WO2013162165A1 - Method for producing large quantities of cellulase using palm by-products - Google Patents

Abstract

The present invention provides a method for producing large quantities of cellulase, comprising the steps of: (a) adding water to pulverized palm by-products, inoculating Aspergillus oryzae, which produces cellulase, and adding a nitrogen source; and (b) carrying out solid state fermentation at 27-33 ℃ for 2-12 days.

Description

Mass production method of cellulase using palm byproducts

The present invention relates to a method for mass production of cellulase using palm byproducts, and more particularly, (a) inoculating Aspergillus oryzae to produce cellulase after adding water to pulverized palm byproducts, Adding a; And (b) relates to a mass production method of cellulase comprising the step of solid phase fermentation for 2 to 12 days at 27 ~ 33 ℃.

Palm trees are palm trees, and are generally grown in tropical countries such as Southeast Asia. Each year, 8-15 fresh fruit bunches (FFBs) are held on a single palm tree, with 1,000-1,300 berries in each fruit tree. The harvested palm fruit is immediately fed into the milking process at the palm oil plant, which produces various forms of palm byproducts. At the first stage of the milking process, the palm fruits are sterilized by steam at high temperature and high pressure. The sterilized fruit clusters are passed through a rotary drum thresher, and the palm fruits fall off, and the empty fruit bunch (EFB) and fruits are used as by-products. Fibers are formed. In addition, palm by-products generated during the milking process include palm kernel shell, palm kernel cake and palm oil mill effluent (POME). There are also palm byproducts, such as the leaves that are removed from the bottom of the stems periodically to grow palm trees, and the trunks of trees that are cut off with long life. The amount of empty by-products of these palm by-products amounts to 22% of the processed palm fruit by-products, and a proper utilization method for treating such by-products of palm trees has not been developed.

There has been a lot of research for glycosylating cellulose and xylan for a long time, and a lot of research has been carried out, especially with the development of glycosylation enzymes. As a result, the use of these saccharifying enzymes has been variously developed and commercialized in various fields, and new applied researches are actively being conducted. Cellulase is widely used in the textile industry, the paper industry, the detergent industry, and the feed industry. In addition to the food industry, the cellulase has been applied to various uses such as the production of low-calorie foods and the fermentation of food waste. On the other hand, xylanase (xylanase) has been widely applied to the paper industry, food industry and feed industry.

In the past, the production of these enzymes mainly used fungi, and especially in the industrial aspect, the production of enzymes mainly used Aspergillus and Trichodelma. Cellulase-producing strains were mainly studied by Trichodelmar lyse, and xylanase was mainly studied by bacteria of the genus Bacillus and Aspergillus. In general, enzyme production is mainly produced by culturing cellulase producing strain in the liquid phase. However, there was a problem that the concentration and activity of enzymes were not sufficient to meet the industrialization, and the production cost was due to the use of expensive culture medium containing expensive substances such as glucose as a substrate for the production of these enzymes. There was a high issue.

Korean Patent No. 0449170 discloses 'Aspergillus Niger KK2 strain producing cellulase and xylanase and enzymes and solid cultures produced therefrom', and Korean Patent Publication No. 2002-0004003 In the present invention, 'Aspergillus CＸ-1 strain producing a hydrolase and a hydrolase produced by the strain' are disclosed, but as described in the present invention, a mass production method of cellulase using palm byproducts is disclosed. none.

The present invention is derived from the above requirements, the present inventors added aspergillus duck material, a strain that produces cellulase using palm byproducts as a carbon source, and after 30 ° C. for 10 days of solid phase fermentation, the cellulase was a large amount. In particular, beta-glucosidase production yield, which is a kind of cellulase, was confirmed to be significantly higher than that of other strains when using Aspergillus duck, thereby completing the present invention.

In order to solve the above problems, the present invention

(a) inoculating Aspergillus oryzae producing cellulase after adding water to the crushed palm byproducts and adding a nitrogen source; And

(b) provides a cellulase mass production method comprising the step of solid phase fermentation at 27 ~ 33 ℃ for 2 to 12 days.

The present invention provides a method of mass-producing cellulase through solid phase fermentation after adding aspergillus duck material using a palm by-product that is discarded without proper use as a carbon source. It is an industrially useful invention.

1 shows solid phases in three strains of Aspergillus oryzae (GDX02), Penicillium oxalicum (GDX01) and Trichoderma longibrachiatum using palm by-products as carbon sources. The result of comparing the cellulase production yield through fermentation is shown.

Figure 2 shows the results of comparing the yield of cellulase production through the solid phase fermentation of Aspergillus oryzae using palm by-products and rice straw as a carbon source.

Figure 3 shows the result of comparing the cellulase production yield according to the initial water content of palm by-products.

Figure 4 shows the result of comparing the yield of cellulase production through the solid phase fermentation of Aspergillus duck ash ( Aspergillus oryzae ) using the yeast extract and chaejongbak as a nitrogen source.

5 is a result of examining the activity of the cellulase produced through the present invention (AS is a cellulase produced using Aspergillus duck material).

In order to achieve the object of the present invention, the present invention

(a) inoculating Aspergillus oryzae producing cellulase after adding water to the crushed palm byproducts and adding a nitrogen source; And

(b) provides a cellulase mass production method comprising the step of solid phase fermentation at 27 ~ 33 ℃ for 2 to 12 days.

In the method according to an embodiment of the present invention, the palm by-product of step (a) may be EFB (Empty Fruit Bunch), fruit fiber (fiber), palm tree leaves (frond), palm tree trunk (trunk), Preferably it may be EFB, but is not limited thereto.

In the method according to an embodiment of the present invention, the addition of water in the step (a) is to add the water content of the palm by-product is 30 to 70%, preferably the water content of the palm by-product is added to 55 to 65%. Most preferably, the water content of the palm byproduct is added to 60%, but is not limited thereto.

In the method according to an embodiment of the present invention, the nitrogen source of step (a) may be chaejongbak, preferably 15-20% by weight of chaejongbak based on the crushed palm by-product added water, more preferably Preferably it may be 17.5 to 20% by weight of chaejongbak, most preferably may be 17.5% by weight of chaejongbak, but is not limited thereto.

In the method according to an embodiment of the present invention, the cellulase of step (b) may be endoglucanase, exoglucanase, or beta-glucosidase, preferably Preferably beta-glucosidase, but is not limited thereto.

Thus, the present invention preferably

(a) grinding the palm byproducts;

(b) adding water to the crushed palm byproducts of step (a) such that the water content is 30 to 70%;

(c) inoculating Aspergillus oryzae producing cellulase to the palm by-product to which the water of step (b) is added, and adding 15-20% by weight of chaejongbak, which is a nitrogen source; And

(d) provides a beta-glucosidase mass production method comprising the step of solid-phase fermentation at 27 ~ 33 ℃ for 2-12 days,

More preferably

(a) grinding the palm byproducts;

(b) adding water to the pulverized palm byproduct of step (a) such that the water content is 60%;

(c) inoculating Aspergillus oryzae producing cellulase to the palm by-product to which the water of step (b) is added, and adding 17.5% by weight of chaejongbak, which is a nitrogen source; And

(D) it can provide a beta-glucosidase mass production method comprising the solid phase fermentation at 30 ℃ for 10 days, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Experiment method

1. Management and Storage of Strains

Aspergillus oryzae ( KCTC 6983T) , obtained from the Korea Life Resource Center, is a fungus that secretes cellulase. In general, 3-4 days of incubation at 28 ° C. in a medium of PDA (potato dextrose agar), and a safe cryopreservation method was used for storage of the strain. The grown fungus was stored in -70 ° C in 6 pieces of 15% glycerol in agar block state. In preparation, several were prepared and stored in different cryogenic freezers. In addition to the cryopreservation method, a one-year storage method was used using a slope medium.

2. Palm by-product solid phase fermentation and production of cellulase

① Solid Fermentation Medium Using Palm By-products

Among the palm byproducts, EFB (empty fruit bunch) was a fiber type lignocellulose (lignocellulose) from which palm fruits were removed, which was crushed to a length of 5 to 10 mm using a cutter mill. 40g EFB 40g or rice straw was added to a 500ml bottle and additionally water and nitrogen sources. Subsequent experimental methods conducted on EFB and rice straw were performed identically. Water is an important factor for fungi to secrete cellulase. Therefore, the initial water content was 40-80%, and water was added according to the conditions of optimal cellulase activity. Nitrogen source was 17.5% rapeseed. The fungus incubated for about a week was inoculated into a solid fermentation medium and cultured at an optimum temperature of 30 ° C. Inoculation of the fungus was carried out by cutting a PDA medium containing a sufficiently grown Aspergillus duck material with a round agar cutter to a diameter of 0.5 cm and adding 5 pieces. The culture was completed on day 3 by measuring cellulase activity and used for cellulase production.

② Cellulase Elution

When the solid phase fermentation is completed, the solid material is mixed with mold, cellulase secreted by the fungus, and other media components. Cellulase was eluted with 0.05M sodium citrate buffer (pH 4.8) because it was entangled with fungal cells and EFB. 4-5 times the volume of the solid phase fermentation medium and the buffer was mixed well. This was put in a 30 ℃ shake incubator and allowed to react for one hour. Finally, cellulase was dissolved in the buffer to prepare an enzyme in liquid form.

③ Cellulase Isolation and Concentration

Separation was performed to obtain only cellulase secreted by pure liquid fungus. First, to separate the EFB and fungal cells was centrifuged for 1 hour at 8000rpm using a large-capacity centrifuge. This was again centrifuged at 13000 rpm for 20 minutes and then used MF (membrane filtration) to completely separate fungal cells. In general, since microorganisms are filtered through a 0.2 μm filter, only cellulase in liquid form was obtained by vacuum filtration using this filter. Finally, to increase the concentration of cellulase was concentrated using UF (ultrafiltration) method. Since the filter filters out components with a molecular weight of 30,000 or less, only the cellulase that is continuously filtered out and finally concentrated 10 times or more is obtained.

3. Measurement of Cellulase Activity

Cellulase is mainly classified as endoglucanase (endoglucanase), exoglucanase (exoglucanase), beta-glucosidase (beta-glucosidase) and when they act in a suitable ratio, the conversion from cellulose to glucose. Three kinds of substrates were used to determine cellulase activity, and the activity of endoxylanase was also measured to determine the resolution of hemicellulose. There is a method of directly inserting cellulose into a substrate and measuring reducing sugars degraded by an enzyme using an OD value. A filter paper assay was used to measure the activity of total cellulase, which puts the substrate into filter paper which is cellulose. In addition, CMCase activity can be confirmed through CMC, and beta-glucosidase activity can be confirmed using cellobiose, a dimer of glucose, as a substrate. The unit used to measure activity was based on 1 μmole of product made per minute of IU (international unit).

① Total Cellulase Measurement

Filter paper assays are a method of measuring the activity of total cellulase. 1x6 cm size of Whatman no.1 filter paper was used as the substrate. This is the equivalent of 50 mg of glucose. 1 ml of 0.05M sodium citrate buffer was placed in a centrifuge tube (Microcentrifuge tube), and the supernatant of the solid phase fermentation sample containing cellulase was added to 500 μl. The filter paper was added and not added thereto and reacted at 50 ° C. for 1 hour. Dinitro salicylic acid (DNS) analysis was used to identify the reducing sugars formed on ice immediately after the reaction and decompose the substrate. 200 μl of the reaction solution and 700 μl of the DNS solution were mixed and then boiled for 10 minutes. After cooling on ice, absorbance was measured at a wavelength of 540 nm. Prior to this, a standard curve was made using glucose 1mmole, and the generated sugar was calculated. The unit used was U, which was based on 1 μmole of sugar produced by breaking down the filter paper per minute.

② CMCase measurement

CMC (carboxy methylcellulose) is a cellulose-like substance in which cellulose ends are substituted with carboxyl groups and methyl groups, respectively. Thus endoglucanase acts specifically. 1% CMC was used as substrate and proceeded on a principle similar to filter paper analysis. First, 100 μl of 0.1 M potassium phosphate buffer (pH 6.0) was added to a centrifuge tube, and 50 μl of the solid phase fermentation supernatant containing 1% CMC and cellulase was added to make a total 200 μl reaction solution. 50 μl of 0.1 M potassium phosphate buffer was added instead of 50 μl of 1% CMC as a negative control. In addition, the blank was removed from the spectrophotometer (spectrophotometer) and the enzyme was removed with 1% CMC instead. When the reaction solution was completed, the reaction was carried out for 30 minutes in a 50 ℃ incubator, and the activity was measured by DNS analysis as in the filter paper analysis.

③ beta-glucosidase measurement

In general, the activity of beta-glucosidase is measured using pNPG. However, using CB (cellobiose) directly as a substrate allows simpler activity measurement. The reaction solution was prepared by adding 500 μl of sodium citrate buffer to the solid phase fermentation supernatant containing 500 μl of cellulase. A buffer containing 15 mM CB was used as the substrate. The reaction was carried out in a 50 ° C. incubator for 30 minutes, and boiled immediately for 10 minutes to stop the reaction. The amount of degraded glucose was measured using the glucose KIT of Youngdong Pharmaceutical. 10 μl of the reaction solution was added to 1 ml of KIT and reacted at 37 ° C. for 10 minutes. Absorbance was measured at OD _{600 nm} with a blank sample without the reaction solution. Similar to DNS analysis, glucose was used to draw a standard curve.

④ Endozylanase measurement

Unlike hexasaccharide, cellulose, hemicellulose is broken down by xylanase. Substrate was used 1% Birchwood xylan, the experimental method was performed in the same manner as the CMC analysis.

4. Validation through saccharification of cellulase produced

Biomass without pretreatment did not saccharify well even after treatment with cellulase, so that the saccharification process was performed after pretreatment according to certain conditions. After mixing EFB in a natural state with a solid solution ratio of 1: 6, the reaction mixture was 150 ° C. for 30 minutes. This removes the lignin component and converts it into a cellulose form that is susceptible to glycosylation. The glycosylation ability was confirmed by measuring the rate of conversion to glucose when cellulase was treated using pre-treated EFB as a material for saccharification. Cellulase 10FPU (filter paper unit) was added to 1 g of pre-treated EFB, and the remainder was filled with 0.05 M sodium citrate buffer to obtain a high liquid ratio of 1: 100. This was reacted at 50 rpm suspension incubator for 72 hours at 150 rpm. 1 ml was taken in the middle of reaction and glucose amount was measured. Glucose was measured using high performance liquid chromatography (HPLC).

Example 1 Determination of Enzyme Activity Isolated from Aspergillus Duckwood

Cellulase production through solid phase fermentation was compared in three strains of Penicillium oxalicum (GDX01), Aspergillus oryzae (GDX02) and Trichoderma longibrachiatum (Fig. One). FPase showed the highest activity in all three strains, but the two strains, Aspergillus duck and Trichoderma longgibraquiatum, showed rapid activity. In addition, beta-glucosidase (beta-glucosidase) activity can be seen that the Aspergillus duck material is significantly higher (Fig. 1).

The cellulase productivity according to the type of carbon source was compared (FIG. 2). EFB and rice straw were used as carbon sources. FPase, beta-glucosidase and xylanase were more produced in rice straw and CMCase activity was little different between the two carbon sources (FIG. 2). Through this, it was confirmed that the cellulase productivity using EFB was similar to that of rice straw.

In order to find the optimum conditions for solid state fermentation using Aspergillus duck material, experiments were performed by changing the factors affecting solid state fermentation (FIG. 3). Initial yield was an important factor in solid phase fermentation, and cellulase productivity was varied from 30% to 80%. The activity was measured at 2, 3, and 4 days after the cultivation. There was no significant difference in the FPase production from 40 to 60% of the initial water content after 3 days of cultivation, and it started to decrease from 70% to about 80% at the 80% initial content. Decreased. Meanwhile, beta-glucosidase showed a similar pattern (FIG. 3).

Nitrogen source was changed from 10% to 20% of chaejongbak as an important factor in solid phase fermentation and compared with 5% of yeast extract and cellulase productivity was investigated (FIG. 4). At 3 and 4 days after incubation, the FPase production was increased up to 10% -17.5% of the cultivars at 3 days after incubation, and higher than 5% of the yeast extract at 17.5%. On the other hand, Veda-glucosidase showed higher results in 10% -20% of chaejongbak than 5% of yeast extract (Fig. 4).

Glycosylation experiments were performed using pre-treated EFB to see if cellulase produced under optimized conditions was effective for actual saccharification (FIG. 5). At this time, Cellullast (Celluclast 1.5L) sold by Novozymes was used as a control. In addition, the experiment was carried out by mixing the cellulose and the enzyme produced in Aspergillus duck. As a result, the enzyme produced by Aspergillus duck alone was found to be less glycosylated than cellulose. However, when the two enzymes were mixed, the rate of saccharification as well as the rate of saccharification was faster than that of cellulose. This suggests that the Aspergillus duck enzyme contains a protein that promotes glycosylation. In particular, aspergillus duck enzyme has a strong xylanase activity. In fact, when comparing the xylose glycosylation rate, it can be seen that xylose glycosylation rate is highest when the aspergillus duck enzyme is used alone (FIG. 5). ).

Claims (6)

1. (a) inoculating Aspergillus oryzae producing cellulase after adding water to the crushed palm byproducts and adding a nitrogen source; And

   (B) a cellulase mass production method comprising the step of solid phase fermentation at 27 ~ 33 ℃ for 2 to 12 days.
2. The method of claim 1, wherein the palm by-product of step (a) is EFB (Empty Fruit Bunch), fruit fiber (fiber), palm tree leaves (frond) or palm tree trunk (trunk).
3. The method of claim 1, wherein the addition of water in step (a) is characterized in that the addition of the water content of the palm by-product is 30 to 70%.
4. The method of claim 1, wherein the nitrogen source of step (a) is characterized in that the seed.
5. The method according to claim 1, wherein the cellulase of step (b) is endoglucanase, exoglucanase, or beta-glucosidase.
6. The method of claim 1,

   (a) grinding the palm byproducts;

   (b) adding water to the crushed palm byproducts of step (a) such that the water content is 30 to 70%;

   (c) inoculating Aspergillus oryzae producing cellulase to the palm by-product to which the water of step (b) is added, and adding 15-20% by weight of chaejongbak, which is a nitrogen source; And

   (d) a method for mass production of beta-glucosidase comprising the step of solid-phase fermentation at 27-33 ° C. for 2-12 days.

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