KR102246866B1 - Method for Saccarification of Pine Tree Biomass Using Cellulose Degrading Enzyme - Google Patents

Abstract

The present invention relates to a saccharification method of pine biomass, characterized in that the saccharification of pine biomass is treated with cellulose derived from Trichoderma genus KMF006 strain (KCTC13500BP) for saccharification. According to the present invention, saccharification was difficult. Since the treated pine biomass can be more effectively saccharified, it can be usefully used in biofuel production.

Description

Method for Saccarification of Pine Tree Biomass Using Cellulose Degrading Enzyme}

The present invention relates to a method for saccharifying pine biomass using a cellulose degrading enzyme, and more particularly, to saccharify pine biomass by treating with cellulose derived from KMF006 strain (KCTC13500BP) of Trichoderma genus KMF006 strain (KCTC13500BP). It relates to a method of saccharifying biomass.

Bioenergy is attracting attention as an energy that can replace fossil fuels according to global warming and high oil prices due to consumption of fossil fuels, and technology for producing ethanol from plant biomass resources is being developed.

Starch-based raw materials such as sugar cane and corn, which are the main raw materials of currently commercialized bioethanol, have emerged as a social issue, causing problems in the price and supply of the food market. For this reason, wood-based biomass with sufficient fuel supply, reproducible and environmentally friendly, is emerging as a next-generation biofuel raw material for the production of biofuels including bioethanol.

However, lignocellulosic biomass is characterized by a hard bond and high strength as the main constituents of cellulose, hemicellulose, and lignin, so it is difficult to decompose and requires a complicated process to separate it into monosaccharides, resulting in a problem of high cost. In the treatment of lignocellulosic biomass, first, the pretreatment process that effectively removes lignin from the lignocellulosic biomass, reduces the crystallinity of cellulose, increases the surface area of the biomass, and helps efficient saccharification, and the saccharification process to decompose cellulose and hemicellulose into usable monosaccharides. I need this. The saccharification process is an existing commercial technology, such as acid saccharification using concentrated sulfuric acid or dilute acid. However, as they are carried out at high temperatures, fermentation inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are formed, causing problems such as environmental pollution. In addition, since it corrodes metals, saccharification technology using enzymes has become the mainstream in recent years. Enzymatic saccharification has a disadvantage in that it takes time to saccharify, but it is very advantageous in terms of energy saving and environmental aspects because it does not use chemicals and does not require chemical recovery or neutralization process.

Enzymatic hydrolysis of fibrin-based materials is performed by a hydrolysis reaction with a cellulose complex enzyme including Endo-1,4-β-glucanase (EG), β-glucosidase (BGL) and cellobiohydrolase (CBH). In general, cellulase complexes are often obtained from organisms using woody biomass as a nutrient source. In particular, a number of studies on the development of cellulose derived from microorganisms capable of efficiently hydrolyzing cellulose have been conducted. These microorganism-derived celluloses can be expressed differently in their activity depending on various culture conditions, and the carbon source and nitrogen source play an important role in the production of lignin-degrading enzymes.

However, saccharification of wood-based biomass known to date is mostly a method of saccharifying straw, rice bran grass, etc., which are relatively easy to saccharify, and the establishment of saccharification enzymes or conditions that can efficiently saccharify wood is difficult, especially coniferous trees. There is no situation in establishing conditions for saccharifying phosphorus pine biomass.

Accordingly, the present inventors screened a new strain of Trichoderma genus KMF006 that produces cellulose having saccharification activity on wood from which lignin has not been removed, and applies the cellulose isolated from the strain to pre-explosion-treated pine chips, It was confirmed that the saccharification yield was significantly higher than that when the conventional commercial enzyme was treated, and the present invention was completed.

An object of the present invention is to provide a method for saccharifying pine biomass.

In order to achieve the above object, the present invention comprises the steps of (a) pre-treating pine biomass; And (b) it provides a saccharification method of pine biomass comprising the step of saccharifying the pretreated pine biomass by treating with cellulose derived from Trichoderma genus KMF006 strain (KCTC13500BP).

According to the present invention, it is possible to more effectively saccharify the explosively processed pine biomass, which was difficult to saccharify, and thus can be usefully used in biofuel production.

1 shows the main effect on the saccharification rate (%) of pine trees subjected to steam-explosion pretreatment (25kgf/cm _{2, 13 minutes).}
Figure 2 shows a contour diagram of the saccharification rate (%) of a pine tree subjected to steam-explosion pretreatment (25kgf/cm _{2, 13 minutes).}
3 shows the surface reactivity of the saccharification rate (%) versus enzyme concentration (FPU) and substrate concentration (%) of the pine tree subjected to steam-explosion pretreatment (25kgf/cm _{2, 13 minutes) (X: substrate concentration, Y:} Enzyme concentration).
Figure 4 shows the surface reactivity of the saccharification rate (%) versus Tween80 (mg/g, glucan), and the substrate concentration (%) of the pine tree subjected to steam-explosion pretreatment (25kgf/cm _{2, 13 minutes) (X: substrate} Concentration, Y: Tween80 concentration).
5 shows the surface reactivity of the saccharification rate (%) vs. Tween80 (mg/g, glucan), and enzyme concentration (FPU) of the pine tree subjected to steam-explosion pretreatment (25kgf/cm _{2, 13 minutes) (X: enzyme} Concentration, Y: Tween80 concentration).
6 shows the optimum conditions for saccharification of pine trees subjected to steam-explosion pretreatment (25kgf/cm _{2, 13 minutes).}
7 shows the main effect on the saccharification rate (%) of pine trees subjected to steam-explosion pretreatment (25kgf/cm _{2, 7 minutes).}
8 shows a contour diagram of the saccharification rate (%) of pine trees subjected to steam-explosion pretreatment (25kgf/cm _{2, 7 minutes).}
9 shows the surface reactivity of the saccharification rate (%) versus enzyme concentration (FPU) and substrate concentration (%) of the pine tree subjected to steam-explosion pretreatment (25kgf/cm _{2, 7 minutes) (X: substrate concentration, Y:} Enzyme concentration).
Figure 10 shows the surface reactivity of the saccharification rate (%) vs. Tween80 (mg/g, glucan), and the substrate concentration (%) of the pine tree subjected to steam-explosion pretreatment (25kgf/cm _{2, 7 minutes) (X: substrate} Concentration, Y: Tween80 concentration).
Figure 11 shows the surface reactivity of the saccharification rate (%) vs. Tween80 (mg/g, glucan), and enzyme concentration (FPU) of the pine tree subjected to steam-explosion pretreatment (25kgf/cm _{2, 7 minutes) (X: enzyme} Concentration, Y: Tween80 concentration).
12 shows the optimum conditions for saccharification of pine trees subjected to steam-explosion pretreatment (25kgf/cm _{2, 7 minutes).}
13 shows the saccharification rate (%) of pine and cellulose pretreated with steam-explosion using an enzyme derived from KMF006 and a commercial enzyme (Cellic CTec2) under optimal condition 1.
14 shows the saccharification rate (%) of pine and cellulose pretreated with steam-explosion using an enzyme derived from KMF006 and a commercial enzyme (Cellic CTec2) under optimal condition 2.

In the present invention, an attempt was made to develop a method for effectively saccharifying pine biomass, which is a lignocellulosic biomass, and cellulase derived from the novel Trichoderma genus KMF006 strain (Korea Patent Application 2018-0069387) having excellent saccharification enzyme activity was added to the pre-explosive pine biomass By treatment, it was confirmed that when saccharification was performed, pine biomass could be saccharified with higher efficiency than a commercial enzyme.

Accordingly, the present invention, in one aspect, (a) the step of pre-treating the pine biomass; And (b) saccharifying the pine biomass by treating the pre-treated pine biomass with cellulose derived from Trichoderma genus KMF006 (KCTC13500BP).

The term'saccharification', as used herein, refers to a reaction that converts high molecular weight carbohydrates such as starch and fiber into low molecular weight (monosaccharide or disaccharide) saccharides by hydrolyzing them by the action of enzymes or acids. do.

In the present invention, the strain KMF006 of the genus Trichoderma can produce all of endo-β-1,4-glucanase, cellobiohydrolace, and beta-glucosidase required for the process of decomposing cellulose into monosaccharides (glucose).

As used herein, the term'cellulase' is a generic term for an enzyme that hydrolyzes cellulose, and'cellulose' refers to glucose as β-1,4 glucoside (β-1,4-glucosde) It means a homopolymer connected by a bond.

Cellulase of the present invention is characterized in that it is obtained from a culture medium obtained by culturing the strain KMF006 of the genus Trichoderma.

The cellulose is endo-β-1,4-glucanase (endo-β-1,4-glucanase; EC 3214), cellobiohydrolase (EC 32191), and beta-glucosidase. ; EC32121) can be selected from the group consisting of.

The endo-beta-1,4-glucanase is an enzyme that randomly hydrolyzes the β-1,4-glucoside bond of cellulose, and generally uses soluble carboxymethyl cellulose (CMC) as a substrate. It is also called CMC degrading enzyme (CMCase) because it is used as

Cellobiohydrolace hydrolyzes the reducing and non-reducing ends of the product hydrolyzed by endo-beta-1,4-glucanase to produce cellobiose, a glucose disaccharide. The generated cellobiose inhibits the activities of endo-beta-1,4-glucanase and cellobiohydrolace. Because beta-glucosidase decomposes cellobiose into glucose (glucose), it eliminates the inhibition of the activity of endo-beta-1,4-glucanase and cellobiohydrolace by cellobiose.

'Cellulase' of the present invention may be characterized by including all of the culture of the KMF006 strain (KCTC13500BP) of the genus Trichoderma, cell lysates, purified enzymes, and concentrates.

The concentrate of the present invention means that the culture or cell-free culture of the trichoderma genus KMF006 strain (KCTC13500BP) is concentrated.

In the present invention, the cell-free culture medium means removing the cultured microorganisms from the culture medium in which the microorganisms are cultured, and the culture means containing the cultured microorganisms.

In the present invention, the pretreatment of the pine biomass is preferably detonation pretreatment, more preferably steam-detonation pretreatment.

In the present invention, the bombardment may be characterized in that it is performed for 3 to 20 minutes under the condition ^{of 20 to 30 kgf/cm 2, and preferably, it may be performed for 5 to 13 minutes.}

In one aspect of the present invention, the steam-explosion pretreatment process was performed based on Masonite technology, and the steam-explosion treatment was treated by adjusting the treatment time to 3, 5, 7, 13 minutes at a pressure of ^{25 kgf/cm 2,} The steam-crushed pine trees were collected in a cyclone, cooled to about 40°C, and filtered for solid recovery. After filtration, the remaining residue was analyzed for chemical composition and used as a substrate for saccharification.

In the present invention, the step (b) may be characterized in that the saccharification enhancer is additionally treated, and nonionic Tween 80 and PEG may be used as the saccharification promoter, and Tween 80 may be preferably used. have.

In the present invention, when using Tween 80 as the saccharification enhancer, it may be characterized in that treatment with _{10 to 800 mg/g glucan , preferably 100 to 500 mg/g glucan,} may be used.

In the present invention, the cellulase may be characterized in that it is treated with 10 to 80 FPU, preferably 20 to 70 FPU, and more preferably 30 to 60 FPU.

In the present invention, in the saccharification treatment, the substrate concentration of the explosive material may be 3 to 10%, more preferably 5 to 8%, and most preferably 7%.

In one aspect of the present invention pine width swaejae (25kgf / cm ^2, 13min) when sikyeoteul glycosylation using KMF006 strain derived cellulose raised, pine width swaejae factor three kinds of substrate concentration of ^{(25kgf / cm 2, 13min)} 7% , Enzyme concentration of about 33.74FPU, saccharification enhancer (Tween80) concentration of 500 mg/g, it was found that the maximum predicted glycation rate was about 105.69% in glucan.

In another aspect of the present invention pine width swaejae (25kgf / cm ^2, 13min) when sikyeoteul glycosylation using KMF006 strain derived cellulose raised, pine width swaejae factor three kinds of substrate concentration of ^{(25kgf / cm 2, 13min)} 7% , At the enzyme concentration of about 33.74FPU, 60FPU, and the saccharification enhancer (Tween80) concentration of 342.42 mg/g, glucan), the maximum predicted glycation rate was found to be about 93.75%%.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1: Preparation of cellulase enzyme solution

Trichoderma genus KMF006 strain (KCTC13500BP) was cultured in MEA plates for 5 to 7 days, and mycelium was inoculated in 50 ml of PDB, and pre-cultured for 5 days while shaking at 150 rpm at 30°C.

This culture was carried out by preparing a liquid medium under the conditions described in Table 1 below. The prepared liquid medium was inoculated with 5% (w/v) of the pre-culture solution, and incubated at 30° C. at 150 rpm for about 4 weeks. During incubation, 500 µl of the culture solution was collected every day, centrifuged to remove colonies, and then used as an enzyme solution.

ingredient density Avicel (g/L) 30 Yeast extract (g/L) 15 K ₂ HP 0 ₄ (g/L) 5 KH ₂ P0 ₄ (g/L) 5 MgS0 ₄ (g/L) 3 pH 6.0

Example 2: Cellulase enzyme activity measurement

Beta-glucosidase activity, endo-beta-1,4-glucanase (EG) activity and cellobiohydrolace of enzyme solution derived from the strain KMF006 of the genus Trichoderma obtained in Example 1 The activity of (cellobiohydrolase, CBH) was measured.

2-1: Beta-glucosidase (BGL) activity measurement

0.1 M sodium acetate (NaAC) buffer solution (pH 5.0) in 0.8 ml of 10 mM p-nitrophenyl-β-D-glycopyranoside (p-nitrophenyl-β-D-glycopyranoside, pNPG) 0.1 ml and the above 0.1 ml of the main culture solution was added and reacted at 50° C. for 15 minutes. Thereafter, 0.1 ml of a 2M sodium carbonate (Na2CO3) solution was added to stop the reaction, and absorbance was measured at 405 nm to confirm the amount of p-nitrophenol produced. The enzyme activity unit, 1 unit (U), was defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol for 15 minutes under certain conditions.

2-2: endo-beta-1,4-glucanase activity

An enzyme reaction solution was prepared by dissolving sodium carboxymethylcellulose at a concentration of 2% (v/v) in 0.1M sodium acetate buffer (pH 5.0). 5 µl of the culture solution was added to 45 µl of the enzyme reaction solution, followed by reaction at 50°C for 30 minutes, and 50µl of a copper solution was added, followed by heating at 100°C for 10 minutes to stop the reaction. The amount of reducing sugar produced was measured by the Somogyi-Nelson method (latest experimental microbiology, p253, 2001). 1 unit (U), which is a unit of enzyme activity, is the amount of enzyme required to produce 1 μmol of glucose for 30 minutes under certain conditions. It was defined as a quantity.

2-3: cellobiohydrolace activity

To 08 ml of 0.1M sodium acetate buffer (pH 5.0), 0.1 ml of p-nitrophenyl-β-D-cellobioside (pNPC) and 0.1 ml of the culture solution were added. It was allowed to react for 15 minutes. Thereafter, 0.1 ml of 2M sodium carbonate solution was added to stop the reaction, and absorbance was measured at 405 nm to confirm the amount of p-nitrophenol produced. The enzyme activity unit, 1 unit (U), was defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol for 15 minutes under certain conditions.

The enzyme activity unit FPU (filter paper unit) was measured according to the method of measuring cellulose activity (Measurment of Cellulose Activity, NREL/TP-510-42628) using cellulose derived from the KMF006 strain of Trichoderma genus. 50mg Whatman No. 1 Using filter paper, 100mM sodium citrate buffer pH 4.5 was used. Each buffer and enzyme solution were added to a test tube containing 50 mg of filter paper as a substrate and reacted at 50° C. for 60 minutes. Thereafter, 3mL DNS (3, 5 Dimitrosalicylic acid, Alfa Aesar) reagent was added, put in boiling water for 5 minutes, and the reaction was stopped and cooled to room temperature. After being settled by centrifuging the pulped filter paper at 13500 rpm for 10 minutes, it was mixed with distilled water at a certain ratio and absorbance was measured at 540 nM.

Beta-glucosidase activity, endo-beta-1,4-glucanase (EG) activity, and cellobiohydrolase of the enzyme solution derived from the KMF006 strain measured by the above method. The activity of CBH) is shown in Table 2.

Enzyme activity (U/ml) Type of enzyme Beta-glucoside
S Endo-beta-1,4-glu
Canace Cello Bio
Hydro race Enzyme solution derived from KMF006 strain 216.04±1094 202.13±785 8.9±034

Example 3: Pretreatment of explosives of pine biomass

For woody biomass, Korean pine ( Pinus densiflora ) that had been pretreated with steam-explosion was used. For the used pine, wood chips produced from logs harvested in Jinju City, Gyeongsangnam-do were purchased and used. Cellulose (DAEJUNG, 20-100 microns) was used as a comparative substrate. The steam-explosion pretreatment process was performed in a customized batch pilot unit (Yurim Hitech, Korea) based on Masonite technology. The steam-blasting treatment was ^{treated by adjusting the treatment time to 3, 5, 7, 13 minutes at a pressure of 25kgf/cm 2} , and the steam-crushed pine trees were collected in a cyclone and cooled to about 40°C. Then, it was filtered to recover the solid. After filtration, the remaining residue was analyzed for chemical composition and used as a substrate for saccharification.

In order to analyze the chemical composition of the pre-explosion-treated pine wood, according to the Determination of structural carbohyrates and lignin in biomass method (NREL/TP 510-42618), glucan, xylan, and gallbladder were used. Analysis experiments were performed on lactan arabinan, mannan, lignin, and Ash, and the results are shown in Table 3.

Chemical composition of steam-explosive pine (25kgf/cm ² , 5 minutes) Component Rate of component ( % w/w) Glucan Xylan Galactan Arabinan Mannan Lignin Ash Pine explosives
(25kgf/cm ² , 7min) 37.00 1.50 - - 2.05 47.77 0.32 Pine explosives
(25kgf/cm ² , 13min) 41.02 0.44 - - - 50.60 0.28

Example 4: Establishment of optimal saccharification conditions of pine biomass pre-explosion treatment

In Example 3 using an enzyme derived from the KMF006 strain, the optimal saccharification conditions of the pine biomass pretreated with steam-explosion were established.

4-1: Pine tree bombardment condition 25kgf/cm ² , Optimum conditions of experiment design for deriving optimal saccharification conditions of 13min.

The experimental design for deriving the optimal conditions for saccharification of pine trees pre-detonated under the conditions of 25kgf/cm ^{2 and 7min. was used by the Box-behnken method among the response surface analysis methods.} Among the factors affecting the saccharification rate, the influencing factors were substrate concentration, enzyme concentration, and saccharification enhancer concentration as three factors, and the concentration range of each factor was set as shown in Table 2, and the test was conducted. The design was performed using the 3-factor 3-level perfect factor design method in the Box-behnken method, and the number of repeated experiments was set as 2 times and the center point 1, and a total of 26 experiments were composed, and this design is shown in Table 3 in a coded format. I got it.

Tween80 (Sigma-aldrich) was used as a saccharification enhancer, and it was set at a level of 1 to 5%.

Factor levels for response surface analysis Factor Levels of factor - 0 + One Substrate concentration(%)
steam-explosion pine (25kgf/cm ² , 13min) 3 5 7 2 Enzyme concentration
(FPU, KMF006) 10 35 60 3 Tween80 (mg/g,glucan) 100 300 500

Experimental design coding format of variables Order Run Substrate concentration Enzyme concentration Tween80
(mg/g,glucan) 13 One + 0 - 25 2 - - 0 5 3 - 0 + 19 4 0 - - 21 5 + 0 + 24 6 0 0 0 28 7 0 0 0 4 8 0 + + 2 9 0 - + 7 10 - 0 - 29 11 0 + - 20 12 - + 0 17 13 + + 0 6 14 0 0 0 26 15 + - 0 27 16 + 0 - 22 17 - - 0 15 18 - 0 + 8 19 0 - - 23 20 + 0 + 14 21 0 0 0 10 22 0 0 0 18 23 0 + + 3 24 0 - + 11 25 - 0 - 12 26 0 + - 9 27 - + 0 One 28 + + 0 16 29 0 0 0 30 30 + - 0

As a result, the results of deriving the optimal saccharification conditions of the pine wood pre-detonation treatment using the response surface analysis method (Box-behnken) are shown in FIGS. 1 to 6. The main effect according to the three factors affecting saccharification, substrate concentration, enzyme concentration, and saccharification enhancer concentration, as shown in FIG. 7, showed the greatest main effect of the substrate concentration. It showed a tendency to see. The substrate concentration showed the highest saccharification rate at 7%. In addition, in the case of the enzyme concentration, the higher the concentration, the higher the saccharification rate, but the 60FPU tended to decrease. In the case of Tween80, the saccharification rate was highest at 500 mg/g, glucan concentration, and the saccharification rate tended to increase as the saccharification enhancer concentration increased. In addition, the same trend was exhibited in the contour diagrams and surface diagrams shown in FIGS. 2 to 5.

Based on these results, as shown in FIG. 6, the three factors of the ^{pine explosive material (25kgf/cm 2} , 13min) are substrate concentration 7%, enzyme concentration: about 33.74FPU, saccharification enhancer (Tween80) concentration 500 mg/ The maximum predicted saccharification rate in g,glucan was found to be about 105.69%.

4-2: Pine tree bombardment condition 25kgf/cm ² , Optimal conditions of experiment design for derivation of optimal saccharification conditions of 7min.

The experimental plan for deriving the optimal conditions for saccharification of pine trees pre-detonated under the conditions of 25kgf/cm ^{2 and 13min. was used by the Box-behnken method among the response surface analysis methods.} The influencing factors were three factors: substrate concentration, enzyme concentration, and saccharification enhancer concentration among the factors that affect the saccharification rate, and the concentration range of each factor was set as shown in Table 4, and the test was carried out. The design was performed using the 3-factor 3-level perfect factor design method in the Box-behnken method, and a total of 26 experiments were composed by setting the number of replicates to 2 times and center point 1, and this design is shown in Table 5 in a coded format. I got it.

Factor levels for response surface analysis Factor Levels of factor - 0 + One Substrate concentration
steam-explosion pine (25kgf/cm ² , 7min) 3 5 7 2 Enzyme concentration
(FPU, KMF006) 10 35 60 3 Tween80 (mg/g,glucan) 100 300 500

Experimental design coding format of variables Order Run Substrate concentration Enzyme concentration Tween80
(mg/g,glucan) 18 One + 0 - 7 2 - - 0 14 3 - 0 + 9 4 0 - - 27 5 + 0 + 4 6 0 0 0 15 7 0 0 0 23 8 0 + + 25 9 0 - + One 10 - 0 - 13 11 0 + - 19 12 - + 0 17 13 + + 0 21 14 0 0 0 5 15 + - 0 6 16 + 0 - 11 17 - - 0 16 18 - 0 + 24 19 0 - - 22 20 + 0 + 28 21 0 0 0 2 22 0 0 0 3 23 0 + + 20 24 0 - + 30 25 - 0 - 29 26 0 + - 26 27 - + 0 10 28 + + 0 12 29 0 0 0 8 30 + - 0

The results of deriving the optimal saccharification conditions of the pine wood ^{pre-detonation treatment (25kgf/cm 2} , 7 minutes) using the response surface analysis method (Box-behnken) are shown in FIGS. The main effect according to the three factors affecting saccharification, the substrate concentration, the enzyme concentration, and the saccharification enhancer concentration, as shown in FIG. 12, showed the greatest main effect of the substrate concentration, and the higher the substrate concentration, the higher the saccharification rate. It showed a tendency to see. The substrate concentration showed the highest saccharification rate at 7%. In addition, in the case of the enzyme concentration, the higher the concentration, the higher the saccharification rate, and the tendency to decrease in 60FPU. In the case of Tween80, the highest saccharification rate was shown at 342.42mg/g, glucan concentration. In addition, the same trend was also exhibited in the contour diagrams and the surface diagrams shown in FIGS.

Based on these results, as shown in FIG. 12, the ^{three factors of pine explosives (25kgf/cm 2} , 7min) are substrate concentration 7%, enzyme concentration 60FPU, saccharification enhancer (Tween80) concentration 342.42 mg/g, glucan ), the maximum predicted saccharification rate was derived to be about 93.75%.

Example 5: saccharification of pine biomass by optimal conditions

The same treatment as in Example 3 was used in the range of 3 to 7% of the substrate concentration as a pre-treated pine explosive material (25 kgf/cm ^{2 ,13 minutes).} Table 8 shows the chemical composition of the pine explosives.

Component Rate of component ( % w/w) Glucan Xylan Galactan Arabinan Mannan Lignin Ash Pine explosives
(25kgf/cm ² , 3min) 36.79 2.61 1.60 - 4.81 44.47 0.40 Pine explosives
(25kgf/cm ² , 7min) 37.00 1.50 - - 2.05 47.77 0.32 Pine explosives
(25kgf/cm ² , 13min) 41.02 0.44 - - - 50.60 0.28 1) Acid insoluble lignin

After concentration, the enzyme activity of KMF006-derived cellulose was 3,241 ± 50.07 units/mℓ for endo-β-glucanase activity, 3,587 ± 41.92 unit/mℓ for β-glucosidase activity, and 256.92 ± 12.46 unit/mℓ for cellobiohydrolase activity, respectively. As a control, the commercial enzyme Cellic CTec2 (Novozyme) was used as a comparative enzyme.

Tween 80 (Sigma-aldeich) was used as the saccharification enhancer, and saccharification was performed under the following conditions, which are the optimal conditions established in Example 4.

Pre-explosion-treated pine trees optimal condition [1]: substrate concentration 7%, enzyme concentration 33.74 FPU, Tween80 500mg/g, glucan)

Pre-explosion-treated pine trees optimal condition [2]: substrate concentration 7%, enzyme concentration 60 FPU, Tween80 342.43mg/g, glucan)

Saccharification conditions: 72 hours, 5.0 pH, 250 rpm

As a result of applying the optimum condition [1], as shown in FIG. 13, the enzyme derived from KMF006 has a saccharification rate of 80.13% for cellulose (DAEJUNG, 20-100 microns), which is a comparative substrate, and a commercial enzyme (Cellic CTec2) of 76.55%. It showed a high saccharification rate compared to that of the saccharification rate.

The saccharification rate of the pine explosives was different according to the exploding time. In the case of pine trees without saccharification enhancer, the saccharification rate of pine trees pretreated with 3, 5, and 7 minutes of exploding time was about 50-76%, and about 90% of the saccharification rate in the case of 13 minutes exploding pretreatment. And showed similar results to the commercial enzyme

In the case of pine trees without saccharification enhancer, the saccharification rates of pine trees pretreated with 3 minutes and 7 minutes of detonation time were 21% and 33%, respectively, and about 76% in pine samples with 13 minutes pre-detonation treatment time. However, when the saccharification enhancer was used, the saccharification rate of pine trees with pre-explosion treatment time of 7 minutes and 13 minutes was 100%, respectively. According to this result, it was found that the optimum condition [1] derived from the saccharification optimum condition of the present invention can be applied to pine trees subjected to pre-detonation treatment. In particular, in the case of commercial enzymes, the performance of KMF006 under optimal conditions is considered to be considerably high in the possibility of future commercialization, compared to the low saccharification rate of about 33% even for samples with a pre-explosion treatment time of 13 minutes at the same enzyme concentration as KMF006.

As a result of applying the optimum condition [2], as shown in Fig. 14, the KMF006-derived enzyme had a saccharification rate of 90.79% for Cellulose, which is a comparative substrate, compared with a commercial enzyme (Cellic CTec2) showing an 80.50% saccharification rate, which is higher than that of the commercial enzyme (Cellic CTec2). Expressed the rate of hwaryul

In the case of pine trees without saccharification enhancer, the saccharification rates of pine trees pretreated with 3 minutes and 7 minutes of detonation time were 29.43% and 38.97%, respectively, and about 90.55% in pine samples with 13 minutes pre-detonation treatment time. However, when the saccharification enhancer was used, the saccharification rate was 98.00 and 100%, respectively, in pine samples with 7 minutes and 13 minutes of explosive treatment time. According to this result, it was found that the optimum condition [2] derived from the saccharification optimum condition of the present invention can be applied to pine trees subjected to pre-detonation treatment. In particular, in the case of commercial enzymes, the performance of KMF006 under optimal conditions is considered to be considerably higher for future commercialization, compared to KMF006 and a low saccharification rate for samples with a pre-explosion treatment time of 13 minutes.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

Saccharification method of pine biomass comprising the following steps:
(a) step of steam-blasting pretreatment for 3 to 20 minutes under conditions of 20 to 30 kgf/cm ^{2 of pine biomass;} And
(b) Endo-beta-1,4-glucanase (endo-β-1,4-glucanase), beta-glucanase from Trichoderma genus KMF006 strain (KCTC13500BP) that degrades lignin from the pretreated pine biomass Saccharification by treatment with a cellulase enzyme solution containing β-glucosidase and cellobiohydrolase and a saccharification enhancer.
delete delete delete The method of claim 1, wherein the saccharification enhancer is Tween 80.
delete The method of claim 5, wherein the saccharification enhancer is treated _{with 10 to 800 mg/g glucan of Tween 80.}
The method of claim 1, wherein the cellulase is treated with 10 to 80 FPU.

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