CELLULASE-PRODUCING NOVEL STRAIN AND SACCHARIFICATION METHOD USING THE SAME

Abstract

The present invention relates to the novel strain Pholiota adiposa SKU714, a method for producing cellulase from the strain and a method for saccharifying cellulose using the produced cellulase. Since the cellulase produced by the novel strain according to the present invention exhibits better saccharification yield than the existing saccharification enzymes, it can be used in various applications, including bioenergy production, textile industry, papermaking industry, detergent industry, feed industry, food industry, production of low-calorie foods, fermentation of food wastes, or the like.

Claims

1. A method for saccharifying cellulose, comprising saccharifying a cellulose substrate using cellulase produced by culturing the Pholiota adiposa SKU714 deposited under Accession No. KCCM 11187P.

2. The method for saccharifying cellulose according to claim 1, wherein the cellulose substrate is poplar, rice straw or a mixture thereof.

3. The method for saccharifying cellulose according to claim 1, wherein the saccharification is performed under the condition of a substrate concentration of 5-25 wt %, a cellulase concentration of 1-45 FPU/g substrate, pH 4-7 and a temperature of 50-80° C.

4. The method for saccharifying cellulose according to claim 1, wherein the culturing is performed in a medium of pH 4.5-5.5 containing corn steep powder (5-10 g/L), yeast extract (1-5 g/L), potassium dihydrogen phosphate (3-7 g/L), potassium hydrogen phosphate (3-7 g/L), magnesium sulfate heptahydrate (1-5 g/L), thiamine hydrochloride (0.01-0.03 g/L) and a carbon source (10-30 g/L).

5. The method for saccharifying cellulose according to claim 1, wherein the carbon source is selected from a group consisting of cellulose, cellobiose, rice straw and avicel.

6. The method for saccharifying cellulose according to claim 1, wherein the culturing is performed under the condition of a stirring rate of 100-200 rpm, an aeration rate of 0.8-1.2 vvm and a culturing temperature of 25-30° C.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0023] FIG. 1 shows a result of analyzing the genetic relationship between the ITS-5.8S rDNA sequence of the strain of the present invention and similar species.

[0024] FIG. 2a shows a result of measuring the β-glucosidase activity (--), cellobiohydrolase activity (-◯-), endoglucanase activity (-.box-tangle-solidup.-) and glucose production (-Δ-) through breakdown of filter paper per unit enzyme amount of the Pholiota adiposa SKU714 strain with culturing time.

[0025] FIG. 2b shows a result of measuring the xylanase activity (--), laccase activity (-◯-), mannanase activity (-.box-tangle-solidup.-), and lignin peroxidase activity (-Δ-) of the Pholiota adiposa SKU714 strain with culturing time.

[0026] FIG. 3a shows the activity of β-glucosidase produced by the Pholiota adiposa SKU714 strain depending on pH.

[0027] FIG. 3b shows the activity of β-glucosidase produced by the Pholiota adiposa SKU714 strain depending on temperature.

BEST MODE FOR CARRYING OUT INVENTION

[0028] The present invention will be described in more detail through examples.

[0029] However, the scope of this invention is not limited by the examples.

EXAMPLE 1

Screening of Cellulase-Producing Strain

[0030] For screening of cellulase-producing strains, 10 μL of a mushroom culture was suspended in 10 mL of physiological saline. 10 μL of the resulting suspension (1×10.sup.4 cfu mL.sup.−1) was plated onto potato dextrose agar containing 2% carboxymethyl cellulose and incubated at 27° C. for 3 days. After colony was formed on the solid agar medium, the plate was stained with 0.1% Congo red and then destained with 1 M sodium chloride. Then, cellulase-producing mushroom strains were screened by selecting ones having halos produced by hydrolysis of cellulose around the colony.

[0031] Primary strains (S1-S6) were screened through this procedure. From the screened strains, the S4 strain exhibiting the best cellulose degrading ability was selected after testing on the solid agar medium containing carboxymethyl cellulose using the existing producing strain Trichoderma reesei ZU-02 as control (C).

Example 2

Identification of Strain

[0032] For identification of the S4 strain screened in Example 1, the ITS-5.8S rDNA sequence was analyzed by the Korean Culture Center of Microorganisms. The ITS-5.8S rDNA sequence of the S4 strain was named as SEQ ID NO 1.

[0033] As a result of analyzing the genetic relationship between the ITS-5.8S rDNA sequence of the S4 strain with similar species, the S4 strain was identified as Pholiota adiposa (FIG. 1).

[0034] The S4 strain was named as ‘Pholiota adiposa SKU714’ and deposited at the Korean Culture Center of Microorganisms on April 20, 2011 with Accession No. KCCM 11187P under the Budapest Treaty.

Example 3

Optimization of Medium for Producing Cellulase

[0035] (1) Cellulase Activity Depending on Carbon Source

[0036] Cellulase-producing activity of the Pholiota adiposa SKU714 strain depending on carbon source was tested in a 7-L fermentation tank. Cellulose, glucose, lactose, maltose, cellobiose, carboxymethyl cellulose, sucrose, xylan, rice straw and avicel were used as carbon source.

[0037] After inoculating the Pholiota adiposa SKU714 strain in a 50-mL flask containing 50 mL of a whole culture medium (potato starch 4 g/L, dextrose 20 g/L), the strain was cultured in a shaking incubator at 150 rpm and 25° C. for 5 days. 50 mL of the culture was inoculated a 50-mL flask containing a growth medium (corn steep powder 8 g/L, yeast extract 2 g/L, potassium dihydrogen phosphate 5 g/L, potassium hydrogen phosphate 5 g/L, magnesium sulfate heptahydrate 3 g/L, thiamine hydrochloride 0.02 g/L and carbon source 20 g/L, pH 5) and cultured at 150 rpm, 25° C. and pH 5 for 7 days.

[0038] The result of measuring the β-glucosidase activity and glucose production of the Pholiota adiposa SKU714 strain for each carbon source is shown in Table 1.

TABLE-US-00001 TABLE 1 Glucose production through breakdown of filter β-Glucosidase paper per unit enzyme Carbon source (20 g/L) activity (U/mL) amount (U/mL) Cellulose 16.4 0.63 Glucose 3.45 0.15 Lactose 4.10 0.14 Maltose 8.60 0.31 Cellobiose 10.6 0.43 Carboxymethyl 3.52 0.13 cellulose Sucrose 3.42 0.12 Xylan 5.30 0.26 Rice straw 15.0 0.52 Avicel 14.9 0.61

[0039] As seen from Table 1, superior cellulase activity was achieved when cellulose, cellobiose, rice straw and avicel were used as carbon source. The maximum cellulase activity was achieved when cellulose was used as the carbon source.

[0040] (2) Cellulase Activity Depending on Nitrogen Source

[0041] Cellulase-producing activity of the Pholiota adiposa SKU714 strain depending on nitrogen source was tested in a 7-L fermentation tank. Yeast extract, peptone, corn steep powder, urea, ammonium sulfate, potassium nitrate, sodium nitrate and tryptone were used as nitrogen source.

[0042] A result of measuring the β-glucosidase activity and glucose production of the Pholiota adiposa SKU714 strain for each carbon source at a concentration of 5 g/L is shown in Table 2.

TABLE-US-00002 TABLE 2 Glucose production through breakdown of filter β-Glucosidase paper per unit enzyme Nitrogen source (5 g/L) activity (U/mL) amount (U/mL) Yeast extract 18.6 0.72 Peptone 11.8 0.45 Corn steep powder 18.5 0.70 Urea 14.5 0.31 Ammonium sulfate 6.0 0.24 Potassium nitrate 11.1 0.36 Sodium nitrate 10.7 0.34 Tryptone 14.6 0.69

[0043] As seen from Table 2, superior cellulase activity was achieved when yeast extract, corn steep powder and tryptone were used as nitrogen source. The maximum cellulase activity was achieved when yeast extract was used as the nitrogen source.

Example 4

Optimization of Culturing Condition for Production of High-Activity Enzyme

[0044] (1) Optimization of Culturing Condition when Poplar is Used as Substrate

[0045] Culturing condition was optimized in a 7-L fermentation tank using a medium containing corn steep powder (8 g/L), yeast extract (2 g/L), potassium dihydrogen phosphate (5 g/L), potassium hydrogen phosphate (5 g/L), magnesium sulfate heptahydrate (3 g/L), thiamine hydrochloride (0.02 g/L) and poplar (20 g/L). Cellulase activity was compared while varying pH from 3 to 7 and changing culturing temperature from 20 to 35° C. The maximum cellulase activity was achieved at pH 5 and 25-30° C.

[0046] Also, the activity of each cellulase with culturing time was measured at the optimized culturing condition (pH 5, 25° C.) in the medium containing the poplar substrate (FIGS. 2a and 2b). FIG. 2a shows a result of measuring β-glucosidase, cellobiohydrolase and endoglucanase activities and glucose production through breakdown of filter paper per unit enzyme amount with culturing time. FIG. 2b shows a result of measuring the change in xylanase, laccase, mannanase and lignin peroxidase activities with culturing time.

[0047] (2) Optimization of Cellulase Production and Activity when Rice Straw is Used as Substrate

[0048] Culturing condition was optimized in a 7-L fermentation tank using a medium containing corn steep powder (8 g/L), yeast extract (2 g/L), potassium dihydrogen phosphate (5 g/L), potassium hydrogen phosphate (5 g/L), magnesium sulfate heptahydrate (3 g/L), thiamine hydrochloride (0.02 g/L) and rice straw (20 g/L). Cellulase production was compared while varying pH from 3 to 7 and changing culturing temperature from 20 to 35° C. The maximum cellulase production was achieved at pH 5 and 25-30° C.

[0049] Also, β-1,4-glucosidase activity was compared while varying pH from 3 to 7.5 and changing temperature from 40 to 85° C. The result is shown in FIGS. 3a and 3b. As seen from FIGS. 3a and 3b, the maximum cellulase activity was achieved at pH 5 and 65° C.

Example 5

Analysis of Saccharification Yield

[0050] In general, lignocellulose contained in a plant cannot be saccharified at high yield only with enzymatic hydrolysis. For this reason, lignin and hemicellulose are fragmented prior to enzymatic hydrolysis through a pretreatment process in order to increase the cellulose hydrolysis efficiency by cellulase. In Example 5, for the pretreatment, 10 g of rice straw was added to a flask containing 40 mL of 2 wt % sodium hydroxide solution and reacted at 85° C. for 1 hour. Then, the rice straw was filtered through a 0.45-uM filter and dried at 65° C.

[0051] In order to find the optimized saccharification condition, experiment was conducted while varying enzyme concentration, substrate concentration, reaction temperature and reaction pH.

[0052] First, the pretreated rice straw at various concentrations was added to 20 mL of 0.1 M sodium acetate buffer (pH 5.0) together with cellulase at various concentrations. After reaction at 15-55° C. and 150 rpm for 72 hours, the reaction mixture was boiled at 100° C. for 3 minutes to remove denatured enzyme, which was then cooled to room temperature and centrifuged at 4000 rpm for 15 minutes. Enzyme activity was measured by the reducing sugar method from the supernatant.

[0053] Saccharification yield was determined according to Equation 1 by measuring the weight decrease of the rice straw after drying at 105° C. for 24 hours per gram of the rice straw.

Saccharification yield (%)=[(Weight of produced reducing sugar/g substrate)×0.9/Weight of carbohydrate in rice straw]×100   [Equation 1]

[0054] (1) Saccharification Yield Depending on Enzyme Concentration

[0055] Saccharification was performed using Pholiota adiposa SKU714 strain at 65° C. and pH 6 while varying enzyme concentration. The result is shown in Table 3.

TABLE-US-00003 TABLE 3 Enzyme concentration (FPU/g substrate) Saccharification yield (%) 1 9.7 5 23.9 17.5 83.0 30 81.2 42.5 80.6

[0056] As seen from Table 3, the best saccharification yield was achieved when the enzyme concentration was 15-45 FPU/g substrate.

[0057] (2) Saccharification Yield Depending on Substrate Concentration

[0058] The effect of substrate concentration on the saccharification of poplar by the saccharification enzyme produced by the Pholiota adiposa SKU714 strain was investigated. Saccharification yield was measured while varying the initial concentration of the poplar substrate from 1 to 27 wt %. The result is shown in Table 4.

TABLE-US-00004 TABLE 4 Substrate concentration (wt %) Saccharification yield (%) 1 43.0 2 63.5 11 81.2 20 83.1 27 51.0

[0059] As seen from Table 4, superior saccharification yield was achieved when the initial concentration of the poplar substrate was 10-25 wt %. The best saccharification yield was achieved when the poplar concentration was 20 wt %.

[0060] (3) Saccharification Yield Depending on Temperature

[0061] The effect of temperature on the saccharification of poplar by the saccharification enzyme produced by the Pholiota adiposa SKU714 strain was investigated. Saccharification yield was measured at different reaction temperatures of 20, 35, 50, 65 and 80° C. The result is shown in Table 5.

[0062] [Table 5]

TABLE-US-00005 TABLE 5 Reaction Saccharification temperature yield (° C.) (%) 20 33.2 35 45.0 50 75.8 65 82.1 80 71.2

[0063] As seen from Table 5, superior saccharification yield was achieved when the saccharification temperature was 50-80° C. The best saccharification yield was achieved at 65° C.

[0064] (4) Saccharification Yield Depending on pH

[0065] The effect of pH on the saccharification of poplar by the saccharification enzyme produced by the Pholiota adiposa SKU714 strain was investigated. Saccharification yield was measured at different reaction pH's of 1, 3, 5, 7 and 9. The result is shown in Table 6.

TABLE-US-00006 TABLE 6 pH Saccharification yield (%) 1 20.0 3 62.6 5 81.4 7 84.0 9 39.4

[0066] As seen from Table 6, superior saccharification yield was achieved at pH 4-7. The best saccharification yield was achieved at pH 7.

Example 6

Saccharification Under Optimized Condition

[0067] (1) Saccharification of Poplar Using Pholiota adiposa Cellulase

[0068] Saccharification of poplar was conducted under the optimized condition using the cellulase produced by the Pholiota adiposa SKU714 strain. Saccharification was performed under the condition of a substrate concentration 10 wt %, an enzyme concentration 25 FPU/g substrate, pH 6 and a temperature 65° C. The saccharification yield of the cellulase produced by the Pholiota adiposa SKU714 strain is compared with that of Novozymes' cellulase derived from Trichoderma reesei(Celluclast 1.5L) in Table 7.

TABLE-US-00007 TABLE 7 Sugar production Saccharification Cellulase (mg/g poplar) yield (%) Pholiota adiposa 672 84 SKU714 Celluclast 1.5 L 242 35

[0069] (2) Saccharification of Rice Straw Using Pholiota adiposa Cellulase

[0070] Saccharification of rice straw was conducted under the optimized condition using the cellulase produced by the Pholiota adiposa SKU714 strain. Saccharification was performed for 24 hours under the condition of a substrate concentration 10 wt %, an enzyme concentration 16 FPU/g substrate, pH 6 and a temperature 65° C. The saccharification yield of the cellulase produced by the Pholiota adiposa SKU714 strain is compared with those of Novozymes' cellulase derived from Trichoderma reesei (Celluclast 1.5L) and cellulase derived from the Pholiota nameko KTCC26163 strain in Table 8.

TABLE-US-00008 TABLE 8 Sugar production Saccharification Cellulase (mg/g rice straw) yield (%) Pholiota adiposa SKU714 690 88 Trichoderma reesei 582 76 Pholiota nameko 420 56

[0071] Although the Pholiota nameko and Pholiota adiposa strains belong to the same genus Pholiota, they exhibit quite different cellulose saccharification effects. Whereas the Pholiota nameko strain is limited for use as a saccharification enzyme due to low protein productivity and low enzyme activity, the Pholiota adiposa strain according to the present invention is suitable for commercial application owing to production of various biomass-degrading enzymes including cellulase, high protein productivity, high enzyme activity and good thermal stability.

[Accession No.]

[0072] Deposition agency: Korean Culture Center of Microorganisms

[0073] Accession No.: KCCM 11187P

[0074] Accession date: 20110420