JP2011083274A - Biomass saccharification technology using earthworm powder or extract - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve biomass saccharification simply and at a low cost. <P>SOLUTION: The composition for cellulose saccharification comprises powder of Lumbricus rubellus or its extract. The cellulase enzyme is purified from Lumbricus rubellus. The composition for cellulose saccharification comprises the enzyme. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a technique for realizing biomass saccharification easily and at low cost.

  Currently, oil-free energy has been actively proposed. In particular, the development of new energy technologies is urgent due to soaring global petroleum resources. As a new energy technology, bioethanol production from cellulosic biomass has attracted worldwide attention.

  Ethanol production from biomass is mainly composed of two steps: a saccharification step for producing monosaccharides from biomass and an ethanol production step for ethanol fermentation using the produced monosaccharides. If the saccharification efficiency of cellulose is low, the overall yield of ethanol fermentation is reduced. Therefore, efficient saccharification technology of biomass is desired.

  The filamentous fungus Tricoderma reesei (T. reesei) has high cellulase secretion ability, and the cellulase derived from T. reesei shows better activity than cellulases produced by other fungi (see Non-Patent Document 1). For this reason, enzymatic saccharification of cellulosic biomass using this enzyme has been actively studied.

JP 2008-72985 A (published April 3, 2008) JP 2009-28037 A (published February 12, 2009)

Ng, T. K. and Zeikus, J. G. 1981. Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414. Appl. Environ, Microbiol. 42: 231-240. Aira, M., Monroy, F., and Domienguez, J. 2007. Earthworms strongly modify microbial biomass and activity triggering experimental activities during vermicomposting independently of the application rates of pig slurry. Sci. Total Environ. 385: 252-61. Nakajima, N., Mihara, H., and Sumi, H. 1993. Characterization of potent fibrinolytic enzymes in earthworm, Lumbricus rubellus. Biosci. Biotechnol. Biochem. 57: 1726-1730.

  However, the cost of the enzyme used for the saccharification of cellulose in the production process is a problem. Cellulase produced by T. reesei has been actively researched from the viewpoint of excellent enzyme activity, but there are still many problems in terms of practical use.

  This invention is made | formed in view of said problem, The objective is to provide the technique which implement | achieves biomass saccharification simply and at low cost.

  Earthworms, also known as earth dragons, have long been loved as a traditional Chinese medicine that is effective in relieving fever and analgesia. Moreover, in order to reduce the amount of garbage, composting of garbage by mixing with worms has been performed for a long time (see Non-Patent Document 2, etc.). In this way, earthworms are deeply involved in our lives.

  As described above, it has been suggested that the earthworm is capable of hydrolyzing raw garbage, but no studies have been known that examined its saccharification ability.

  In recent years, rumbling kinase, a thrombolytic enzyme, has been found from the red earthworm Lumbricus rubellus (L. rubellus) (see Non-Patent Document 3), and its function has attracted attention. As a result of examining the functions of red earthworms in more detail, the present inventors found for the first time that cellulase activity is present in the crude extract of red earthworm powder and succeeded in purifying the cellulase activity fraction. And it confirmed that biomass could be directly saccharified if this cellulase-derived cellulase active fraction was used, and came to complete this invention as a new biomass saccharification technique.

  The composition for saccharification of cellulose according to the present invention is characterized by containing a dry powder of red earthworm or an extract thereof. The solvent used for extraction is preferably an aqueous solvent from the viewpoint of convenience in various operations, but may be an organic solvent.

  Patent Document 1 describes that an amylase important for composting by the earthworm is purified. However, amylase is an enzyme that hydrolyzes starch and cannot use cellulose as a substrate. In addition, the cellulose degradation according to the present invention has nothing to do with the starch degradation described in Patent Document 1 in terms of action and function, and can be said to be a completely different technique.

  As described above, research on thrombolytic enzymes has progressed for red earthworms, and those skilled in the art have never anticipated that a cellulase activity fraction can be obtained from such red earthworms. Patent Document 2 examines cellulase contained in the earthworm and confirms its properties, but does not examine saccharification ability. In addition, a technique that uses red earthworms conventionally is thrombus decomposition, and a technique that conventionally uses red earthworms is composting of garbage. A person skilled in the art cannot easily make use of knowledge obtained in a specific technical field in a technical field that is completely unrelated.

  The composition for cellulose saccharification according to the present invention is more preferably used for directly saccharifying cellulosic biomass.

  The kit for cellulose saccharification according to the present invention is characterized by comprising a red earthworm powder or an extract thereof.

  The cellulose saccharification method according to the present invention is characterized by including a step of incubating a cellulose-containing substance with a dry powder of red earthworm or an extract thereof. In the cellulose saccharification method according to the present invention, the cellulose-containing substance is preferably cellulosic biomass.

  Furthermore, the present inventors have completed the present invention by purifying an enzyme, which is considered to be involved in biomass saccharification, contained in the dry powder of red earthworm or its extract and identifying its properties.

  The cellulose saccharifying enzyme according to the present invention is derived from red earthworm, has an estimated molecular weight of 53.3 kDa, an optimum pH of 6.0, and an optimum temperature of 45 ° C. Moreover, it is preferable that the cellulose saccharifying enzyme which concerns on this invention is used with respect to the cellulose containing substance (preferably cellulosic biomass) containing lichenan, autospertoxylan, (beta) -glucan, or these arbitrary mixtures.

  The cellulose saccharifying enzyme according to the present invention is derived from red earthworm, has an estimated molecular weight of 57.5 kDa, an optimum pH of 5.5, and an optimum temperature of 40 ° C. The cellulose saccharifying enzyme according to the present invention is based on a cellulose-containing substance (preferably cellulosic biomass) containing laminaran, lichenan, β-glucan, β-1,3-glucan, or any mixture thereof. It is preferable to be used.

  Patent Document 2 examines cellulase contained in the earthworm and confirms its properties, but does not examine saccharification ability. Moreover, the properties of the enzyme fractionated by column chromatography are clearly different between the enzyme of the present invention and the enzyme described in Patent Document 2.

  The composition for cellulose saccharification according to the present invention is characterized by containing the enzyme. The kit for cellulose saccharification according to the present invention is characterized by comprising the above-mentioned enzyme.

  The cellulose saccharification method according to the present invention includes a step of incubating a cellulose-containing substance with the enzyme. In the method according to the present invention, the cellulose-containing substance is preferably cellulosic biomass.

  If this invention is used, biomass saccharification can be implement | achieved simply and at low cost. Furthermore, if this invention which can saccharify various biomass directly is used, biomass saccharification which usually requires a several process can be performed simply. Still further, with the present invention, biomass degradation can be achieved under relatively mild conditions. In particular, when red bran is used as biomass, the effect is superior to that of a T. reesei cellulase solution known as a useful enzyme.

It is a figure which shows the property of the cellulase contained in the crude enzyme solution extracted from Lumbricus rubellus. It is a figure which shows the result of Congo red dyeing | staining using the crude enzyme solution extracted from Lumbricus rubellus. It is a figure which shows the result of the azo dye-type plate assay using the crude enzyme solution extracted from Lumbricus rubellus. It is a figure which shows the result of having performed the saccharification test of various biomass using the crude enzyme solution extracted from Lumbricus rubellus. It is a figure which shows the result of the anion exchange column chromatography of the crude enzyme solution extracted from Lumbricus rubellus. It is a figure which shows the result of SDS-PAGE about the enzyme (CMC I and II) refine | purified from Lumbricus rubellus. It is a figure which shows the property about the enzyme (CMC I) refine | purified from Lumbricus rubellus. (A) shows the optimum temperature of each enzyme, (b) shows the optimum pH of each enzyme. It is a figure which shows the result of having measured the time-dependent change of the amount of produced | generated reducing sugars when the crude enzyme solution prepared from Lumbricus rubellus was made to act on red bran. It is a figure which shows the result of having measured the time-dependent change of the amount of produced | generated reducing sugars when the secretory enzyme solution prepared from T. reesei was made to act on red bran.

[1. Red earthworm powder or extract thereof
The present invention provides a composition for saccharification of cellulose and a kit. The composition for saccharification of cellulose according to the present invention is characterized by containing a dry powder of red earthworm or an extract thereof. The cellulose saccharification kit according to the present invention is characterized by comprising a red earthworm powder or an extract thereof.

  As used herein, the term “composition” is intended to include embodiments in which multiple ingredients are contained in a single composition. In addition, as used herein, the term “kit” may be an embodiment in which a plurality of components are contained in separate containers in a single substance (kit). That is, the composition according to the present invention is a form further including a reaction solution used for cellulose saccharification reaction and other components, and the kit according to the present invention includes a reaction solution used for cellulose saccharification reaction and other components. This is a form in which components and the like are provided separately.

  The red earthworm used in the present invention may be a cultured earthworm or a natural earthworm. It is not known at all that an extract from red earthworm, which is well known to contain a serine protease having thrombolytic activity, has excellent cellulase activity. The dried red earthworm powder may be obtained using various methods well known in the art.For example, in order to obtain the dry powder of red earthworm, the earthworm in the digestive tract is discharged by immersing the red earthworm in water, A procedure of lyophilization after crushing using a homogenizer, a mixer, a blender or the like may be employed.

  The solvent (extraction solvent) for obtaining the extract from the dry powder of red earthworm is preferably an aqueous solvent from the viewpoint of convenience in various operations, but may be an organic solvent. As used herein, an “aqueous solvent” has a buffering action at a weakly acidic to weakly alkaline pH (preferably pH 5.0 to 7.5, more preferably pH 5.0 to 7.0). Most preferred are the buffers shown (eg, phosphate buffer, Tris / HCl buffer, Tris / acetate buffer, carbonate buffer, borate buffer, acetate buffer, HEPES buffer, etc.), but with water Alternatively, it may be an aqueous solution containing a water-soluble substance (for example, organic acids, inorganic acids, etc.).

  An extraction process will not be specifically limited if the active ingredient contained in the crushed red earthworm (red earthworm dry powder) is extracted in an aqueous solvent. The obtained extract may be subjected to further purification steps, such as ammonium sulfate precipitation used for general enzyme purification, various chromatography (eg, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration). Etc.), and purification means such as electrophoresis can be appropriately used alone or in combination.

  As will be described later in Examples, a part of the active ingredient contained in the composition for cellulose saccharification according to the present invention has an estimated molecular weight of 41.7 kDa, an optimum temperature of 50 ° C., and an optimum pH of 7.0. It is an endoglucanase. Cellulose saccharification using dilute sulfuric acid is performed in the prior art, but by using the present invention that acts stably near neutrality, it is possible to produce monosaccharides without corroding the production plant with acid. . In general, since an enzyme is deactivated in a high temperature environment, it is necessary to perform an enzyme reaction after lowering the temperature of the reaction solution. If the present invention that retains the enzyme activity at 70 ° C. is used, the enzyme reaction can be started even at a high temperature, so that the total working time can be greatly shortened.

  Although the present invention has been described by taking the extract of red earthworm as an example, the present invention is not limited to the extract, and dry powder of red earthworm can also be suitably used. Those skilled in the art who read this specification will easily understand that cellulose may be incubated with dry powder of red earthworm, particularly when the extraction process of the active ingredient is performed in an environment similar to that of cellulose saccharification. . That is, a composition containing a dry powder of red earthworm or a kit provided with a dry powder of red earthworm is also suitably used for cellulose saccharification, and is therefore included within the scope of the present invention. Of course, it may be used together with a cellulase derived from T. reesei that exhibits good cellulase activity.

  The present invention also provides a method for saccharifying cellulose. The cellulose saccharification method according to the present invention is characterized by including a step of incubating a cellulose-containing substance with a dry powder of red earthworm or an extract thereof. The cellulose-containing substance is not particularly limited, but is preferably a cellulosic biomass mainly composed of cellulose. Cellulosic materials such as waste paper and cardboard, and by-products generated in large quantities during food production (for example, beet pulp) , Wheat bran, waste fungus bed, defatted soybean, defatted lees, etc.), agricultural and forestry waste (for example, rice straw, cedar, sawdust), weeds growing in non-agricultural land, and the like. In addition, when using the woody biomass which has a lignin and a cellulose as a main component, you may perform a delignification process previously as needed.

  Those skilled in the art who read this specification will easily understand that the above-described composition or kit may be used to carry out the cellulose saccharification method according to the present invention. As described above, since the active component of cellulose saccharification in the present invention is an endoglucanase having an estimated molecular weight of 41.7 kDa, an optimal temperature of 50 ° C., and an optimal pH of 7.0, the incubation step is performed in a temperature range of 30 to 50 ° C. However, the temperature and pH used for the incubation are not limited to the above ranges.

[2. Purified cellulase protein
The present invention provides cellulase proteins purified from red earthworms. In the first embodiment, the protein according to the present invention is a protein derived from red earthworms and having an estimated molecular weight of 53.3 kDa. Moreover, the optimal pH of the protein which concerns on this embodiment is 6.0, and the optimal temperature is 45 degreeC. If the protein which concerns on this embodiment is used, a cellulose containing substance can be saccharified. Preferred cellulose-containing substances as a protein substrate according to the present embodiment include cellulosic biomass, more preferably biomass containing lichenan, oat spelled xylan, β-glucan, or any mixture thereof. obtain.

  In the second embodiment, the protein according to the present invention is a protein having an estimated molecular weight of 57.5 kDa derived from red earthworm. Moreover, the optimal pH of the protein which concerns on this embodiment is 5.5, and optimal temperature is 40 degreeC. If the protein which concerns on this embodiment is used, a cellulose containing substance can be saccharified. Preferred cellulose-containing materials include cellulosic biomass, more preferably biomass containing laminaran, lichenan, β-glucan, β-1,3-glucan, or any mixture thereof. As shown in the Examples described later, the N-terminal amino acid sequence of the protein according to this embodiment is identified as “(A / S) ELWISTGDELXQLV” (SEQ ID NO: 1 or 2).

  The present invention further provides compositions and kits for cellulose saccharification. The composition for cellulose saccharification according to the present invention is characterized by containing the protein. The composition for cellulose saccharification according to the present invention may further contain a cellulase derived from T. reesei. The kit for cellulose saccharification according to the present invention is characterized by comprising the above protein, and may further comprise a cellulase derived from T. reesei. As described above, the composition according to the present invention is a form further including a reaction liquid used for cellulose saccharification reaction and other components, and the kit according to the present invention is a reaction liquid used for cellulose saccharification reaction. And other components are provided separately.

  The present invention also provides a method for saccharifying cellulose. The cellulose saccharification method according to the present invention includes a step of incubating a cellulose-containing substance with the protein. The cellulose-containing substance to which the present invention is to be applied may be those mentioned in the above-mentioned section “Dried red earthworm powder or extract thereof”, and preferably may be preferable as a substrate for the protein.

  Those skilled in the art who read this specification will easily understand that the above-described composition or kit may be used to carry out the cellulose saccharification method according to the present invention. That is, an embodiment in which cellulase protein purified from red earthworm is used together with cellulase derived from T. reesei is also within the scope of the present invention. In addition, it is preferable that the temperature range and pH range which should be employ | adopted in an incubation process are performed with reference to the optimal temperature and optimal pH of the said protein.

  The invention is illustrated in more detail by the following examples, but should not be limited thereto. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

[Example 1: Crude enzyme solution]
(Extraction of crude enzyme solution)
Lumbricus rubellus (L. rubellus) lyophilized powder used for extraction of the crude enzyme solution was provided by Waki Pharmaceutical Co., Ltd. 75 g of lyophilized L. rubellus powder was added to 750 mL of 50 mM sodium phosphate buffer (pH 8.0) containing 1 mM DTT, and the mixture was stirred at 4 ° C. for 20 hours. The mixture was centrifuged at 12,000 × g for 60 minutes at 4 ° C., and the supernatant was collected. 350 mL of the above buffer solution was further added to the precipitate fraction, and the mixture was stirred at 4 ° C. for 24 hours. The mixture was centrifuged under the above conditions, and the supernatant was collected. Each collected supernatant was mixed and centrifuged three more times under the above conditions, and the supernatant was used as a crude enzyme solution.

[Ammonium sulfate fraction]
Ammonium sulfate having a final concentration of 25% was added to the obtained crude enzyme solution. The solution was centrifuged at 12,000 × g for 60 minutes at 4 ° C., and the supernatant was recovered. A final concentration of 60% ammonium sulfate was added to the resulting solution. The solution was centrifuged under the same conditions, and the precipitate was collected. The collected precipitate was dissolved in 100 mL of 25 mM sodium phosphate buffer (pH 8.0) containing 1 mM DTT.

[Column chromatography]
All column chromatography was performed at 4 ° C. using AKTAprime plus (GE Healthcare Bioscience). The ammonium sulfate fraction of the crude enzyme solution obtained above was dialyzed using 25 mM sodium phosphate buffer (pH 8.0) (A buffer) containing 1 mM DTT. This solution was applied to a weak anion exchange column DEAE-TOYOPEARL 650M (Tosoh Corp.) pre-equilibrated with A buffer. After washing the column with the same buffer, the adsorbed protein was eluted with a linear concentration gradient method using A buffer containing 0.5 M NaCl, and fractions having cellulase activity confirmed by the method described later were collected. did.

[Gel filtration column chromatography]
The active fraction obtained above was concentrated with a filter and applied to a gel filtration column TOYOPEARL HW-55S pre-equilibrated with 25 mM sodium phosphate buffer (pH 8.0) containing 0.1 M NaCl and 1 mM DTT. Adsorbed protein was eluted with the same buffer.

(Enzyme activity measurement)
Cellulase activity was measured using 1.25% carboxymethylcellulose (CMC) as a substrate. The fraction to be measured was appropriately diluted, and 100 μL was added to 400 μL of 25 mM sodium phosphate buffer (pH 8.0) containing 1.25% CMC and incubated at 40 ° C. for 15 minutes. The amount of reducing sugar released was measured by the Somogyi-Nelson method. After the enzyme reaction, 500 μL of Somogyi copper reagent was added to the reaction solution, boiled for 15 minutes, and left in a water bath for 5 minutes. After adding 500 μL of Nelson color reagent to this, 1 mL of distilled water was added and centrifuged at 1,000 × g for 15 minutes, and the absorbance of the collected supernatant was measured at a wavelength of 520 nm. The amount of enzyme that liberates the amount of reducing sugar corresponding to 1 μmol of glucose per minute was defined as 1 U.

[Congo red plate assay]
Prepare plates so that the final concentrations are 0.1% substrate (CMC, lichenan, laminaran, xyloglucan, oat spelled xylan, PSC), 0.1M sodium phosphate buffer (pH 8.0), and 2% Agar, respectively. did. Each enzyme solution was dropped onto the plate and reacted at 40 ° C. for 2 hours. The plate was washed with 1M Tris-HCl (pH 7.5), added with a congo red staining solution, and allowed to stand for 30 minutes. The plate was then washed with 1M NaCl, 5% acetic acid solution was added, and the halo size was measured.

[Azo dye plate assay]
Final concentrations of 0.1% substrate (amylose, burly β-glucan, protein, mannan, 1,3β-glucan, galactan, arabinan), 0.1M sodium phosphate buffer (pH 8.0), 2% Agar A plate was prepared as follows. Each enzyme solution was dropped onto the plate and reacted at 40 ° C. for 24 hours (color reaction).

[Biomass saccharification test]
Biomass sample obtained by adding 0.1 g of each biomass (red bran, cedar, waste fungus bed) to a 50 cc wide mouth bottle together with 11.7 mL of 50 mM phosphate buffer (pH 8.0) containing 1 mM sodium azide As previously adjusted. Filter-sterilized 3.3 mL crude enzyme solution was added to the biomass sample and allowed to react at 40 ° C. for 24 hours. After the reaction, an appropriate amount of the reaction solution was collected, and the amount of produced reducing sugar contained in the supernatant after centrifugation was quantified by the Somogyi-Nelson method.

[Protein content measurement]
The amount of protein was measured by the Bradford method. Specifically, using a Bio-Rad protein assay reagent manufactured by Bio-Rad, quantification was performed at a wavelength of 595 nm using a Model 680XR microplate reader (Bio-Rad). Bovine serum albumin (BSA) was used to create a calibration curve.

〔result〕
[1. Examination of various properties using crude enzyme)
[1.1. (Investigation of optimum temperature)
The optimum temperature of cellulase contained in the crude enzyme solution was examined using CMC as a substrate. As a result, it was shown that the optimum temperature of the enzyme was around 50 ° C. (FIG. 1 (a)). The activity was measured when the crude enzyme solution was reacted at various temperatures. It was shown that the enzyme stability gradually decreased as the temperature increased, but the activity was maintained at about 20% even at around 70 ° C. (FIG. 1B). Activity was measured when the crude enzyme solution adjusted to various pH was reacted at 40 ° C. As a result, it was shown that the optimum pH was around pH 7.0 (FIG. 1 (c)). The crude enzyme solution adjusted to various pHs was compared with the activity when reacted at pH 7.0 and 40 ° C. As a result, the stability in the acidic region was not good, but it was shown that it was relatively stable in the alkaline region (FIG. 1 (d)).

[2. Result of plate assay by Congo red staining method]
The results of Congo red staining are shown in FIG. Enzyme activity for each substrate was examined by the presence or absence of halo. As a result, it was possible to confirm halo in a medium containing CMC, lichenan, laminaran, xyloglucan, and autospertoxylan. This suggested the presence of β-1,4 endoglucanase and β-1,4 endoxylanase in the crude enzyme solution.

[3. Azo dye plate assay results]
The results of the azo dye-based plate assay are shown in FIG. When the enzyme decomposes the substrate in the medium, the azo dye is solubilized and becomes blue, and the presence or absence of enzyme activity for each substrate was examined by confirming this coloration. Color development could be confirmed with amylose, Burley β-glucan, protein, mannan, and 1,3-β-glucan. This suggested the presence of amylase, β-1,4 endoglucanase, protease, and β-1,4 endomannanase in the crude enzyme solution.

[4. (Biomass saccharification test)
The saccharification test of various biomass (red bran, cedar, waste fungus bed) was performed using the crude enzyme solution. The change over time in the amount of reducing sugar produced was measured using the Somogyi-Nelson method (FIG. 4). a is the amount of reduced sugar produced by red bran, b is the amount of produced reduced sugar of cedar, and c is the amount of produced reduced sugar of the waste fungus bed. As a result, it has been clarified that the enzyme acts efficiently on red bran.

[5. Enzyme purification
Using various chromatographies, enzyme purification was attempted using CMC degradation activity as an index. As a result, the results of gel filtration chromatography and SDS-PAGE suggested that the target enzyme was a monomer with an estimated molecular weight of 41.7 kDa.

  As described above, it was shown that L. rubellus has an endoglucanase with an estimated molecular weight of about 45 kDa. In addition, the presence of β-1,4 endoglucanase, β-1,4 endoxylanase, amylase, protease, β-1,4 endomannanase and the like was also suggested. Furthermore, the crude enzyme solution directly saccharified various biomass. This makes it possible to propose a novel biomass saccharification technology using a crude enzyme solution, and is expected to be applied to industry.

[Example 2: Purification of enzyme]
(Extraction of crude enzyme solution)
Lumbricus rubellus (L. rubellus) lyophilized powder used for extraction of the crude enzyme solution was provided by Waki Pharmaceutical Co., Ltd. 75 g of lyophilized L. rubellus powder was added to 750 mL of 50 mM sodium phosphate buffer (pH 6.0) containing 1 mM DTT, and the mixture was stirred at 4 ° C. for 24 hours. The mixture was centrifuged at 12,000 × g for 30 minutes at 4 ° C., and the supernatant was collected. In order to remove nucleic acid, streptomycin sulfate having a final concentration of 2% was added to the supernatant, stirred at 4 ° C. for 30 minutes, and then centrifuged at 12,000 × g for 30 minutes. It was set as the solution.

[Ammonium sulfate fraction]
Ammonium sulfate was added to the resulting crude enzyme solution to a final concentration of 20%. The solution was centrifuged at 19,000 × g for 30 minutes at 4 ° C., and then ammonium sulfate was added to the collected supernatant to a final concentration of 60%. The solution was centrifuged again under the same conditions, and the precipitate was recovered. The collected precipitate was dissolved in 100 mL of 25 mM sodium phosphate buffer (pH 6.0) containing 1 mM DTT.

[Anion column chromatography]
According to the procedure described above, column chromatography using a weak anion exchange column DEAE-TOYOPEARL 650M (Tosoh Corporation) was performed, and a fraction having cellulase activity was fractionated.

[Multimodal weak cation exchange column chromatography]
The obtained fraction was concentrated with a filter, dialyzed against 50 mM acetate buffer (pH 4.5) containing 1 mM DTT, and then applied to a Capto MMC column pre-equilibrated with the same buffer. After washing the column with the same buffer, the adsorbed protein was eluted with a 50 mM sodium phosphate buffer (pH 7.0) containing 1 mM DTT and 1 M NaCl by a linear concentration gradient method.

[Hydroxyapatite column chromatography]
The obtained fraction was concentrated with a filter, dialyzed against 1 mM sodium phosphate buffer (pH 6.0) containing 1 mM DTT, and then applied to Bio-Scale Mini CHT Type I previously equilibrated with the same buffer. After washing the column with the same buffer, the adsorbed protein was eluted with a 100 mM sodium phosphate buffer (pH 6.0) containing 1 mM DTT by a linear concentration gradient method. This operation was performed only for CMC I. For CMC II, this step was omitted, and purification using a gel filtration column was subsequently performed.

[Gel filtration column chromatography]
The obtained fraction was concentrated with a filter and subjected to a gel filtration column TOYOPEARL HW-55S pre-equilibrated with 25 mM sodium phosphate buffer (pH 6.0) containing 0.1 M NaCl and 1 mM DTT. Adsorbed protein was eluted with the same buffer.

(Enzyme activity measurement)
The activity of CMCase was measured according to the procedure for measuring cellulase activity using CMC as a substrate. Further, β-glucosidase (BGL) activity or cellobiohydrolase (CBH) activity was measured according to the following procedure. To 1 μL of enzyme solution, 4 μL of 25 mM sodium phosphate buffer (pH 7.0) was added, and 45 μL of substrate solution (BGL activity: 4-methylbelliferyl β-D-glucopyroxide (4MU-G) was added to 25 mM sodium phosphate buffer (pH 7). 0.0) (final concentration 0.5 mM), CBH activity: 4-Methylumbelliferyl β-D-cellbioside (4MU-G2) dissolved in 25 mM sodium phosphate buffer (pH 7.0) (Final concentration 0.5 mM)) was further added. After this mixed solution was incubated at 40 ° C. for 30 minutes, a solution to which 150 mM Glycine-NaOH buffer (pH 10) was added was excited at 360 nm and measured at 460 nm. The amount of enzyme that produces 1 μmol of 4-methylbelliferone per minute was defined as 1 U. Further, 4-methylbelliferone was used for preparing a calibration curve.

[Congo red plate assay]
Plates with final concentrations of 0.1% substrate (CMC, laminaran, lichenan, oat spelled xylan, birchwood xylan, xyloglucan), 0.1M sodium phosphate buffer (pH 7.0), 2% Agar, respectively Was made. Each enzyme solution was dropped onto the plate and reacted at 40 ° C. for 2 hours. The plate was washed with 1M Tris-HCl (pH 7.5), added with a congo red staining solution, and allowed to stand for 30 minutes. The plate was then washed with 1M NaCl, 5% acetic acid solution was added, and the halo size was measured.

[Azo dye plate assay]
Final concentration is 0.1% for each substrate (β-glucan, amylose, mannan, 1,3β-glucan, galactan, arabinan, xylan, protein), 0.1M sodium phosphate buffer (pH 7.0), 2% Agar A plate was prepared so that Each enzyme solution was dropped onto the plate and reacted at 40 ° C. for 24 hours (color reaction).

[Synthetic substrate plate assay]
Plates were prepared so that the final concentration was 4 mM MU-G or 4 MU-G2, 0.1 M sodium phosphate buffer (pH 7.0) and 2% Agar, respectively. Each enzyme solution was dropped on this plate, reacted at 40 ° C. for 2 hours, irradiated with 365 nm ultraviolet rays, and the size of halo was measured.

[N-terminal amino acid sequence analysis]
The purified CMC II was subjected to SDS-PAGE and then transferred to a PVDF membrane, and the N-terminal amino acid sequence of the target protein band was analyzed using an amino acid sequencer.

[Protein content measurement]
The amount of protein was measured by the Bradford method. Specifically, using a Bio-Rad protein assay reagent (Bio-Rad) and a Model 680XR microplate reader (Bio-Rad), quantification was performed at a wavelength of 595 nm. Bovine serum albumin (BSA) was used to create a calibration curve.

〔result〕
[1. Anion column chromatography)
The elution results of anion column chromatography are shown in FIG. Two fractions, CMC I and CMC II, were obtained as CMCase active fractions. Two active fractions of BGL were also obtained (FIG. 5). Since CMCase has endoglucanase (EG) activity, this result suggests that the crude enzyme contains at least two types of EG and one type of BGL.

[2. Properties of purified CMC I and II]
CMC I and II were purified by various chromatography, and their properties were examined. The purification results of CMC I and II are shown in Table 1.

  CMC I showed a molecular weight of 48.7 kDa from the results of gel filtration chromatography and a molecular weight of 53.3 kDa (FIG. 6) from the results of SDS-PAGE, suggesting that it is a monomer. Moreover, it was shown that the optimum temperature of the enzyme is around 45 ° C. (FIG. 7A), and the optimum pH is around 6.0 (FIG. 7B).

  In addition, CMC II was found to be a monomer because it showed a molecular weight of 58.9 kDa from the results of gel filtration chromatography and a molecular weight of 57.5 kDa (FIG. 6) from the results of SDS-PAGE. In addition, it was shown that the optimum temperature of the enzyme is around 40 ° C. (FIG. 7A), and the optimum pH is around 5.5 (FIG. 7B).

[3. (Plate assay result)
As a result of the plate assay, CMC I showed activity against 4MU-G, CMC, lichenan, autospertoxylan, and β-glucan. CMC II showed activity against CMC, laminaran, lichenan, β-glucan, β-1,3-glucan.

[4. N-terminal amino acid sequence analysis results]
As a result of N-terminal amino acid sequence analysis of CMC II, it was found to be (A / S) ELWISTGDELXQLV (SEQ ID NO: 1 or 2). This sequence was searched with BLAST, but no sequence that appeared to be related was found to be hit.

  The molecular weight of the purified cellulase protein present in L. rubellus was 50-60 kDa. This was slightly higher than the estimated molecular weight in the crude purification stage, which can be said to be within experimental error. The obtained purified protein had an optimum pH of weakly acidic to near neutral and an optimum temperature of 30 to 50 ° C., and various biomass were directly saccharified under mild conditions. As a result, a novel biomass saccharification technology using purified protein can be proposed, and the application to the industry is greatly expected.

[Example 3: Biomass saccharification test]
What added 1.0 g of biomass (red bran) with 50 mM phosphate buffer (pH 7.0) containing 3 mM sodium azide to a 50 cc wide-mouth bottle was prepared in advance as a biomass sample. The crude enzyme solution having 30 U equivalent of CMCase activity sterilized by filter was added to the biomass sample and reacted at 30-50 ° C. with shaking for 48 hours. After the reaction, an appropriate amount of the reaction solution was recovered, boiled for 10 minutes, the enzyme was inactivated, and the amount of reducing sugar contained in the supernatant after centrifugation was quantified by the Somogyi-Nelson method.

  FIG. 8 shows the results of measuring the change over time in the amount of reducing sugar produced using the Somogyi-Nelson method. As shown in the figure, the crude enzyme solution was found to produce reducing sugar most efficiently at 50 ° C. The amount of reducing sugar produced by the crude enzyme solution exceeded the amount of reducing sugar produced at 50 ° C. when a similar test was performed using a secretory enzyme derived from T. reesei (FIG. 9).

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  When the crude enzyme solution directly saccharified various biomass and red bran was used as the biomass, it showed results exceeding the effect of the T. reesei cellulase solution known as a useful enzyme, and the purified enzyme Since it operates under relatively mild conditions, simpler biomass saccharification can be realized, and industrial application is expected by the new biomass saccharification technology.

Claims (10)

  A composition for cellulose saccharification, comprising a dry powder of red earthworm (Lumbricus rubellus) or an extract thereof.   A kit for cellulose saccharification, comprising a dry powder of red earthworm (Lumbricus rubellus) or an extract thereof.   A cellulose saccharification method comprising a step of incubating a cellulose-containing substance with dry powder of red earthworm (Lumbricus rubellus) or an extract thereof.   The method according to claim 3, wherein the cellulose-containing substance is cellulosic biomass.   A cellulose saccharifying enzyme derived from red earthworm (Lumbricus rubellus) having an estimated molecular weight of 53.3 kDa, an optimum pH of 6.0, and an optimum temperature of 45 ° C.   A cellulose saccharifying enzyme derived from red earthworm (Lumbricus rubellus) having an estimated molecular weight of 57.5 kDa, an optimum pH of 5.5, and an optimum temperature of 40 ° C.   A composition for cellulose saccharification, comprising the enzyme according to claim 5 or 6.   A kit for cellulose saccharification, comprising the enzyme according to claim 5 or 6.   A method for saccharifying cellulose, comprising a step of incubating a cellulose-containing substance with the enzyme of claim 5 or 6.   The method according to claim 9, wherein the cellulose-containing substance is cellulosic biomass.