JPS6117476B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for saccharifying cellulose using cellulase produced by Acremonium bacteria. Cellulase is a general term for a group of enzymes that catalyze the enzymatic reaction system that breaks down cellulose into glucose, cellobiose, and cellooligosaccharides. Depending on their mode of action, cellulase can be classified into _C1 enzyme, _Cx enzyme, β-glucosidase, exo-β-glucanase, End-
There are enzymes that are called by various names, such as β-glucanase and cellobiase, but their true nature is still unclear. This is due to the presence of a wide variety of enzymes with different modes of action on crystalline cellulose, carboxymethyl cellulose (CMC), cellodextrin, cellooligosaccharides, cellobiose, etc., depending on the origin of the microorganism that produces cellulase, and the structural complexity of natural cellulose. caused by.
However, cellulase is a complex enzyme that breaks down cellulose into its constituent sugars through the harmonious interaction of these multiple enzymes. In recent years, cellulases have attracted attention and have been actively researched from the perspective of effectively utilizing biomass resources, but until now, cellulases produced by well-known microorganisms such as Trichoderma and Aspergillus have been There were problems in that the decomposition power for natural cellulose was not sufficient, and cellulose could not be completely decomposed into its constituent sugar, glucose, resulting in the production of large amounts of cellobiose and cellooligosaccharides. Furthermore, conventionally known cellulases usually have poor thermostability, and in long-term saccharification reactions 45 to 50%
Since the reaction could only be carried out at temperatures around °C, there was often a risk of contamination with bacteria during saccharification. The present inventors have searched extensively for microorganisms in the natural world in search of cellulase-producing bacteria that have excellent decomposition power for crystalline cellulose and a strong ability to saccharify glucose into glucose. Cellulase produced by a filamentous fungus identified as belonging to the genus Acremonium has a strong degrading power for crystalline cellulose, and this enzyme also contains β-glucosidase compared to the well-known cellulases produced by Trichoderma reesei etc. Because it is contained in large amounts, it was found that it is an enzyme with extremely excellent saccharification properties, being able to almost completely decompose various cellulose sources into glucose. Furthermore, since the cellulase produced by this bacterium has excellent thermal stability, the reaction can be carried out at a temperature approximately 5 to 10°C higher than the saccharification temperature when using conventional cellulases. For this reason, it has been recognized that the enzyme is highly effective both technically and economically in terms of its use, with almost no bacterial contamination occurring during saccharification. The present invention has been made based on this knowledge. That is, the present invention is characterized in that Acremonium genus bacteria having cellulase-producing ability are cultivated, and the cellulase obtained from the culture is allowed to act on cellulose or cellulose-containing materials. The present invention relates to a method for treating cellulose-containing materials. The present invention will be explained in more detail below. The cellulase produced by the present invention has a high resistance to cellulases with high crystallinity such as Avicel.
This can be almost completely disassembled. That is, the sugar composition of the cellulose saccharide obtained by the present invention is mostly glucose, and hardly any cellobiose or cellooligosaccharides are produced. 1st
The figure shows changes over time in the decomposed products when Avicel was decomposed with the enzyme of the present invention. In other words, in the decomposition products generated from the early stage of the reaction, the amount of reducing sugar measured as glucose, the total amount of sugar measured by the phenol-sulfuric acid method and quantified as glucose, and the amount of glucose determined by the glucose oxidase method are almost the same. . This means that the decomposition products obtained when cellulose is decomposed by the enzyme of the present invention are mostly glucose. On the other hand, for example, it has been reported that the cellulose saccharified product produced by Trichoderma reesei, a well-known cellulase, has a cellobiose:glucose ratio of 1:1 to 1:3. . {Journal of
Fermentation Technology, Vol. 54, p. 267 (1976), etc.} Then, in order to decompose this cellobiose into glucose, β-glucosidase from Aspergillus niger, which has an excellent decomposition ability against cellobiose, is used in combination. It is common practice to use it. {Applied
Microbiology, Vol. 16, p. 419 (1968), etc.).
However, since the cellulase produced according to the present invention contains a sufficient amount of β-glucosidase, there is no need to add β-glucosidase from other sources. Trichoderma reesei exemplified in Table 1
Cellulase produced by QM9414 strain, FPase (which is thought to have the same effect as Avicelase, and a 1 x 6 cm piece of filter paper is used as a substrate instead of Avicelase), CMCase and β of the present invention. - Representative amounts of glucosidase are shown.

[Table] Notes; (1) Cellulase data of Trichoderma reesei QM9414 strain is provided by M. Mandel et al. Biotech, Bioenz.
, XX2009 (1981). (2) FPase is an enzyme titer that decomposes a piece of filter paper (1 x 6 cm) to produce reducing sugar, and the measurement method is as described in J.
Ferm.Tech., Vol. 54, p. 267 (1976), and the activity was expressed in international units (the amount of enzyme that produces a reducing power equivalent to 1 μmol of glucose per minute). As is clear from the table, the cellulase produced by Trichoderma reesei is FPase: CMC
The ratio of enzyme:β-glucosidase is 1:17.5:
0.06, whereas the same ratio of cellulase produced by the present invention is 1:12.1:5.4. Usually, the enzyme activity ratio produced by the present invention is 1:10-20:3-10, and β
- It is characterized by a significantly high glucosidase activity content ratio. This is thought to be an important reason why the saccharified product of cellulose when using the cellulase of the present invention consists almost exclusively of glucose. As is clear from the above, the fact that the product is mostly glucose even when the reaction is carried out under a high concentration of substrate suggests that the cellulase β-glucosidase produced by the present invention is not subject to much product inhibition. It is also important to be able to As described above, the cellulase agent used in the present invention contains extremely highly active β-glucosidase, and this enzyme is a novel β-glucosidase as shown in the enzymatic properties section below. Saccharification of cellulose using the cellulase agent of the present invention can be applied not only to pure cellulose such as Avicel, cellulose powder, and cotton, but also to herbaceous plants such as bagasse, rice straw, rice husk, napia, newspaper, various pulps, and cardboard. Cellulose-containing materials can also be suitably processed by subjecting them to appropriate treatments such as alkali treatment and blasting. In this case, advantageously, since the cellulase produced by the present invention has a strong hemicellulase activity, hemicellulose such as xylan contained in the cellulose-containing material can also be used as a constituent sugar such as xylose. It was found that it was decomposed at a high rate.
Therefore, the enzyme of the present invention is useful not only for the production of glucose from cellulose or cellulose-containing materials, but also for the production of xylose from hemicellulose,
It can also be effectively used to remove the skins and extract active ingredients from grains such as rice, wheat, and beans, fruits, and seaweeds. It can also be used in medicine as a digestive agent. The mycological properties of the cellulase-producing bacteria used in the present invention are as follows. Growth: Growth is fast on malt extract agar at 30℃7
It reaches a diameter of 70mm in days. The colony is initially white and later becomes slightly yellowish. The aerial hyphae are loosely swollen, wool-like, and sometimes form rope-like hyphal bundles.
In the later stages of cultivation, the underside of the colony becomes pinkish-brown to reddish-brown. Almost the same growth was observed on Tuapetsk agar, but the growth of aerial mycelium was smaller. Growth PH
The range is 3.5 to 6.0, with an optimal pH around 4, and a growth temperature range of 15°C to 43°C, with an optimal growth temperature around 30°C. Morphology: The diameter of the hyphae is 0.5 to 2.5 μm, colorless, and septa are observed in the hyphae. In addition, the hyphal surface is smooth. Conidia: The ability to form conidia is extremely unstable and disappears by subculturing on Czapetsk agar and malt extract agar media. When observed during isolation, the conidiophores protrude from the sides of the aerial hyphae and are colorless.
Conidia are subglobular (2.5-5 x 2-4.5 μm), smooth, colorless, and have very loose chains and are easily dispersed. Regarding the above mycological properties, see “Cephalosporiumartige Schimmelpilge” by W. Gams, P84.
G. Fisher (1971) and CHDickinson, Mycol.
As a result of referring to Papers 115 P10 (1968), we considered that it is appropriate to consider this fungus to be a filamentous fungus closely related to the genus Acremonium. In addition,
The genus Acremonium has not been known to have strong cellulase productivity, and the strain of the present invention produces a strong and characteristic cellulase. cellulolyticus). This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-6867. The genus Acremonium is a genus name recently adopted after a detailed study by Gams and a reexamination of the genus previously described as the genus Cephalosporium. Bacteria of the genus Acremonium that produce strong true cellulases capable of acting on cellulose, namely Avicelase and FPase, were completely unknown prior to the present invention. The cellulases produced by this bacterium are represented by enzymes that act on Avicel, which is a highly crystalline cellulase due to its action characteristics, such as _C1 enzymes represented by Avicelase or FPase, and so-called CMCase that acts on carboxymethyl cellulose (CMC). 3, mainly the C _x enzyme that is
It is a complex enzyme system consisting of different enzyme groups. Through the harmonious interaction of these enzymes, natural cellulose can be completely broken down into glucose. The properties of these enzymes are described below. (A) Enzymatic properties of Avicelase (1) Action It acts on highly crystalline insoluble cellulose such as cellulose powder, Avicel, and absorbent cotton to produce reducing sugars such as glucose and cellobiose. (2) Action PH and optimal action PH The action PH range of this enzyme was 2 to 8, and the optimum action PH was found to be approximately 4.5. (Figure 2a) (3) Stable PH The stable PH range was about 3.5 to about 6 when left at 45° C. for 20 hours under a citric acid-phosphate buffer. (Fig. 2c) (4) Action temperature range and optimal action temperature This enzyme acts at high temperatures up to approximately 90°C;
% Avicel, the optimal working temperature is about 65 when reacted for 10 minutes under 0.05M acetate buffer (PH4.5)
℃ was observed. (Figure 2b) (5) Thermostability As a result of heat-treating this enzyme at each temperature for 10 minutes under 0.05M acetate buffer (PH4.5), the enzyme showed almost no stability at temperatures up to about 60°C. Not inactivated, 65
Heating at 70°C for 10 minutes deactivated about 50%, and heating at 70°C for 10 minutes deactivated about 80%. (Figure 2d) (6) Inhibitor Among various heavy metal ions, it is strongly inhibited by mercury ion and copper ion at 1mM or more.
It is also inhibited by approximately 80% at 1 mM by the SH inhibitor parachlormercury benzoate. (7) Purification method This enzyme is desalted and concentrated from the culture filtrate using a holofiber (Amicon HI-P5).
Further purification can be achieved by column chromatography using DEAE-Sepharose (CL-6B) (NaCl 0→1M gradient) and rechromatography using the same column (NaCl 0→0.6M). (8) Molecular weight The molecular weight measured by gel filtration using a Bio-gel (A0.5m) column was approximately 140,000. (9) Activity measurement method Add an appropriate amount of enzyme solution to 0.5ml of 0.5% Avicel suspension (PH4.5) in 0.1M acetate buffer, make the total volume 1.0ml with distilled water, and perform the reaction at 50℃. . The reducing sugar produced was measured by the Somogyi-Nelson method. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit. (B) Enzymatic properties of CMCase (1) Multicomponent nature of CMCase CMCase is separated into at least four components by disk electrophoresis, and each component is distinguished by its molecular weight and isoelectric point. CMCase has a molecular weight of approximately 160,000 and an isoelectric point of 5.08;
160000, 4.95, is approximately 120000, 4.60, is approximately
120,000, 4.48, and CMCase is composed of a complex of these isozymes. (2) A component (CMCase) that acts on soluble cellulose derivatives such as carboxymethylcellulose (CMC) and decomposes it into glucose, cellobiose, etc. and an action that produces very little glucose and decomposes it into cellooligosaccharides larger than cellobiose. There is a component (CMCase,) with (3) Action PH and optimal action PH The action PH range of the CMCase complex is approximately 2 to
Over the course of 8 years, the optimal working PH was found to be approximately 4.5. (Figure 2a) (4) Stable pH Stable pH of CMCase complex when left at 45℃ for 20 hours under citrate-phosphate buffer
The range was about 3.5 to about 6. (Figure 2c) (5) Action temperature range and optimal action temperature This CMCase complex acts at high temperatures up to about 90°C, but under 1% CMC and 0.05M acetate buffer (PH4.5). The optimum working temperature was found to be about 65°C when reacted for 10 minutes. (Figure 2b) (6) Thermostability As a result of heat-treating this enzyme for 10 minutes at each temperature under 0.05M acetate buffer (PH4.5), the enzyme showed almost no stability at temperatures up to about 60°C. Not inactivated, 65
Heating at 70°C for 10 minutes deactivated about 40%, and heating at 70°C for 10 minutes deactivated about 70%. (Figure 2d) (7) Inhibitor Among various heavy metal ions, it is strongly inhibited by mercury ion and copper ion at 1mM or more. (8) Purification method This enzyme is desalted and concentrated from the culture filtrate using a holofiber (Amicon HI-P5).
It can be purified into each component by column chromatography using DEAE-Sepharose (CL-6B) (NaCl 0→1M gradient), rechromatography using the same column, and chromatofocusing. (9) Activity measurement method Add an appropriate amount of enzyme solution to 0.5ml of 1% CMC solution (PH4.5) dissolved in 0.1M acetate buffer, make the total volume 1.0ml with distilled water, and perform the reaction at 50℃. . The reducing sugar produced was measured by the Somogyi-Nelson method. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit. (C) Enzymatic properties of β-glucosidase (1) Actions Salicin, cellobiose, cellotriose,
It acts on cellooligosaccharides such as cellotetraose, cellopentaose, and cellohexaose,
This is broken down into glucose. Additionally, this enzyme acts on polymer cellulose such as Avicel, but has almost no effect on CMC or HEC (hydroxyethylcellulose). The Km values for salicin, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose were 3.40, 2.26, and 1.19, respectively.
They were 0.82, 0.52 and 0.51mM. (2) Action PH and optimal action PH The action PH range of this enzyme was 2 to 8, and the optimum action PH was found to be approximately 4.5. (Figure 2a) (3) Stable PH The stable PH range was about 3.5 to about 5 when left at 45°C for 20 hours under a citric acid-phosphate buffer. (Fig. 2c) (4) Action temperature range and optimal action temperature This enzyme acts at high temperatures up to approximately 90°C;
% salicin, the optimal working temperature when reacted for 10 minutes under 0.05 M acetate buffer (PH4.5) is approximately 65
℃ was observed. (Figure 2b) (5) Thermostability As a result of heat treatment at each temperature for 10 minutes in 0.05M acetate buffer (PH4.5), this enzyme was almost inactivated at high temperatures up to about 65℃. Approximately 40% of the activity was inactivated by heating at 70°C for 10 minutes, and more than 90% was inactivated by heating at 80°C for 10 minutes. (Figure 2d) (6) Inhibitor Among various heavy metal ions, it is strongly inhibited by mercury ion and copper ion at 1mM or more. Furthermore, glucose-δ-lactone acts as a competitive inhibitor against the substrate. (7) Purification method This enzyme was desalted and concentrated from the culture filtrate using a holofiber (Amicon HI-P5).
Column chromatography (NaCl 0→1M gradient) and chromatofocusing (PH6→
4) and gel filtration using Bio-gel (A0.5m), it can be electrophoretically purified to homogeneity. (8) Molecular weight The molecular weight measured by gel filtration using Bio-gel (A0.5m) was approximately 240,000. (9) Activity measurement method An appropriate amount of enzyme solution was added to 0.5 ml of 1% salicin solution (PH4.5) dissolved in 0.1 M acetate buffer, the total volume was made up to 1.0 ml with distilled water, and the reaction was carried out at 50°C.
The produced glucose was then measured by the Somogyi-Nelson method. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit. The β-glucosidase of the present invention has a greater affinity for oligosaccharides such as cellohexaose and cellopentaose than for small molecular substrates such as salicin and cellobiose, and the enzyme has a higher affinity for oligosaccharides such as cellohexaose and cellopentaose than for small molecular substrates such as salicin and cellobiose. It also acts on molecular weight substrates. In particular, the existence of an enzyme with substrate specificity capable of degrading cellobiose and degrading Avicel to a considerable extent was clarified for the first time by the present invention, and the fact that all of the products are glucose indicates that the enzyme produced by this bacterium is β-glucosidase is a novel β-glucosidase that has not been previously known, and the present inventors named this enzyme glucocellulase because it has action characteristics similar to those of glucoamylase on starch. Thus, the cellulase produced by the Acremonium bacterium of the present invention is a novel cellulase complex enzyme agent containing a novel β-glucosidase. In order to produce cellulase using Acremonium bacteria of the present invention, cellulose or cellulose-containing materials such as cellulose, Avicel, cotton, bagasse, and wheat flour are usually used as a carbon source, and nitrates and ammonium salts are added as a nitrogen source. , cultured aerobically at 20 to 40°C for about 2 to 15 days using an organic or inorganic nitrogen source such as urea, peptone, or yeast extract, and a liquid or solid medium containing a small amount of metal salt. Ru. Cellulase is an enzyme produced outside the bacterial body, so in the case of liquid culture, it is the supernatant that is filtered after culturing, and in the case of solid culture, it is extracted with water or an appropriate inorganic salt solution after culturing. is used as an enzyme solution. The saccharification of cellulose according to the present invention usually involves pH3
~6, preferably PH4~5, temperature usually 30~60
Performed at °C. It is better to keep the substrate concentration as high as possible, and it is usually carried out at a concentration of 5 to 20%. Next, the details of the present invention will be explained with reference to Examples. Example 1 Cellulose 4%, KH ₂ PO ₄ 1.2%, Bactopeptone 1%, ZnSO ₄ 7H ₂ O1×10 ^-3 %, KNO ₃ 0.6%,
_MnSO4・_7H2O1 × ^10-3 %, urea 0.2%, _CuSO4・
_5H2O1 × ^10-3 %, KCl0.16%, _MgSO4・_7H2O0.12
%, pH 4.0, was sterilized by a conventional method, inoculated with Acremonium celluloliticus (FERM P-6867), and cultured with aeration at 30°C for 6 days. After culturing, twice the volume of cold acetone was added to the centrifuged supernatant, and the resulting precipitate was collected using a centrifuge and stored dry. Enzyme solution prepared by dissolving 1g of dry enzyme in 150ml of distilled water (Avicerase 10.0u/ml, CMCase 178u/ml)
ml, β-glucosidase 74.0 u/ml) was used in the following saccharification reaction. Add the amount of enzyme listed in Table 2 to 1 g of cellulose powder, make a total volume of 5 ml, and mix at pH 4.5 and 50°C.
Saccharified for 48 hours.

[Table] The results of quantifying reducing sugars in the saccharification solution as glucose are shown in Figure 3. As is clear from the figure, per gram of cellulose, 80 units of avicelase (1426 units of CMCase, 1426 units of β-glucosidase)
589 units), it was almost completely decomposed in about 2 days. As a result of quantifying the reducing sugar in the glycated product by the glucose oxidase method and paper chromatography method, it was found that it was mostly glucose. Example 2 80 units of avicelase (CMCase 1426 units, β-glucosidase 589 units) of the same enzyme used in Example 1 were added to 1 g of cellulose powder, and the mixture was saccharified at pH 4.5 and 50°C. A certain amount was economically taken and the reducing sugar (as glucose), soluble sugar (as glucose) and glucose amount were measured, and the results were as shown in Table 3.

[Table] As is clear from the table, the amount of reducing sugar in the glycated product,
Since the amount of soluble sugar and the amount of glucose are almost the same, it can be said that the product is mostly glucose. The results of analyzing the product by paper chromatography also agreed with this result, and only glucose was detected. Example 3 Pulverized bagasse (approximately 0.5 m/m) was immersed in 1% sodium hydroxide at 30°C for 24 hours, filtered with a glass filter, washed with distilled water, and dried at 105°C. For each 1 g of alkali-treated bagasse, add 3 to 3 to
28 units/g dry matter) was added to make the total volume 10 ml, and the mixture was saccharified at 50° C. for 48 hours at pH 4.5. The results of measuring the reducing sugar content, soluble sugar content, and glucose content in the saccharide were as shown in FIG. Here, reducing sugars were determined by the Somogyi-Nelson method, soluble sugars by the phenol-sulfuric acid method, and glucose by the glucose oxidase method. It was found that almost complete saccharification could be achieved by adding 30 units of Avicelase to each 1 g of alkali-treated bagasse and saccharifying it at 50°C for about 2 days.

[Brief explanation of the drawing]

Figure 1 shows that Acremonium cellulase
The amount of reducing sugar in the saccharide when Avicel is saccharified,
It shows the amount of soluble sugar and glucose. Second
The figure shows the cellulases Avicelase, CMCase, and β-glucosidase produced by Acremonium celluloliticus (FERM P-6867), a.
PH stability and d thermal stability are shown. FIG. 3 shows the relationship between the amount of cellulase added and cellulose saccharification. FIG. 4 shows the relationship between the amount of cellulase added and the saccharification of alkali-treated bagasse.

Claims (1)

[Claims] 1. A method for producing cellulose or cellulose-containing materials using cellulase produced by Acremonium bacteria, which is characterized by culturing Acremonium bacteria having cellulase-producing ability and allowing cellulase obtained from the culture to act on cellulose or cellulose-containing substances. Processing method.