as shown in Table 1, showing the maximum cellulase activity at a cellulose concentration of 20 g / L.

Table 1 is a table showing the production of cellulase using several carbon sources.

(2) Nitrogen source cellulase activity test

In a 7 L fermentation tank, the concentration of rice straw was 20 g / L. When the nitrogen source concentration was incubated at 10 g / L, the activity was shown in Table 2, and the maximum activity was shown in 5 g / L potassium nitrate + yeast extract.

Table 2 is a table showing the activity of the cellulase enzyme at concentrations of various nitrogen sources.

Example 4 : Experiment of Optimum Culture Condition for Highly Active Enzyme Production

(1) Optimal culture conditions of cellulase using North American Aspen as a substrate

The concentrations of peptone, yeast extract, potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate (hexahydrate), chiamine hydrochloride and rice straw in 7L fermenters were 8 g / L, 2 g / L, 5 g / L and 5 g / L, respectively. , 3 g / L, 0.02 g / L, 20 g / L was carried out to optimize the culture environmental conditions. As a result of comparing the endo-glucanase activity by changing the pH 3-6 and the culture temperature to 22-38 ?, the maximum activity was shown at pH 5 and the culture temperature 70? as shown in FIGS. 3A and 3B.

Example 5: Strain Glycation yield  analysis

In general, lignocellulosic contained in plants cannot obtain high glycation yields only by hydrolysis of enzymes. Therefore, the enzyme is subjected to a pretreatment before the hydrolysis process. In the present invention, an alkali treatment method of 2% by weight sodium hydroxide was used. This pretreatment process provides fragmentation of lignin and hemicellulose, resulting in an increase in fibrinase efficiency of the cellulase enzyme. For pretreatment, 10 g of rice straw is placed in a flask containing 40 ml of 2 wt% sodium hydroxide solution, reacted at 85 ° C. for 1 hour, and filtered through a 0.45 uM filter. Thus pretreated and filtered rice straw was used to dry at 65 ℃.

In order to explore the optimum glycosylation conditions of the strain, experiments were carried out for the concentration of enzyme, substrate concentration, reaction temperature, reaction pH. First, pretreated rice straw was added to various concentrations of cellulase in 20 ml of 0.1 M sodium acetate buffer (pH 5.0) by concentration. Cellulase-added buffer was reacted at 150 rpm at 15-55 ° C. for 72 hours, and then the reaction solution was boiled at 100 ° C. for 3 minutes, cooled at room temperature, and then centrifuged at 4000 rpm for 15 minutes to remove denatured enzyme. . Enzyme activity was measured using the supernatant as a reducing sugar measurement. The glycosylation rate was measured based on the weight of 1 g of rice straw reduced after drying the dried rice straw at 105 ° C. for 24 hours.

Quantity of reducing sugars produced / substrate g number X 0.9

Glycation yield% = ----------------------------------------- X 100

The amount of carbohydrates present in rice straw

(1) according to enzyme concentration Glycation yield  Experiment

Saccharification experiment of the brown mulberry mushroom SKU0114 strain according to the enzyme concentration in 500 ml Erlenmeyer flask was performed. As a result of experiments at 50-70 ° C. by treating various concentrations of enzymes, the glycosylation yield was as shown in Table 3, and preferably, when the enzyme of 30 to 42.5 FPU / g-substrate was used, the optimum glycation yield was obtained.

Enzyme Concentration (FPU / g-substrate) Glycation yield (%) One 8.6 5 21.2 17.5 63.0 30 79.00 42.5 71.6

(2) depending on substrate concentration Glycation yield  Experiment

The effect of substrate concentration on the glycosylation of North American Aspen trees by the saccharase produced by the strain SKU0114 strain brown mulberry mushroom of the present invention was confirmed. North American Aspen Tree Table 4 shows the results of culturing with different initial concentrations of 1 to 27% by weight. The saccharification yield was excellent at 2-10 wt%, and the optimal saccharification yield was found at 10 wt% North American Aspen concentration.

Substrate concentration (%) Glycation yield (%) One 59 2 67.2 10 78.3 20 64.3 27 55.0

(3) according to temperature Glycation yield  Experiment

The effect of temperature on the saccharification of North American Aspen tree by the saccharase produced by the strain SKU0114 strain brown mulberry mushroom of the present invention was confirmed. Table 5 shows the results of culturing with different reaction temperatures at 20, 35, 50, 65, and 80 ^° C., respectively. The saccharification yield was excellent at 75.5 ~ 80 ℃, and the optimum saccharification yield was shown at 65 ℃.

Reaction temperature ( ^o C) Glycation yield (%) 20 32.6 35 52.4 50 69.6 65 75.5 80 74.9

(4) pH In accordance Glycation Experiment

The effect of pH on the saccharification of sheeps by the saccharifying enzyme produced by the strain brown mulberry mushroom SKU0114 strain of the present invention was confirmed. Table 6 shows the results of saccharification by varying the reaction pH to 1, 3, 5, 7, 9, respectively. The saccharification yield was excellent at pH 5-7, and the optimum saccharification yield was shown at pH 5.

pH Glycation yield (%) One 23.1 3 52.1 5 78.1 7 76.0 9 51.3

Example  6: at optimum conditions Glycation Experiment

(1) Saccharification Experiment of North American Aspen Tree Using Brown Mulberry Mushroom Cellulase

The saccharification experiment using the glycosylation enzyme of strain SKU0114 strain brown mulberry mushroom of the present invention under conditions optimized in the reactor. Enzyme concentration in the reaction solution was adjusted to 20 ~ 42.5 FPU / g-substrate, pH was 4 ~ 6 and the temperature was adjusted to 50 ~ 70 ℃. Table 7 shows the glycosylation yield of each substrate under optimized conditions. As a result of investigating the saccharification efficiency of North American Aspen by cellulase of brown mulberry mushroom SKU0114 according to the reaction time The maximum glycosylation rate was 79%. Table 7 shows the cellulase produced from the brown mulberry mushroom SKU0114 strain of the present invention and the celluclast 1.5L derived from Novozyme's Tricoderma ressay by comparing the saccharification yields at the optimum conditions.

Strain Sugar yield (mg / g-North American Aspen) Glycation yield (%) Brown Mulberry Mushroom 597 79 Novozyme (Celluclast 1.5L) 282 37.3

Korea Microorganism Conservation Center (overseas) KCCM11186P 20110420

Claims (9)

Brown Mulberry Mushroom ( Arillaria) Producing Cellulase gemina ) (accession number KCCM 11186P). Brown mulberry mushroom of claim 1 ( Armillaria gemina ) (Accession No. KCCM 11186P) or a cellulase production composition comprising the culture medium as an active ingredient. Brown mulberry mushroom of claim 1 ( Armillaria gemina ) (Accession No. KCCM 11186P) culturing in a production medium to produce a cellulase comprising the step of separating the cellulase. According to claim 3, wherein the cellulase production medium is rice straw 10-30 g / L, corn steep liquor 8-10 g / L, yeast extract 2 ~ 5 g / L, potassium monophosphate 3 ~ 5 g / L, Potassium phosphate 3 ~ 5 g / L and thiamine hydrochloride 0.005 ~ 0.01 g / L production method characterized in that it comprises a. The method of claim 3 or 4, wherein the production of the cellulase is carried out at a stirring speed of 250 rpm, aeration rate 0.8 ~ 1.2 vvm, pH 5 and the incubation temperature of 30 ℃. Brown mulberry mushroom ( Arillaria ) of claim 1 on a biomass substrate gemina ) (Accession No. KCCM 11186P) Method for glycosylation of biomass comprising the step of treating the cellulase produced from the strain. The method for saccharifying biomass according to claim 6, wherein the biomass is North American Aspen. The method of claim 6, wherein the saccharification of the biomass is a method of saccharification of biomass, characterized in that carried out at a cellulase concentration 20 ~ 42.5 FPU / g- substrate, pH 4 ~ 6, temperature 50 ~ 70 ℃. Brown mulberry mushroom of claim 1 ( Armillaria gemina ) (Accession No. KCCM 11186P) Biomass glycosylation composition comprising a cellulase produced from the strain as an active ingredient.

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