KR101135178B1 - Cellulase producing Schizophyllum commune and its use for saccharification - Google Patents

Abstract

The present invention relates to a Schizophyllum commune producing a cellulase ( Schizophyllum commune ) and a saccharification method using the same, Cellulase produced from the strain of the present invention shows a better glycation yield than the conventional glycosylation enzymes in the production of bioenergy, textile industry It can be applied to various applications such as paper, paper industry, detergent industry, feed industry and food industry.

Cellulase, Saccharification, Bioenergy

Description

Cellulase producing Schizophyllum commune and its use for saccharification

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

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 diverse due to various side reactions, which makes the separation process difficult and the production cost is high due to the cost of waste disposal and environmentally unfriendly. Holding it.

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 to 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 the production of glycosylating enzyme. Therefore, there is an urgent need for the development of highly active strains that produce cellulase.

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 provides a Schizophyllum commune producing cellulase.

In one embodiment of the present invention, the Schizophyllum commune ( Schizophyllum commune ) is preferably an accession number KMJ820 (KACC93084P), but is not limited thereto.

In another aspect, the present invention provides a method for producing cellulase comprising culturing the Schizophyllum commune of the present invention in a cellulase production medium.

The production method of the present invention is cultivated Schizofil column; It provides a method for producing the enzyme of the present invention, comprising collecting the enzyme of the present invention from a culture.

In one embodiment of the present invention, the production medium of the cellulase is preferably used as a carbon source selected from the group consisting of carboxymethyl cellulose, Avicel, rice straw, sugar cane bagasse, tangerine peel, and cellulose powder, but is not limited thereto. Not.

In one embodiment of the present invention, the carbon source is preferably 10 ~ 40g / L concentration, the production medium of the cellulase is one selected from the group consisting of yeast extract, peptone, potassium monophosphate, potassium diphosphate and magnesium sulfate It is preferable to further include the above materials, but is not limited thereto.

In addition, in a preferred embodiment of the present invention, the culture is preferably incubated at a stirring speed of 200 to 350 rpm, pH 4 to 7 or a culture temperature of 29 to 37 ℃ or a combination thereof, but is not limited thereto.

In another aspect, the present invention provides a composition for producing cellulase comprising the strain of the present invention or a culture solution thereof as an active ingredient.

The present invention also provides a method for saccharifying cellulose comprising treating a cellulase produced from the Schizophyllum commune of the present invention to a substrate.

In a preferred embodiment of the present invention, the concentration of the cellulase is 37.5 ~ 87.5 U / (cellulose) g, the substrate cellulose concentration is 3.5 ~ 5.5% by weight, the reaction pH is 3 ~ 7, the reaction temperature is 25 ~ It is preferred to glycosylate cellulose at 45 ° C. but is not limited thereto.

In one embodiment of the invention, the cellulose source substrate is preferably, but not limited to, one-sided.

Cellulase in the present invention is an enzyme that hydrolyzes β-D-glycosidic bond in cellulose. Cellulolytic enzymes can be classified into three main classes: endo-beta-1,4-glucanase (endo-β-1,4-

glucanase, EC 3.2.1.4, endoglucanase, exoglucanase or cellobiohydrolase (EC 3.2.1.91), and beta-glucosidase (EC 3.2.1.21).

As the culturing method of the strain of the present invention, any method used for culturing ordinary microorganisms, such as batch type, flow batch type, continuous culture, reactor type, or the like can be used.

Incidentally, the culturing temperature accumulates and recovers cellulase in the cells by culturing in the above-mentioned temperature range.

Enzyme proteins of the invention can be isolated or purified. Such isolation or purification involves centrifugation of the cells or supernatants from the culture obtained by culturing the strains of the present invention to disrupt the cells or remove unwanted proteins from whole cells, debris, foreign proteins or the final composition. Separation techniques recognized in the art for example ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation, size exclusion chromatography, filtration, gel electrophoresis or gradient phase separation Or a suitable combination thereof.

Confirmation that the obtained purified substance is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis, western blotting or the like.

Enzymes of the invention may include, for example, activators, anti-inhibitors, preferred ions, compounds that adjust pH or other enzymes, etc. to enhance the desired activity of the invention.

The enzymes of the present invention can be formulated into a variety of forms, such as powders, pellets, or granules of suitable size using common formulators.

The enzyme of the present invention formulated above may be used as it is, or it may be dried at room temperature or dried by freeze drying or the like.

Specific examples of the object that can be hydrolyzed by the enzyme of the present invention include plant stems, leaves, straw, genus, bran, damaged grains, fruits, vegetables and the like after processing the shells, seeds, leaves, clusters, shells, juices, etc. This includes. More specifically, for example, but not limited to corn hull, corn cop, sugarcane bagasse, rice straw rice husk and rice bran Etc. The object can be converted into various sugars by hydrolysis with the enzyme of the present invention. Therefore, there is an advantage that can be recycled these agricultural residues while preventing environmental pollution.

Schizopilum commune KMJ820 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 glycosylation enzymes of bioenergy In the production, textile, paper, detergent, feed, and food industries, it can be applied to a variety of uses, such as the production of low-calorie foods and the fermentation of food waste.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are described for the purpose of illustrating 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, 10ul (1x10 ⁴ cfu ml ^-1 ) of the suspension was taken, and the agar medium (potato dextrose) added with 2% carboxymethyl cellulose was added. agar) and incubated at 27 ° C. for 3 days. 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 S8) through the above search process. After confirming the fibrinolytic ability in the medium, the S6 strain having the best fibrin resolution was selected.

Example  2: Identification of the strain

In order to identify the S6 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 S6 strain is shown in SEQ ID NO: 1.

As a result of analyzing the soft relationship with the analogous species of the ITS-5.8S rDNA sequence of the S6 strain, it was identified as Schizofilum coccum (FIG. 1).

The S6 strain was named as Schizophyllum commune KMJ820, and was deposited with KACC93084P on November 9, 2009 at the Korea Agricultural Microbiological Conservation Center in Gwonseon-gu, Gyeonggi-do, Korea .

Example  3: Medium Optimization Experiment for Cellulase Production of Strains

(One) Carbon source  Cellulase Activity Test by Type

Cellulase production experiments of the strain Schizofilum commun KMJ820 strain of the present invention according to the type of carbon source in a 7L fermenter were performed. For measuring cellulase activity, beta-glucosidase and exo-β-1,4-glucanase are para-nitrophenyl glucose (pNPG), para-nitrogen substrates with p-nitrophenyl (pNP) groups. -Nitrophenyl cellobiose (pNPC) was used, respectively, and the activity of endo-β-1,4-glucanase was measured by using a somogyi-nelson method using a reducing sugar.

Species culture: A single colony of stored Sjozofilum commune was inoculated into a 500 mL flask containing 50 mL of preculture medium (Potato starch 4g / L, Dextrose 20g / L) and incubated at 200 rpm, 30 ° C. for 3 days in a shaker.

Main culture: 200 mL of seed culture medium containing 4L of medium (20g / L carbon source, yeast extract 2g / L, peptone 8g / L, potassium monophosphate 5g / L, potassium diphosphate 5g / L, magnesium sulfate 3g / L) Inoculated in a 7L fermenter in the present culture was carried out for 6 days at a stirring speed of 200rpm, incubation temperature 33 ℃, pH 5.

Carbon source
(20 g / L) Endo-β-1,4-glucanase (U / mg-protein) Exo-β-1,4-glucanase (U / mg-protein) β-glucosidase (U / mg-protein) Carboxymethyl Cellulose 15.1 4.0 14.2 Avicel 6.5 9.7 23.2 Straw 5.1 2.2 8.9 Sugar cane bagasse 4.1 3.7 7.4 Tangerine peel 12.0 7.9 11.2 Cellulose powder 12.7 12.0 23.5

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

(2) Of carbon source (cellulose powder)  Cellulase Activity Test According to Concentration

The cellulose powder concentration in the 7 L fermentation tank was cultured at different concentrations of 5-40 g / L, and the activity was shown in Table 2, and the maximum activity was shown in 20 g / L cellulose powder.

g / L Endo-β-1,4-glucanase (U / mg-protein) Exo-β-1,4-glucanase (U / mg-protein) β-glucosidase (U / mg-protein) Cellulose
powder 5 7.2 4.5 15.4 10 10.0 6.2 20.1 20 12.4 11.8 24.1 30 10.3 10.1 23.2 40 9.6 8.8 18.1

Table 2 is a table showing the activity of the cellulase enzyme at various cellulose powder concentrations.

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

The concentrations of cellulose powder, yeast extract, peptone, potassium monophosphate, potassium diphosphate, and magnesium sulfate in 7L fermenters were set at 20 g / L, 2 g / L, 8 g / L, 5 g / L, 5 g / L, and 3 g / L, respectively. Optimization experiments of cultivation environmental conditions were performed. As a result of comparing the cellulase activity by varying the stirring speed of 200-350rpm, pH 4-7 and the culture temperature at 29-37 ° C, the maximum cellulase activity was shown at the stirring speed of 250rpm, pH 5 and the culture temperature of 35 ° C. 2A, 2B and 2C show changes in time-dependent activity of endo-β-1,4-glucanase, exo-β-1,4-glucanase and beta-glucosidase at the optimum conditions. Respectively indicated in

Endo-β-1,4-glucanase
(U / mg-protein) Exo-β-1,4-glucanase
(U / mg-protein) β-glucosidase
(U / mg-protein) Incubation temperature (℃) 29 9.2 7.9 18.2 31 11.0 10.1 19.8 33 12.4 11.8 23.7 35 13.4 13.3 25.7 37 11.3 12.2 24.3 Stirring Condition (rpm) 200 13.2 13.2 25.9 250 14.0 14.3 27.6 300 12.9 12.5 27.3 350 11.0 10.2 26.2 pH 4 8.1 11.2 23.5 5 14.1 14.6 27.9 6 12.9 13.5 26.7 7 9.4 9.4 22.9

Table 3 is a table of experimental conditions for the optimization of enzyme activity.

Trichoderma Reese ZU-02 was used as an active control bacterium of three enzymes to compare the activity of Schizopilum commune KMJ820 strain. The culture conditions were consistently applied, and as a result of the activity comparison, the cellulase enzymes produced by the strain Sjozofilum cochlear KMJ820 were found to be endo-β-1,4-glucanase 14.1 U / mg-protein, exo-β-1, 4-glucanase 14.6 U / mg-protein, beta-glucosidase 27.9 U / mg-protein was found to have significantly higher cellulase activity compared to the control (Table 4).

Strain Endo-β-1,4-glucanase
(U / mg-protein) Exo-β-1,4-glucanase
(U / mg-protein) β-glucosidase
(U / mg-protein) Tricoderma RJ ZU-02 4.7 0.40 3.4 Szzopilum
KJM820 14.1 14.6 27.9

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 the protein 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. The pretreated and filtered white patches were dried at 65 ° C. and used.

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, the pretreated protein was added to various concentrations of cellulase in 20 ml of 0.1 M sodium acetate buffer (pH 5.0) by concentration. The cellulase-added buffer was reacted at 200 rpm at 25 to 45 ° 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 determined by the following equation, based on the reduced weight of 1 g of the protein, after the reaction was dried for 24 hours at 105 ℃.

(1) according to enzyme concentration Glycation yield  Experiment

The saccharification experiment of the strain Schizofilum commune KMJ820 of the present invention according to the enzyme concentration in a 500 ml Erlenmeyer flask was performed. Experiments at various concentrations of enzymes at 35 ° C. and 200 rpm were shown in Table 5. The optimum glycation yield was obtained when enzymes of 37.5-87.5 FPU / g-substrate, preferably 50 FPU / g-substrate, were used. Indicated.

Enzyme Concentration (FPU / g-substrate) Glycation yield (%) 37.5 21.2 50.0 22.5 62.5 20.0 75.0 16.8 87.5 12.5

(2) depending on substrate concentration Glycation yield  Experiment

The effect of substrate concentration on the glycosylation of glycoproteins produced by the strain Schizozolum colum KMJ820 of the present invention was confirmed. Table 6 shows the results of incubation with different initial concentrations of 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, and 5.5 wt%. The saccharification yield was excellent at 5.0 to 5.5 wt%, and the optimum saccharification yield was found at 5.0 wt% of the protein concentration.

Substrate concentration (%) Glycation yield (%) 3.5 16.3 4.0 18.2 4.5 19.6 5.0 25.7 5.5 23.5

(3) according to temperature Glycation yield  Experiment

The effect of temperature on the glycosylation of rice straw by the glycosylation enzyme produced by the strain Schizozolum Colum KMJ820 of the present invention was confirmed. Table 7 shows the results of incubation at different reaction temperatures of 25, 30, 35, 40, and 45 ° C. The saccharification yield was excellent at 30 ~ 40 ℃, the optimum saccharification yield was appeared at 40 ℃.

Reaction temperature (℃) Glycation yield (%) 25 15.4 30 18.3 35 20.7 40 25.3 45 16.3

(4) pH In accordance Glycation Experiment

The effect of pH on glycosylation of glycoprotein by the glycosylase produced by the strain Schizozolum columbium KMJ820 of the present invention was confirmed. Table 8 shows the results of culturing with different reaction pHs of 3, 4, 5, 6 and 7. The saccharification yield was excellent at pH 3 ~ 5, and the optimum glycation yield was found at pH 4.

pH Glycation yield (%) 3.0 24.9 4.0 28.6 5.0 21.3 6.0 19.4 7.0 18.0

Example  6: at optimum conditions Glycation Experiment

Glycosylation experiment using the glycosylation enzyme of the present invention Schizopilum commune KMJ820 under conditions optimized in the reactor. Enzyme concentration in the reaction solution was adjusted to 50 FPU / g-substrate, substrate concentration 5.0%, pH was 4 and the temperature was adjusted to 40 ℃. As a result of the experiment under the optimized conditions, the glycation yield according to the reaction time is shown in FIG. 3.

As a result of investigating the glycosylation efficiency of the protein by the cellulase of Schizopilum commune KMJ820 according to the reaction time implemented in the present invention, using an enzyme concentration of 50 FPU / g-substrate, a substrate of 5.0%, pH 4, 40 ℃ When reacted with time, the maximum glycosylation rate was 32.6%.

In addition, in the present invention, for the production of cellulase, the stirring speed is preferably 200 to 350 rpm, pH 4 to 7, and the incubation temperature is 29 to 37 ° C.

Meanwhile, the cellulase produced in the Sjozofilum coquette KMJ820 strain of the present invention and the cellulase (trade name celluclast) of Novozyme are shown in Table 9 by comparing the glycation yield under the optimum conditions.

Strain Sugar output (mg / g-cylinder) Glycation yield (%) Schizophyllum commune 295 ± 10 32 ± 1.1 Novozyme (celluclast) 241 ± 8 26 ± 1.0

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 (a) is a graph showing the endo-β-1,4-glucanase activity of the Schizopilum comb strain compared to the culture time.

Figure 2 (b) is a graph showing the comparison of exo-β-1,4-glucanase activity of Schizopilum combs strain according to the culture time.

Figure 2 (c) is a graph showing the beta-glucosidase activity of Schizopilum comb strain compared to the culture time.

Figure 3 is a graph showing the glycosylation rate of each protein by reaction time under the optimum conditions using the glycosylation enzyme produced by Schizofilum comb strain.

<110> Konkuk University Industrial Cooperation Corp. <120> Cellulase producing Schizophyllum commune and its use for          saccharification <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 615 <212> DNA <213> Schizophyllum commune <400> 1 cctgcggaag gatcattaac gaatcaaaca agttcatctt gttctgatcc tgtgcacctt 60 atgtagtccc aaagccttca cgggcggcgg atgactacgt ctacctcaca ccttaaagta 120 tgttaacgaa tgtaatcatg gtcttgacag accctaaaaa gttaatacaa ctttcgacaa 180 cggatctctt ggctctcgca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat 240 tgcagaattc agtgaatcat cgaatctttg aacgcacctt gcgccctttg gtattccgag 300 gggcatgcct gtttgagtgt cattaaatac catcaaccct cttttgactt cggtctcgag 360 agtggcttgg aagtggaggt ctgctggagc ctaacggagc cagctcctct taaatgtatt 420 agcggatttc ccttgcggga tcgcgtctcc gatgtgataa tttctacgtc gttgaccatc 480 tcggggctga cctagtcagt ttcaatagga gtctgcttct aaccgtctct tgactgagac 540 tagcgacttg tgcgctaact tttgacttga cctcaaatca ggtaggacta cccgctgaac 600 ttaagcatat caata 615  

Claims (12)

Schizophyllum commune KMJ820 (Accession No. KACC93084P), which produces cellulase. delete The method of producing a cellulase comprising the step of culturing the skim colum commun KMJ820 of claim 1 in a cellulase production medium. The method of claim 3, wherein the carbon source of the cellulase production medium is one selected from the group consisting of carboxymethyl cellulose, Avicel, rice straw, sugar cane bagasse, tangerine peel, and cellulose powder. The method of claim 4, wherein the carbon source is 10 ~ 40g / L concentration method of producing a cellulase. The method of claim 3, wherein the culturing is performed at a stirring speed of 200 to 350 rpm, pH 4 to 7 or at a culture temperature of 29 to 37 ° C, or a combination thereof. Cellulase production composition comprising the strain of claim 1 or a culture thereof as an active ingredient. A method of saccharification of cellulose comprising treating a cellulase produced from Schizophyllum commune of claim 1 to a substrate. The method of claim 8, wherein the concentration of the cellulase is 37.5 ~ 87.5 U / (cellulose) grams, characterized in that the cellulose saccharification method. 9. The method of claim 8, wherein the substrate cellulose concentration is 3.5 to 5.5% by weight of cellulose saccharification method. The method of claim 8, wherein the reaction pH is 3 to 7, and the reaction temperature is 25 to 45 ℃ characterized in that the cellulose saccharification method. 9. The method of claim 8, wherein said cellulose source substrate is one-sided protein.

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