KR101946882B1 - Composition for saccharificaton of cellulosic biomass and method for enhancing enzymatic saccharification by using the same - Google Patents

Abstract

The present invention relates to a composition for saccharifying cellulosic biomass and a method for improving the saccharification rate using the cellulosic biomass. In particular, the present invention relates to a cellulosic biomass comprising a cellulase and a hemicellulase as saccharification enzymes and a polyethylene glycol Thereby providing an effect of improving the saccharification rate of cellulose.

Description

TECHNICAL FIELD The present invention relates to a composition for saccharification of cellulose-based biomass and a method for improving the saccharification rate using the same.

The present invention relates to a composition for cellulosic biomass saccharification which improves the glycation ratio of cellulosic biomass using cellulase, hemicellulase, and polyethylene glycol, and a method for improving the saccharification rate using the same.

As the development and use of excessive fossil fuel resources cause global environmental problems, it is urgent to search for environmentally friendly renewable energy sources to replace existing fossil fuels. In particular, Korea is highly dependent on fossil fuels (85%) and most of them are imported from foreign countries. Therefore, the importance of searching for renewable energy sources is very high.

Biomass as an alternative to fossil fuel means starch-based biomass derived from agricultural products and forest products, and plant organisms such as lignocellulose. It is close to carbon neutral in that it recycles carbon dioxide through photosynthesis . Among them, lignocellulose, a biomass of the second generation, is attracting attention as it is the most common carbohydrate on the planet, mainly forest products (herbaceous, woody) and agricultural products. In addition, there is no ethical problem of consuming food resources compared with starch-based biomass. Among these lignocellulosics, the palm fronds (EFB) are maternal products primarily produced in the tropics and are considered biomass suitable for the production of biochemicals and biofuels because of their high carbohydrate content (up to 60%) and abundant yields.

Processes for producing materials and fuels from lignocellulosic biomass can be divided into pretreatment of biomass, enzymatic saccharification and fermentation process. In particular, the enzyme saccharification process is the bottleneck step of the entire process, accounting for 20-25% of the total process cost. Therefore, the development of technologies for reducing the production cost of enzymes and the amount of enzyme input is being actively carried out all over the world. As a representative example, Novozymes, a global enzyme company in Denmark, produces 1 gallon of ethanol through enzyme development Efforts are underway to reduce the required enzyme cost of $ 0.5 to $ 0.3 by 2015.

In recent years, the search and development of accessory proteins or accessory agents has been recognized as an important part of efforts to reduce enzyme costs. Various saccharide supplements act to change cellulosic forms with high accessibility of enzymes, increase the stability of enzymes, or lower the viscosity and help the activity of cellulase. It is reported that if the amount of glycosylation enzyme can be reduced by a factor of 2 by addition of an adjuvant, the total process cost can be reduced by 1.7-1.9 times. In fact, Novozymes has developed Cellic CTec2, a commercial enzyme cocktail containing glycated synergy protein, to increase the sugar conversion rate and to reduce the total amount of enzyme needed.

 Koppram, R. et al. Trends Biotechnol, vol.32, pp.46-53 (2014)  Cara, C. et al. Proc Biochem, vol. 42, pp. 1003-1009 (2007)

An object of the present invention is to provide a composition for cellulosic biomass saccharification which uses cellulase and hemicellulase as saccharifying enzymes of cellulosic biomass and improves saccharification rate of high concentration of lignocellulose by using polyethylene glycol as an auxiliary, And a method for improving the rate.

In order to accomplish the above object, the present invention provides a pharmaceutical composition comprising: a cellulase; Hemicellulase; And a composition for cellulosic biomass saccharification comprising polyethylene glycol.

The present invention also provides a method for improving the glycation of cellulosic biomass, comprising saccharifying the cellulosic biomass using the composition for cellulosic biomass saccharification.

The present invention is based on the finding that the use of cellulase and hemicellulase as saccharifying enzymes of cellulosic biomass and the use of polyethylene glycol as an adjuvant improves the glycosylation rate of high concentration of lignocellulose and reduces the amount of cell wall degrading enzyme Thereby providing an effect of constructing a sugar production process that is easier and more efficient than the saccharification process.

Figure 1 shows the change in glycation of the hydrothermally treated palm fronds with varying concentrations of cellulase.
Figure 2 shows the change in glycation of the hydrothermally treated palm fronds according to the addition of various concentrations of hemicellulase.
Fig. 3 shows the change in the glycation ratio of the hydrothermally treated palm fronds with addition of polyethylene glycol.
FIG. 4 shows changes in the glycation rate of the palm frutescens by addition of cellulase, hemicellulase and polyethylene glycol at different concentrations of the substrate. FIG. 4 (a) shows the change in the concentration of the substrate when only 10 FPU / g glucan cellulase (Cellic CTec2) (B) is the change of the glycation rate according to the concentration of the substrate when Cellic HTEC2 (2.7 mg / g glucan), which is hemicellulase, is added to the cellulase, and (c) (D) is the change of the glycation rate according to the concentration of the substrate when hemicellulase and polyethylene glycol are added to the cellulase.
FIG. 5A shows the effect of increasing the glycosylation rate and protein-reducing effect of the parenchyma by using cellulase or cellulase and hemicellulase / polyethylene glycol at a low concentration substrate (1.8%, w / v), FIG. 5B shows the effect of high concentration substrate (21.7% In comparison with the effect of increasing the glycation rate and reducing the protein content of the parenchyma by the use of cellulase or cellulase and hemicellulase / polyethylene glycol.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a cellulase; Hemicellulase; And a composition for cellulosic biomass saccharification comprising polyethylene glycol.

The present invention also provides a method for improving the glycation of cellulosic biomass, comprising saccharifying the cellulosic biomass using the composition for cellulosic biomass saccharification.

One of the cautions in glycation of lignocellulosites is that the enzyme should be used in combination with the optimal combination according to the characteristics of the substrate according to the pretreatment method and that the amount of the enzyme which occupies a large proportion in the saccharification process cost should be minimized as much as possible .

The present invention relates to a method for producing a cellulosic biomass, which comprises using a cellulase and a hemicellulase as saccharification enzymes suitable for glycosylation of pretreated cellulosic biomass, polyethylene glycol as an adjuvant for increasing sugar conversion efficiency, .

The term "biomass" is intended to encompass all types of energy such as ethanol, hydrocarbons, gasoline, natural gas, methane, biodiesel and hydrogen gas, electricity, plastics, polymers, nutrients And livestock), fertilizers or other products, or a combination of these materials.

The term "cellulosic or lignocellulosic biomass" refers to any plant-derived organic material (wood or non-wood) that is available for energy on a substantially environmentally friendly basis. For example, herbaceous or woody biomass such as rice straw, giant herring, sugarcane by-products, corn by-products, switchgrass, poplar, empty fruit bunches (EFB), oak or energy crops can be used , And is not particularly limited thereto.

The term " pretreatment of biomass "means changing the bonding state between constituents in the biomass to make a form that is easy to decompose into monosaccharides. For example, the chemical bonding, arrangement, or stereomorphism of the constituents may be altered to remove non-sugar moieties and make the tissues impost to facilitate subsequent saccharification of the enzyme. Further, it is also possible to change the molecular structure, oxidize the constituent components in the biomass, change the average molecular weight, change the average crystallinity, change the surface area, change the degree of polymerization, change porosity, change branching degree, , Or a change in overall area size.

According to one embodiment of the present invention, cellulosic biomass Empty fruit bunches (EFB) can be used, and they can be pulverized to have a particle size of several millimeters or less.

In addition, the pretreatment can be carried out by a hydrothermal treatment, for example, mixing the palm room and water at a ratio of 1: 10-15 (w / v) and heating at 180 to 210 ° C for 10 minutes to 20 minutes Hydrothermal treatment can be performed. The hydrothermal treatment is not particularly limited because it can be suitably used in accordance with the type of the cellulosic biomass.

After the hydrothermal treatment, washing with water and solid-liquid separation may be performed to obtain a solid content.

The solid-liquid separation step may be performed through centrifugation, but is not limited thereto.

Next, the solid content of the cellulosic biomass and the cellulase; Hemicellulase; And saccharifying the cellulose-based biomass saccharifying composition comprising polyethylene glycol to the reactor to perform saccharification.

The present invention uses cellulase and hemicellulase as glycosylation enzymes. The saccharification enzymes are collectively referred to as enzymes that hydrolyze biomass to produce saccharides, and may be separated and purified from microorganisms producing saccharification enzymes, produced or separated by recombinant microorganisms or artificial chemical synthesis methods through genetic engineering recombinant techniques, Commercialization products can be adopted without limit.

The cellulase may be included at a concentration of 10 to 30 FPU, more specifically 10 to 12.5 FPU, more specifically 10 FPU per gram of glucan in the cellulosic biomass. If the concentration is less than 10 FPU, the final glucose yield is significantly lowered due to insolubility of the biomass. If the concentration exceeds 30 FPU, the enzyme cost, which accounts for more than 20% of the total biomass utilization process, It becomes difficult to commercialize the process.

In addition, the hemicellulase may be included at a concentration of 2.7 to 10.6 mg per gram of glucan in the cellulosic biomass, more specifically, 2.7 to 5.3 mg, more specifically, 2.7 mg. When the concentration is less than 2.7 mg, the final glucose yield is remarkably lowered due to the insolubility of the biomass. When the concentration exceeds 10.6 mg, the enzyme cost, which accounts for more than 20% of the total biomass utilization process, The possibility of commercialization of the process is eliminated.

Further, polyethylene glycol may be used as an adjuvant for maintaining the activity of the saccharifying enzyme for a long period of time and enhancing the sugar conversion rate through reaction with lignin, cellulose and the like.

The polyethylene glycol may have a weight average molecular weight of 4,000 to 8,000, more specifically 6,000 to 8,000, more specifically 8,000.

The polyethylene glycol may be included at a concentration of 15.6 to 62.5 mg per gram of glucan in the cellulosic biomass, more specifically 31.2 to 62.5 mg, more particularly 62.5 mg. If the concentration is less than 15.6 mg, the final glucose saccharification yield may be lowered, and if the concentration exceeds 62.5 mg, polyethylene glycol is added unnecessarily.

The cellulosic biomass is used as a substrate for saccharification based on the concentration of glucan, and 1% (w / v) of glucan means 0.1 g of glucan / 10 mL. Thus, preferably, the glucan in the cellulosic biomass is between 1 and 12% (w / v), more specifically between 3 and 12% (w / v), more specifically between 9 and 12% . ≪ / RTI > If it is outside the above range, the decomposition rate of the substrate may be significantly lowered.

Preferably, the saccharification is 10 to 12.5 FPU of cellulase, 2.7 to 5.3 mg of hemicellulase, and 31.2 to 62.5 of hemicellulase per gram of glucan in the cellulosic biomass relative to 1 to 12% (w / v) of glucan in the cellulosic biomass. mg. < / RTI >

The saccharification may be performed at a pH of 4 to 6, 45 to 55 ° C and 150 to 250 rpm for 50 to 80 hours, but is not particularly limited thereto.

In addition, the saccharification may be carried out with stirring at 100 to 500 rpm, more specifically 150 to 300 rpm, more specifically 150 to 250 rpm, for proper mixing of biomass and enzyme.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the scope of the present invention is not limited by the following Examples.

< Example  1> Heat number  Pretreatment and component analysis

The palm chamber was pulverized to have a particle size of 850 μm and then pretreated with water at a concentration of 1: 11.5 (w / v) at 190 ° C. for 15 minutes using a 30 L scale reactor . The approximate method is as follows. The mixture was over-nitrified at 800 ° C in a chamber of palm and 16 L of deionized water at 50 ° C and then centrifuged to remove excess water. The immersed farm balls and chambers were transferred to a reaction vessel, and then deionized water was added to a total volume of 10 L, followed by pretreatment at 190 ° C for 15 minutes. The pre - treated palm chamber was filtered with ultrafine filter (6 ㎛ or less) and centrifuged to remove water. After the freeze-drying process, the palm room and the room were stored at -20 ° C. The pretreated or raw samples were analyzed for constituents according to the NREL standard method (Table 1). The contents of pre - pretreated glucan, hemicellulose and lignin were 37.1 wt%, 24.7 wt% and 22.2 wt%, respectively, and the pretreated contents were 55.4 wt%, 7.6 wt% and 31.1 wt%, respectively. A rapid decrease in hemicellulose, a typical characteristic of hydrothermal pretreatment, was confirmed, and the recovery rate of glucan, which is the main substrate of glycation in the solid fraction after the pretreatment, was confirmed to be 100%.

< Example  2> Determination of proper concentration of commercial enzyme

The saccharification of hydrothermally pretreated farm chambers was performed according to the NREL standard method. The substrate was subjected to hydrothermal treatment in the same manner as in Example 1 at a concentration of 1% (w / v) glucan (= 0.1 g of glucan / 10 mL). Cellulase was treated with 10-30 FPU / g glucan And the change of the glycation rate was confirmed. The saccharification reaction was carried out in a reaction volume of 10 mL at a temperature of 50 ° C, pH 4.8 (using 50 mM sodium citrate buffer) and 200 rpm for 72 hours. The product of the enzyme reaction was analyzed and quantified using high performance liquid chromatography (HPLC) (using Aminex HPX-87H column).

As shown in FIG. 1, it was confirmed that the glycation ratios were increased to 51.1%, 70.7%, and 75.4% when the enzyme concentration was 10, 20, 30 FPU / g glucan. However, considering the economical feasibility of commercial approach, it was considered appropriate to increase the glycosylation rate of the adjuvant based on the cellic CTec2 concentration of 10 FPU.

< Example  3> Hemicellulase  Addition by concentration In the glycation rate  Impact

The reaction conditions are the same as in Example 2. However, in order to optimize the concentration of hemicellulase, Cellic HTec2 (0-120 mg / g glucan) was combined with the cellulase concentration 10 FPU / g glucan determined in Example 2.

As shown in FIG. 2, it was confirmed that the glycation rate was rapidly increased by increasing the protein concentration of Cellic HTec2. However, considering that the protein concentration of Cellic CTec2 of 10 FPU / g glucan is 18.7 mg / g glucan, it is difficult to determine the concentration of Cellic HTec2 only by the increase of the glycation rate. For example, at a Cellic HTec2 concentration of 60 mg, the glycation rate reaches a maximum (76.3%). Thus, the cellic HTec2 concentration of 2.7 mg / g glucan, which is the most economical concentration with the largest increase in the glycation rate per protein, was determined as the optimal value. On the other hand, the increase of the glycation rate per protein was 16.2%, 23.2% and 32.6% when Cellic HTec2 concentration was 2.7 mg, 5.3 mg and 10.6 mg / g glucan, respectively.

< Example  4> Of polyethylene glycol  Addition by concentration In the glycation rate  Impact on

Effectiveness of polyethylene glycol The experimental conditions were the same as in Example 2 except that polyethylene glycol 8000 was added as a saccharification aid. Experiments were conducted at various concentrations (0-250 mg / g glucan) to optimize the concentration of polyethylene glycol.

As shown in Fig. 3, the optimum value was determined to be 62.5 mg / g glucan, which is the minimum concentration capable of obtaining the maximum glycation rate (64.6%).

< Example  5> Palm Room  By concentration Glycation  Effect of supplementary combination

The saccharification experiments were carried out using both of the hemicellulase (2.7 mg / g gluan) and polyethylene glycol 8000 (62.5 mg / g glucan), all of the above-mentioned saccharide supplements. The reaction conditions are the same as in Example 2. Optimum concentrations of each of the adjuvants determined in Examples 3 and 4 were applied, and the concentrations of the five concentrations of palm and air (1.8, 5.4, 10.8, 16.3, 21.7% w / v) substrate corresponded to a glucan content of 1-12% (w / v)) (FIG. 4, Table 2).

As a result, it was confirmed that the saccharification rate by the addition of the adjuvant at the low substrate concentration of 1.8% was 75.2%, which was 47.2% higher than the saccharification rate of 51.1% without the adjuvant. These effects were more pronounced as the substrate concentration increased. In the case of 16.3% and 21.7% of substrates, which were high glycosylation conditions (more than 15% substrate), 75.4% and 72.5% It was confirmed that the maximum glycosylation rate of 50.1% was increased compared to the glycosylation rate in the absence of the adjuvant.

Finally, the low concentration (1.8% (w / v) substrate corresponds to 1% (w / v) glucan content) and the high concentration substrate (21.7% ), The cellic CTec2 concentration was increased to a maximum of 30 FPUs, followed by glycation, followed by addition of 10 FPU-based additives (Cellic HTec2 at 2.7 mg / g glucan and PEG 8000 at 62.5 mg / g glucan) As shown in Fig.

As a result, in the case of low concentration (1.8% substrate) saccharification, the addition rate of 10 FPU-based adjuvant (21.4 mg total protein) was confirmed to be almost the same as that of cellulase only when using 30 FPU (56.1 mg total protein) vs. 75.3%) and the total used protein was reduced by about 62% (Fig. 5A). In the case of high concentration (21.7% substrate) glycation, the rate of glycation was 72.5% when the adjuvant was used under the same conditions, and the rate of glycation at 30 FPU enzyme was 65.4% 5b).

Claims (12)

delete delete delete delete delete delete For 1 to 12% (w / v) of glucan in the cellulosic biomass,
45 to 55 占 폚, 150 to 250 rpm, at a pH of 4 to 6, by mixing a cellulase 10 to 12.5 FPU per gram of the glucan in the cellulosic biomass, a hemicellulase 2.7 to 5.3 mg / g glucan and a polyethylene glycol 31.2 to 62.5 mg / To 50% by weight of the total weight of the cellulosic biomass.
8. The method of claim 7,
The cellulosic biomass may be selected from the group consisting of cellulosic biomass saccharification rate, which is one of rice straw, giant aphid, sugarcane by-product, corn by-product, switchgrass, poplar, Empty fruit bunches (EFB), oak, How to improve.
8. The method of claim 7,
Wherein the cellulosic biomass is hydrothermally pretreated, followed by washing and solid-liquid separation steps.
10. The method of claim 9,
Wherein the hydrothermal pretreatment is performed by mixing the cellulosic biomass and the water at a ratio of 1:10 to 15 (w / v) and heating at 180 to 210 ° C for 10 to 20 minutes, thereby improving the saccharification rate of the cellulosic biomass .
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