JP2011244756A - Cellulose decomposition promoter and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new substance useful for activity promotion of cellulase, its application and the like.SOLUTION: It is revealed that the genus Rhizopus and Mucor microorganisms produce a polypeptide showing a cellulolytic promotion activity. A cellulolytic promotion agent containing the polypeptide is provided.

Description

  The present invention relates to a cellulose degradation accelerator, a method for producing the same, and uses of the cellulose degradation accelerator.

  Cellulose is an organic substance having the most abundance on the earth, and has a structure in which glucose molecules are polymerized in a straight chain with β-1,4 bonds. In recent years, cellulose has attracted attention as a biomass. In particular, development of ethanol production technology using cellulose as a raw material has been energetically advanced.

  Cellulase is used in one of the methods for degrading cellulose. However, when woody materials are used, cellulase alone has low degradation efficiency, so it is necessary to search for cellulase with high degradation activity and to improve cellulase activity. Has been done. As a result, cellulose degradation promoting substances typified by swollenin have been found so far (see, for example, Patent Documents 1 to 3).

Special table 2009-509546 gazette JP 2009-207368 A Japanese Patent No. 42610044

  Amid growing interest in environmental issues such as global warming and the stable supply of energy, there is a great need for technologies that make effective use of cellulose materials. In order to make effective use of cellulose materials, it is necessary to decompose cellulose efficiently. However, since various research groups have been searching for cellulase in the past, it is more than cellulase reported so far. It is unlikely that cellulases with excellent properties will be found. Moreover, at the present time when many cellulases are available, provision of a substance that exhibits an effect of enhancing the action of cellulase (synergistic effect) is not a new cellulase in the sense of utilizing these cellulases. This is what is desired. Then, this invention makes it a subject to provide a novel substance useful for acceleration | stimulation of decomposition | disassembly of a cellulose, its use, etc.

  In order to find a substance capable of enhancing the activity of cellulase, the present inventors conducted screening on a huge number of microorganisms. At that time, it is noted that many existing enhancing substances (see, for example, Patent Documents 1 to 3) themselves have cellulolytic activity, and in addition to the index of cellulose degradation promoting activity, the cellulose degradation possessed by itself Using the activity as an index, substances produced by each microorganism were evaluated. In addition, because biomass degradation in nature is carried out by so-called “solid culture” using microorganisms, enzymes and factors necessary for biomass degradation are more likely to be produced in solid culture than in liquid culture. An evaluation system based on solid culture was adopted.

As a result of screening, it has been found that microorganisms of the genus Rhizopus and the genus Mucor produce substances having desired characteristics, and microbial strains that produce particularly active substances have been identified. As a result of further investigations, it has been clarified that the substance expected to be highly useful exhibits a synergistic effect on various existing cellulases and is excellent in versatility. The present invention described below is mainly based on these results.
[1] A cellulose degradation accelerator comprising a polypeptide produced by a microorganism belonging to the genus Rhizopus or Mucor and exhibiting a cellulose degradation promoting activity.
[2] The microorganism of the genus Rhizopus is a microorganism of a species selected from the group consisting of Rhizopus oryzae and Rhizopus microsporus var. Chinensis, and the microorganism of the genus Mucor is Mucor plumeus (Mucor plumbeus), mucor sarcineroides varieties sarcineroides (Mucor circinelloides f. circinelloides), mucor racemosus, mucor subtilissimus and mucor ramannianus The cellulose degradation accelerator according to [1], which is a certain type of microorganism.
[3] The Rhizopus genus microorganism is a Rhizopus oryzae identified by the receipt number: NITE ABP-945, and the Mucor genus microorganism is the Mucor sarcineroides varieties Salcineroides NBRC4570 or the receipt number: NITE ABP-946. The cellulose degradation accelerator according to [1], which is moss.
[4] The cellulose degradation accelerator according to [1], wherein the polypeptide has the following properties:
Molecular weight: approx. 35 kDa (according to SDS-PAGE).
[5] The cellulose degradation accelerator according to any one of [1] to [4], wherein the polypeptide has low cellulolytic activity.
[6] The cellulose degradation accelerator according to any one of [1] to [4], wherein the polypeptide has substantially no cellulolytic activity.
[7] The cellulose degradation accelerator according to any one of [1] to [4], wherein the synergistic effect ratio calculated by the following formula is 1.2 to 2.5 for the polypeptide:
Synergistic effect ratio = (Cellulolytic activity in the presence of cellulase and the polypeptide) / {(Cellulolytic activity of the polypeptide alone) + (Cellulolytic activity of cellulase alone)}
[8] The cellulose degradation accelerator according to any one of [1] to [7], wherein the polypeptide has low β-glucosidase activity.
[9] The cellulose degradation accelerator according to any one of [1] to [7], wherein the polypeptide has substantially no β-glucosidase activity.
[10] The cellulose degradation promoter according to any one of [1] to [9], which comprises the culture product or culture extract of the microorganism.
[11] The cellulose degradation accelerator according to any one of [1] to [10], further containing cellulase.
[12] A method for producing a cellulose degradation accelerator comprising the following steps (1) and (2):
(1) culturing a microorganism belonging to the genus Rhizopus or Mucor, which produces a polypeptide exhibiting a cellulose degradation promoting activity, under conditions under which the polypeptide is produced;
(2) recovering the polypeptide from the culture product.
[13] The production method according to [12], wherein the medium used in step (1) is a cellulose-containing solid medium.
[14] The production method according to [13], wherein step (2) includes removing a solid component after adding a solvent to the medium after step (1).
[15] The production method according to [14], wherein the polypeptide is purified after removing a solid component.
[16] The microorganism of the genus Rhizopus is a microorganism of a species selected from the group consisting of Rhizopus oryzae and Rhizopus microsporus var. Chinensis, and the microorganism of the genus Mucor is Mucor Purmbeus (Mucor plumbeus), mucor sarcineroides varieties sarcineroides (Mucor circinelloides f. circinelloides), mucor racemosus, mucor subtilissimus and mucor ramannianus The production method according to any one of [12] to [15], which is a certain type of microorganism.
[17] The Rhizopus genus microorganism is a Rhizopus oryzae identified by the receipt number: NITE ABP-945, and the Mucor genus microorganism is the Mucor sarcineroides varieties Salcineroides NBRC4570 or the receipt number: NITE ABP-946. The production method according to any one of [12] to [15], which is Mothus.
[18] A method for decomposing cellulose, comprising treating a cellulose material with cellulase in the presence of the cellulose degradation accelerator according to any one of [1] to [11].
[19] The cellulose decomposing method according to [18], wherein the cellulase is a cellulase derived from Trichoderma, Aspergillus, or Acremonium.
[20] A method for producing ethanol, comprising a step of converting the sugar produced by the cellulose decomposition method according to [18] or [19] into ethanol.
[21] The method for producing ethanol according to [20], wherein the treatment with cellulase and the ethanol conversion are performed in parallel.
[22] A microorganism that produces a polypeptide exhibiting cellulose degradation-promoting activity, which is Rhizopus oryzae identified by NITE ABP-945 or Mucor racemosas identified by NITE ABP-946.

It is the figure which showed the average value of the reducing sugar density | concentration in cellulase T "Amano" 4 single, test sample single, and both coexistence. A gel stained with CBB by SDS-PAGE of the fraction derived from the culture extract of Rhizopus oryzae APC3504 strain that promotes cellulolytic activity by cellulase T “Amano” 4 is shown. Lane 1: molecular weight marker, lane 2: fraction showing a synergistic effect.

(the term)
As used herein, the term “isolated” is used interchangeably with “purified”. “Isolated” when used in reference to the polypeptide of the present invention is substantially free of components other than the polypeptide in the natural material when the polypeptide of the present invention is derived from the natural material. It refers to a state (particularly substantially free of contaminating proteins). Specifically, for example, in the isolated polypeptide of the present invention, the content of contaminating protein is less than about 20%, preferably less than about 10%, more preferably less than about 5%, even more in terms of weight. Preferably it is less than about 1%.

  “Cellulose degradation promoter” refers to a substance or composition that promotes the action of cellulase, that is, cellulose degradation (activity) when used in combination with cellulase. In the present invention, a polypeptide derived from a microorganism constitutes a cellulose degradation accelerator. Unless otherwise specified, when simply expressed as “polypeptide” in the present specification, it means a polypeptide exhibiting cellulose degradation promoting activity. In the present specification, the action or effect of the cellulose degradation accelerator is referred to as “cellulose degradation promoting activity” or “synergistic effect”.

1. Cellulose degradation accelerator The first aspect of the present invention relates to a cellulose degradation accelerator. The cellulose degradation promoter of the present invention contains a polypeptide produced by a microorganism belonging to the genus Rhizopus or Mucor. The polypeptide exhibits cellulose degradation promoting activity. As shown in the Examples below, as a result of large-scale screening by the present inventors, Rhizopus oryzae and Rhizopus microsporus var. Chinensis, which are microorganisms of the genus Rhizopus, are cellulose. It was revealed that a polypeptide excellent in degradation promoting activity was produced. Based on this finding, in a preferred embodiment of the present invention, a microorganism belonging to the genus Rhizopus is any of these. On the other hand, the microorganisms of the genus Mucor, Mucor plumbeus, mucor sarcineroides varieties Sarcineeroides (Mucor circinelloides f. Circinelloides), mucor racemosus, mucor subtilis muss ( It has been revealed that Mucor subtilissimus and Mucor ramannianus produce polypeptides having excellent cellulose degradation promoting activity. Based on this finding, in a preferred embodiment of the present invention, a microorganism belonging to the genus Mucor is any of these. Rhizopus oryzae APC3504 and Mucor racemosus APC4090 are deposited with a predetermined depository as follows and are easily available. NBBC4570 is a NRBC culture collection (Mucor circinelloides f. Circinelloides) NBRC4570, Biotechnology Division, Biotechnology Headquarters, National Institute of Technology and Evaluation, Kazusa Kamazu, Kisarazu, Chiba, Japan 292-0818 2-5-8) and can be obtained from the National Institute of Product Evaluation Technology Biotechnology Headquarters, Biogenetic Resources Division (NBRC) through a designated procedure.
(Rhizopus oryzae APC3504)
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Deposit date (Receipt date): May 21, 2010 Receipt number: NITE ABP-945

(Mucole Racemos APC4090)
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Deposit date (Receipt date): May 21, 2010 Receipt number: NITE ABP-946

As described below, the present inventors have clarified the molecular weight of the polypeptide produced by Rhizopus oryzae APC3504 (details are described in the Examples section below).
Molecular weight: Approximately 350,000 Da (by SDS-PAGE)

The polypeptide constituting the cellulose degradation accelerator of the present invention has a feature of enhancing the action of cellulase. That is, the polypeptide according to the present invention exhibits a synergistic effect on cellulase. This feature is referred to herein as “cellulose degradation promoting activity”. The degree of cellulose degradation promoting activity can be expressed by the synergistic effect ratio calculated by the following formula. The cellulose degrading activity can be determined by the method shown in the examples described later.
Synergistic effect ratio = (Cellulolytic activity in the presence of cellulase and test polypeptide) / {(Cellulolytic activity of test polypeptide alone) + (Cellulolytic activity of cellulase alone)}

  The synergistic effect ratio of the polypeptide according to the present invention is preferably 1.2 to 3.0, more preferably 1.2 to 2.5.

  One of the characteristics of the polypeptide constituting the cellulose degradation accelerator of the present invention is that the cellulolytic activity is low. That is, the polypeptide according to the present invention does not have high cellulolytic activity by itself, but exhibits a synergistic effect when used in combination with cellulase and promotes cellulose degradation. In a preferred embodiment, the polypeptide according to the present invention has substantially no cellulolytic activity. The cellulose degrading activity can be evaluated by the method shown in the below-mentioned examples. “Low cellulose degrading activity” means that the activity ratio calculated by the method is 0.2 or less. “Substantially has no degradation activity” means that the activity ratio is 0 to 0.1 (a negative value is also assumed due to measurement error).

  The polypeptide constituting the cellulose degradation accelerator of the present invention preferably has a low β-glucosidase activity. In a preferred embodiment, the polypeptide according to the present invention has substantially no β-glucosidase activity. In addition, β-glucosidase activity can be evaluated by the method shown in the examples described later. “Low β-glucosidase activity” means that the β-glucosidase activity calculated by the method is 1 U / mL or less. The phrase “having substantially no β-glucosidase activity” means that the β-glucosidase activity is 0 to 0.2 U / mL (it is also assumed that a negative value is shown due to a measurement error).

  As a polypeptide constituting the cellulose degradation accelerator of the present invention, the polypeptide produced by Rhizopus oryzae APC3504 is particularly useful in that the synergistic effect ratio is high and the cellulose degradation activity itself is low. Similarly, the polypeptide produced by Mucor racemosus APC4090 is particularly useful in that β-glucosidase activity is low.

  The cellulose degradation accelerator of the present invention may contain excipients, buffers, suspension agents, stabilizers, preservatives, preservatives, physiological saline and the like in addition to the active ingredient (polypeptide). As the excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  In one aspect, the cellulose degradation accelerator of the present invention contains cellulase. That is, it is provided as a composition containing cellulase in addition to a polypeptide exhibiting cellulose degradation promoting activity. Cellulase can be arbitrarily selected. Here, “cellulase” is an enzyme that hydrolyzes the glucoside bond of β-1,4 glucan. The cellulase used is not particularly limited. For example, a cellulase derived from a microorganism can be used. Cellulases derived from microorganisms belonging to the genus Trichoderma as cellulases derived from microorganisms (for example, manufactured by Meiji Seika Co., Ltd., Novozymes, manufactured by Genencor, manufactured by Amano Enzyme), cellulases derived from Aspergillus microorganisms (for example, manufactured by HIBI, manufactured by Amano Enzyme), Cellulases derived from Acremonium microorganisms (for example, manufactured by Meiji Seika Co., Ltd.) are known. Not only natural (wild-type) cellulase but also recombinant cellulase can be used. Two or more cellulases may be used in combination. A commercially available cellulase or cellulase preparation may be used. Examples of commercially available cellulases or cellulase preparations are: Acceleratorase (Genencore), NS22074 (Novozymes), Mecerase, Acremozyme (Meiji Seika), Cellulosin AC (Hetchby), Cellulase A, Cellulase T (Amano) Enzyme).

  A substance exhibiting a cellulose degradation promoting action (a cellulose degradation accelerator other than the polypeptide according to the present invention) may be further contained. Examples of the substance include swollenin and expansin.

  In one aspect, the cellulose degradation promoter of the present invention is provided in the form of a culture product or culture extract of a microorganism that produces the polypeptide of the present invention. That is, in this embodiment, after subjecting the culture product obtained by culturing the microorganism capable of producing the polypeptide according to the present invention as it is or to the extraction step (for details, refer to the column of the method for producing a cellulose degradation accelerator). And used as a cellulose degradation accelerator of the present invention. What passed through the refinement | purification process (refer the column of the manufacturing method of a cellulose degradation accelerator for details) is good also as a cellulose degradation accelerator of this invention.

2. Method for Producing Cellulose Degradation Accelerator A further aspect of the present invention provides a method for producing a cellulose degradation accelerator. In the production method of the present invention, a step of culturing a microorganism belonging to the genus Rhizopus or Mucor that produces a polypeptide exhibiting cellulose degradation-promoting activity under conditions under which the polypeptide is produced (step (1)) And a step (step (2)) of recovering the polypeptide from the culture product.

  As the microorganism of the genus Rhizopus in step (1), a microorganism of a species selected from the group consisting of Rhizopus oryzae and Rhizopus microsporus var. Chinensis may be used. Preferably Rhizopus oryzae APC3504 is used. On the other hand, Mucor plumbeus (mucor plumbeus), mucor sarcineroides varieties salcineeroides (Mucor circinelloides f. Circinelloides), mucor racemosus, mucor subtilissimus (mucor) and mucor subtilissimus (mucor) A microorganism of a species selected from the group consisting of Mucor ramannianus may be used. Preferably, Mucor racemosus APC4090 is used. The culture method and culture conditions are not particularly limited as long as the target polypeptide is produced. That is, on the condition that a polypeptide exhibiting cellulose degradation promoting activity is produced, a method and culture conditions suitable for culturing the microorganism to be used can be appropriately set. Hereinafter, examples of the culture conditions include a medium, a culture temperature, and a culture time.

  As the medium, a medium in which the microorganism to be used can grow is employed. For example, carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein Nitrogen sources such as hydrolysates, bran and meat extracts, and further added with inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts and zinc salts can be used. In order to promote the growth of the microorganisms to be used, vitamins, amino acids and the like may be added to the medium. Preferably, a medium containing cellulose is used to induce production of the polypeptide of interest. Further, in order to increase the production efficiency of the target polypeptide by making the environment close to the environment of biomass degradation in nature, it is preferable to use a solid medium. The pH of the medium is adjusted to, for example, about 3 to 8, preferably about 5 to 7, and the culture temperature is usually about 10 to 50 ° C., preferably about 25 to 35 ° C. for 1 to 15 days, preferably 3 to Incubate under aerobic conditions for about 7 days. As the culture method, for example, stationary culture, shaking culture, and aerobic deep culture using jar fermenter can be used.

  After culturing under the above conditions, the target polypeptide is recovered from the culture product (step (2)). When solid culture is employed, for example, a solvent (water, physiological saline, buffer solution, etc.) is added to the cultured medium, and then stirred as necessary. And after predetermined time progress, a solid component is removed and it is set as a culture extract. Filter filtration, centrifugation, etc. can be used to remove the solid component. The culture extract thus obtained is used as the cellulose degradation accelerator of the present invention as it is (that is, without any special treatment) or after additional treatment. Examples of “additional treatment” include disruption of cells (pressure treatment, ultrasonic treatment, etc.), concentration (concentration with ultrafiltration membrane, etc.), purification (salting out, various chromatography, etc.), etc. Addition, dilution and drying of the components. Two or more processes may be performed as the additional process. The final form may be liquid or solid (including powder).

3. Use of Cellulose Degradation Accelerator A further aspect of the present invention provides the use of the cellulose degradation accelerator. One of the applications provided by the present invention is a cellulose decomposition method. In the cellulose decomposition method of the present invention, a step of treating the cellulose material with cellulase in the presence of the cellulose decomposition accelerator is performed. In principle, the treatment conditions at this time may be set so that the cellulase used works well (preferably the optimum conditions). However, it is preferable to adjust the processing conditions in consideration of the conditions under which the effect of the cellulose degradation accelerator is enhanced. Such adjustment can be easily identified through preliminary experiments using the cellulose material to be treated and the cellulase used. In addition, if the example of processing conditions is shown, temperature will be 30 degreeC or more and 70 degrees C or less, pH will be about 2 or more and 6 or less, and processing time is about 1 hour or more and 24 hours or less. Processing conditions are not particularly limited.

  The cellulase used is not particularly limited. For example, a cellulase derived from a microorganism can be used. Cellulases derived from microorganisms belonging to the genus Trichoderma as cellulases derived from microorganisms (for example, manufactured by Meiji Seika Co., Ltd., Novozymes, manufactured by Genencor, manufactured by Amano Enzyme), cellulases derived from Aspergillus microorganisms (for example, manufactured by HIBI, manufactured by Amano Enzyme), Cellulases derived from Acremonium microorganisms (for example, manufactured by Meiji Seika Co., Ltd.) are known. Not only natural (wild-type) cellulase but also recombinant cellulase can be used. Two or more cellulases may be used in combination. A commercially available cellulase or cellulase preparation may be used. Examples of commercially available cellulases or cellulase preparations are Accelerase (manufactured by Genencor), NS22074 (manufactured by Novozymes), Meicelase, Acremozyme (manufactured by Meiji Seika Co., Ltd.), Cellulosin AC (manufactured by Etvy), Cellulase A, Cellulase T (Amano Enzyme) Company-made).

  Cellulose material refers to a material containing cellulose as one of the constituent components. Preferably, a material mainly composed of cellulose is used as the cellulose material. As long as it contains cellulose, not only wood but also various plants or processed and processed products thereof (for example, corn fiber, rice bran, bagasse, paper), cellulose produced by some microorganisms (bacterial cellulose), etc. as cellulose materials It can also be adopted.

  Cellulose material is subjected to pretreatment as required. Examples of pretreatment are crushing, pulverization, steaming, heat treatment, chemical treatment with various treatment agents (alkali, organic solvent, hydrous organic solvent, sulfur dioxide, etc.), and treatment with lignin-decomposing organisms.

  Other cellulose degradation accelerators may be used in the treatment with the cellulose degradation accelerator of the present invention. Examples of other cellulose degradation accelerators include swollenin and expansin.

  On the other hand, in addition to or separately from other cellulose degradation accelerators, other enzymes or enzyme agents may be used in combination. Examples of other enzymes herein include hemicellulase, endoglucanase, pectinase, protease, laccase, and esterase. Moreover, as an enzyme agent, the enzyme agent containing 1 type or multiple types of these enzymes can be illustrated.

  The cellulose decomposition method of the present invention can be used in various fields (for example, energy field, biomass processing field, food processing field). Specific application examples include so-called bioethanol production, waste material treatment, vegetable / fruit processing, oligosaccharide production, cellulose fiber processing / modification, bread making, and detergent. As a typical use of the cellulose decomposition method of the present invention, an example of a method for producing bioethanol is shown below. This production method is roughly divided into four steps: pretreatment, saccharification, fermentation and recovery. In the pretreatment, crushing, pulverization, steaming, chemical treatment, and the like are performed so that cellulose in the cellulose material is easily saccharified. The cellulose decomposition method of the present invention is applied to the saccharification step. Typically, saccharification is carried out in a fermentation tank (preferably with a stirring function). In the fermentation process following the saccharification process, filamentous fungi, yeast and the like are added to convert the sugar produced in the saccharification process into ethanol (ethanol fermentation). You may make it perform this saccharification process in parallel with a fermentation process. From the fermentation liquid after the saccharification step, ethanol is recovered by a treatment such as distillation or dehydration (recovery step).

1. Search for microorganisms that produce cellulolytic accelerators In order to find new substances that enhance cellulase activity, they are classified into the genus Rhizopus, Mucor, Fusarium, and Aspergillus. Screening was performed by the following method for molds (1000 strains or more). In addition, since biomass degradation in nature is carried out by so-called “solid culture” by microorganisms, it is considered that enzymes and factors necessary for biomass degradation are produced more abundantly in solid culture than in liquid culture. An evaluation system based on solid culture was adopted.

(1) Preparation of culture extract Each microorganism was inoculated in a solid medium, and statically cultured at 30 ° C. for 3 days. After completion of the culture, 90 mL of tap water was added to the medium and allowed to stand overnight at 4 ° C. to prepare a culture extract from which solid components were removed. The composition of the solid medium was 4.5 g of bran, 0.5 g of cellulose powder, and 1.5 mL of deionized water. In addition, it was decided to add cellulose powder in the hope that production induction of a cellulose degradation promoting substance would occur.

(2) Comparison of synergistic effect ratio and cellulolytic activity of culture extracts
100 μL of 50 mM citrate buffer solution (pH 4.8) was added to a 1.5 mL plastic tube, and a filter paper measuring 2.6 cm in length and 0.3 cm in width was added to the evaluation area. In this evaluation area, 25 μL of test culture extract and 25 μL of cellulase T “Amano” 4 (manufactured by Amano Enzyme) dissolved in 50 mM citrate buffer (pH 4.8) and adjusted to 2 mg / mL were mixed. A filter paper saccharification reaction was performed in the presence of the test culture extract and cellulase T “Amano” 4 at 50 ° C. for 1 hour. Simultaneously, 25 μL of 50 mM citrate buffer (pH 4.8) was added instead of the cellulase T “Amano” 4 solution and the test culture extract, respectively, as the saccharification reaction of the test culture extract alone and the cellulase T “Amano” 4 alone. I went there. The test culture extract, cellulase T “Amano” 4 was added without putting filter paper into the evaluation area, and the respective solutions that were allowed to stand at 50 ° C. for 1 hour were used as blanks. Add 300 µL of 3,5-dinitrosalicylic acid (DNS) reagent (0.5 g of DNS, 20 mL of 2N sodium hydroxide solution, 30 g of Rochelle salt in distilled water to a volume of 100 mL) to the reaction solution and its blank. After boiling for 5 minutes, the absorbance at 540 nm was measured. From each ΔA ₅₄₀ (the value of the sample A _{540 minus} the value of the blank A ₅₄₀ ), the synergistic effect ratio and the cellulolytic activity ratio of the test culture extract were calculated. The synergistic effect ratio of the test culture extract, as expressed by the following formula, is the ΔA obtained by coexistence with the total of ΔA ₅₄₀ obtained with the test culture extract alone and cellulase T “Amano” 4 alone. The ratio was ₅₄₀ . A test culture extract having a higher synergistic effect ratio promotes cellulolytic activity due to a synergistic effect with cellulase T “Amano” 4.
Synergy ratio = (cellulase ^{* 1} and .DELTA.A ₅₄₀ ^* 2 in the coexistence of a test culture extract) / {(test culture extract .DELTA.A ₅₄₀ ^* 2 alone) + (Cellulase ^{* 1} alone in .DELTA.A ₅₄₀ ^{* 2} )}
* 1: Cellulase T “Amano” was used in this screening.
* 2: A parameter indicating cellulolytic activity.

On the other hand, cellulolytic activity ratio of the test culture extract, the following equation expressed as in, cellulase T "Amano" 4 alone resulting in .DELTA.A ₅₄₀ obtained in test culture extract alone against .DELTA.A ₅₄₀ Ratio. The cellulolytic activity ratio is a value indicating the strength of the cellulolytic activity of the test culture extract as a ratio of the cellulase T “Amano” 4 activity, and the low cellulase decomposition activity ratio is the cellulolytic activity ratio of the test culture extract. It indicates that the activity is low.

Cellulolytic activity ratio = (ΔA ₅₄₀ ^{* 2} with test culture extract alone) / (ΔA ₅₄₀ ^{* 2} with cellulase ^{* 1} alone)
* 1: Cellulase T “Amano” was used in this screening.
* 2: A parameter indicating cellulolytic activity.

  As a result of comparing the synergistic effect ratio and cellulolytic activity of each test culture extract, a total of 20 types of lysops genus 13 and mucor genus 7 types were shown as culture extract showing a high synergistic effect ratio and a low cellulolytic activity ratio Those derived from filamentous fungi were found (Table 1).

2. Comparison of β-glucosidase activity of culture extracts Five kinds of culture extracts shown in Table 1 that were considered to be particularly useful were selected and p-nitrophenyl-β in 50 mM citrate buffer (pH 4.8). The β-glucosidase activity of each culture extract was measured using the degradation reaction of D-glucopyranoside at a reaction temperature of 50 ° C. as an index. Table 2 shows the β-glucosidase activity of each culture extract.

3. Confirmation of reproducibility of synergistic effect Rhizopus oryzae APC3504 strain has low cellulolytic activity and β-glucosidase activity and excellent synergistic effect with cellulase T “Amano” 4 among the above five culture extracts An extract was selected and a reproducibility test was performed. First, the extract was prepared again. Specifically, three solid media were prepared, each of which was inoculated with Rhizopus oryzae APC3504 strain, and statically cultured at 30 ° C. for 3 days. After completion of the culture, 90 mL of tap water was added to each medium and left overnight at 4 ° C. to prepare a culture extract from which solid components were removed. The same amount of the three obtained culture extracts was mixed to prepare a test sample. A filter paper saccharification reaction was carried out in the presence of the test sample alone, cellulase T “Amano” 4 alone and in the presence of both, and the concentration of reducing sugar released from cellulose into the evaluation region was measured based on the glucose calibration curve and ΔA ₅₄₀ of the reaction solution. The same measurement was performed three times. As a result, it was not possible to detect the release of reducing sugar from cellulose in the test sample alone three times. In addition, from the average value of each reducing sugar concentration, as the synergistic effect ratio by reducing sugar, cellulase T “Amano” 4 alone, cellulase T “Amano” 4 with respect to the total reducing sugar concentration in the test sample alone, The ratio of reducing sugar concentration was also calculated. The synergistic effect ratio calculated in this way was 2.14 ± 0.34, and it was confirmed that the culture extract derived from Rhizopus oryzae APC3504 strain promotes the degradation of cellulose by a synergistic effect. In addition, the average value of the concentration of reducing sugar in the cellulase T “Amano” 4 alone, the test sample alone, and the coexistence of both is shown in FIG.

4). Synergistic effect on various cellulases The synergistic effect with cellulases other than cellulase T “Amano” 4 was examined on the test sample derived from Rhizopus oryzae APC3504 strain prepared in the above example. Above 1. Accelerase 1500 (GENENCOR) diluted 1000-fold instead of adding cellulase T “Amano” 4 to the evaluation area, NS22074 (Novozymes) diluted 1000-fold, 0.2 mg / mL Mecellase (Meiji Seika Co., Ltd.) or 0.25 mg / mL Acremozyme (Meiji Seika Co., Ltd.) was added. For dilution of each cellulase and preparation of the solution, 50 mM citrate buffer (pH 4.8) was used. The reducing sugar concentration in the presence of the test cellulase alone and the test sample was measured. For each test cellulase, the synergistic effect ratio of the test sample was 1.2 or more, and it was revealed that the test sample showed a synergistic effect on any cellulase. Table 3 shows the reducing sugar concentration and the synergistic effect ratio in the presence of the test cellulase alone and the test sample.

5). Purification of Cellulose Degradation Promoting Factor Cellulose degradation promoting factor (synergistic effect conferring factor) contained in the culture extract derived from Rhizopus oryzae APC3504 strain was purified. First, Rhizopus oryzae APC3504 strain was inoculated into a solid medium composed of 1800 g of bran, 200 g of cellulose powder, and 600 mL of deionized water, and cultured at 30 ° C. for 4 days. After completion of the culture, extraction was performed with 12 L of tap water, and 9 L of culture extract was collected. The precipitate obtained by concentrating 9 L culture extract to 1 L with an ultrafiltration membrane and adding 125.7 g ammonium sulfate to 224 mL concentrate to 80% saturation was added to 10 mM sodium phosphate buffer (pH 6.0). ) And passed through an anion exchange column packed with Q Sepharose Fast Flow (Pharmacia Biotech). Fractionation was by elution with a linear concentration gradient of sodium chloride from 0 to 0.5M. For the fraction that promoted the cellulolytic activity of cellulase T “Amano” 4, the buffer was replaced with 50 mM sodium phosphate-150 mM sodium chloride buffer (pH 7.2), and HiLoad 16/60 Superdex 200 prep grade (Amersham Biosciences Fractionation by gel filtration chromatography. From each fraction, a fraction that promoted the cellulolytic activity of cellulase T “Amano” 4 was obtained. The synergistic effect ratio using this fraction was 1.29, and reducing sugar released from the filter paper could not be detected with the fraction alone. The gel subjected to SDS-PAGE and CBB staining for this fraction is shown in FIG. SDS-PAGE results indicate that the approximately 35 kDa protein that appears in this fraction imparts a synergistic effect on the filter paper saccharification reaction with cellulase T “Amano” 4.

  According to the cellulose degradation accelerator of the present invention, degradation of cellulose material by cellulase can be promoted and made efficient. Polypeptides constituting the cellulose degradation accelerator of the present invention showed enhancing activity against various cellulases. This fact confirms that the cellulose degradation accelerator of the present invention is excellent in versatility. On the other hand, it was suggested that this polypeptide enhances cellulase activity by an action different from that of a cellulose degradation promoting substance found in the past. Therefore, it can be said that the cellulase activity can be further enhanced by using an existing cellulose degradation promoting substance in combination. Also in this respect, the industrial utility value of the present invention is high. The cellulose degradation accelerator of the present invention is used in various applications utilizing cellulase degradation, such as bioethanol production, waste material treatment, vegetable / fruit processing, oligosaccharide production, cellulose fiber processing / modification, bread making It can be applied to detergents.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

As a result of screening, it has been found that microorganisms of the genus Rhizopus and the genus Mucor produce substances having desired characteristics, and microbial strains that produce particularly active substances have been identified. As a result of further investigations, it has been clarified that the substance expected to be highly useful exhibits a synergistic effect on various existing cellulases and is excellent in versatility. The present invention described below is mainly based on these results.
[1] A cellulose degradation accelerator comprising a polypeptide produced by a microorganism belonging to the genus Rhizopus or Mucor and exhibiting a cellulose degradation promoting activity.
[2] The microorganism of the genus Rhizopus is a microorganism of a species selected from the group consisting of Rhizopus oryzae and Rhizopus microsporus var. Chinensis, and the microorganism of the genus Mucor is Mucor plumeus (Mucor plumbeus), mucor sarcineroides varieties sarcineroides (Mucor circinelloides f. circinelloides), mucor racemosus, mucor subtilissimus and mucor ramannianus The cellulose degradation accelerator according to [1], which is a certain type of microorganism.
[3] The Rhizopus microorganism is Rhizopus oryzae identified by accession number: NITE BP-945 , and the Mucor genus microorganism is Mucor sarcineroides varieties Salcineroides NBRC4570 or the accession number: NITE BP-946. The cellulose degradation accelerator according to [1], which is moss.
[4] The cellulose degradation accelerator according to [1], wherein the polypeptide has the following properties:
Molecular weight: approx. 35 kDa (according to SDS-PAGE).
[5] The cellulose degradation accelerator according to any one of [1] to [4], wherein the polypeptide has low cellulolytic activity.
[6] The cellulose degradation accelerator according to any one of [1] to [4], wherein the polypeptide has substantially no cellulolytic activity.
[7] The cellulose degradation accelerator according to any one of [1] to [4], wherein the synergistic effect ratio calculated by the following formula is 1.2 to 2.5 for the polypeptide:
Synergistic effect ratio = (Cellulolytic activity in the presence of cellulase and the polypeptide) / {(Cellulolytic activity of the polypeptide alone) + (Cellulolytic activity of cellulase alone)}
[8] The cellulose degradation accelerator according to any one of [1] to [7], wherein the polypeptide has low β-glucosidase activity.
[9] The cellulose degradation accelerator according to any one of [1] to [7], wherein the polypeptide has substantially no β-glucosidase activity.
[10] The cellulose degradation promoter according to any one of [1] to [9], which comprises the culture product or culture extract of the microorganism.
[11] The cellulose degradation accelerator according to any one of [1] to [10], further containing cellulase.
[12] A method for producing a cellulose degradation accelerator comprising the following steps (1) and (2):
(1) culturing a microorganism belonging to the genus Rhizopus or Mucor, which produces a polypeptide exhibiting a cellulose degradation promoting activity, under conditions under which the polypeptide is produced;
(2) recovering the polypeptide from the culture product.
[13] The production method according to [12], wherein the medium used in step (1) is a cellulose-containing solid medium.
[14] The production method according to [13], wherein step (2) includes removing a solid component after adding a solvent to the medium after step (1).
[15] The production method according to [14], wherein the polypeptide is purified after removing a solid component.
[16] The microorganism of the genus Rhizopus is a microorganism of a species selected from the group consisting of Rhizopus oryzae and Rhizopus microsporus var. Chinensis, and the microorganism of the genus Mucor is Mucor Purmbeus (Mucor plumbeus), mucor sarcineroides varieties sarcineroides (Mucor circinelloides f. circinelloides), mucor racemosus, mucor subtilissimus and mucor ramannianus The production method according to any one of [12] to [15], which is a certain type of microorganism.
[17] The Rhizopus genus microorganism is a Rhizopus oryzae identified by accession number: NITE BP-945 , and the Mucor genus microorganism is Mucor sarcineroides varieties Salcineroides NBRC4570 or the accession number: NITE BP-946. The production method according to any one of [12] to [15], which is Mothus.
[18] A method for decomposing cellulose, comprising treating a cellulose material with cellulase in the presence of the cellulose degradation accelerator according to any one of [1] to [11].
[19] The cellulose decomposing method according to [18], wherein the cellulase is a cellulase derived from Trichoderma, Aspergillus, or Acremonium.
[20] A method for producing ethanol, comprising a step of converting the sugar produced by the cellulose decomposition method according to [18] or [19] into ethanol.
[21] The method for producing ethanol according to [20], wherein the treatment with cellulase and the ethanol conversion are performed in parallel.
[22] Microorganism producing a polypeptide exhibiting cellulose degradation promoting activity, which is Rhizopus oryzae specified by accession number: NITE BP-945 or Mucor racemosus specified by accession number: NITE BP-946 .

1. Cellulose degradation accelerator The first aspect of the present invention relates to a cellulose degradation accelerator. The cellulose degradation promoter of the present invention contains a polypeptide produced by a microorganism belonging to the genus Rhizopus or Mucor. The polypeptide exhibits cellulose degradation promoting activity. As shown in the Examples below, as a result of large-scale screening by the present inventors, Rhizopus oryzae and Rhizopus microsporus var. Chinensis, which are microorganisms of the genus Rhizopus, are cellulose. It was revealed that a polypeptide excellent in degradation promoting activity was produced. Based on this finding, in a preferred embodiment of the present invention, a microorganism belonging to the genus Rhizopus is any of these. On the other hand, the microorganisms of the genus Mucor, Mucor plumbeus, mucor sarcineroides varieties Sarcineeroides (Mucor circinelloides f. Circinelloides), mucor racemosus, mucor subtilis muss ( It has been revealed that Mucor subtilissimus and Mucor ramannianus produce polypeptides having excellent cellulose degradation promoting activity. Based on this finding, in a preferred embodiment of the present invention, a microorganism belonging to the genus Mucor is any of these. Rhizopus oryzae APC3504 and Mucor racemosus APC4090 are deposited with a predetermined depository as follows and are easily available. NBBC4570 is a NRBC culture collection (Mucor circinelloides f. Circinelloides) NBRC4570, Biotechnology Division, Biotechnology Headquarters, National Institute of Technology and Evaluation, Kazusa Kamazu, Kisarazu, Chiba, Japan 292-0818 2-5-8) and can be obtained from the National Institute of Product Evaluation Technology Biotechnology Headquarters, Biogenetic Resources Division (NBRC) through a designated procedure.
(Rhizopus oryzae APC3504)
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Contract date : May 21, 2010
Accession Number: NITE BP-945

(Mucole Racemos APC4090)
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Contract date : May 21, 2010
Accession Number: NITE BP-946

Claims (22)

  A cellulose degradation promoter comprising a polypeptide produced by a microorganism belonging to the genus Rhizopus or Mucor, which exhibits a cellulose degradation promoting activity.   The microorganism of the genus Rhizopus is a microorganism selected from the group consisting of Rhizopus oryzae and Rhizopus microsporus var. Chinensis, and the microorganism of the genus Mucor is a Mucor plumbeus, A species selected from the group consisting of Mucor circinelloides (Mucor circinelloides f. Circinelloides), Mucor racemosus, Mucor subtilissimus and Mucor ramannianus The cellulose degradation promoter according to claim 1, which is a microorganism.   The Rhizopus genus microorganism is a Rhizopus oryzae identified by receipt number: NITE ABP-945, and the Mucor genus microorganism is a Mucor sarcineroides varieties Sarcineroides NBRC4570 or a receipt number: Mucor racemosas identified by NITE ABP-946. The cellulose degradation promoter of Claim 1 which is a certain. The cellulose degradation promoter according to claim 1, wherein the polypeptide has the following properties:
Molecular weight: approx. 35 kDa (according to SDS-PAGE).   The cellulose degradation accelerator according to any one of claims 1 to 4, wherein the polypeptide has a low cellulolytic activity.   The cellulose degradation promoter according to any one of claims 1 to 4, wherein the polypeptide has substantially no cellulolytic activity. The synergistic effect ratio calculated with the following calculation formula is 1.2-2.5 in the said polypeptide, The cellulose degradation promoter as described in any one of Claims 1-4:
Synergistic effect ratio = (Cellulolytic activity in the presence of cellulase and the polypeptide) / {(Cellulolytic activity of the polypeptide alone) + (Cellulolytic activity of cellulase alone)}   The cellulose degradation accelerator according to any one of claims 1 to 7, wherein the polypeptide has low β-glucosidase activity.   The cellulose degradation promoter according to any one of claims 1 to 7, wherein the polypeptide has substantially no β-glucosidase activity.   The cellulose degradation promoter according to any one of claims 1 to 9, comprising a culture product or a culture extract of the microorganism.   The cellulose degradation promoter as described in any one of Claims 1-10 which further contains a cellulase. A method for producing a cellulose degradation accelerator comprising the following steps (1) and (2):
(1) culturing a microorganism belonging to the genus Rhizopus or Mucor, which produces a polypeptide exhibiting a cellulose degradation promoting activity, under conditions under which the polypeptide is produced;
(2) recovering the polypeptide from the culture product.   The production method according to claim 12, wherein the medium used in step (1) is a cellulose-containing solid medium.   The production method according to claim 13, wherein step (2) includes removing a solid component after adding a solvent to the medium after step (1).   The method according to claim 14, wherein the polypeptide is purified after removing a solid component.   The microorganism of the genus Rhizopus is a microorganism selected from the group consisting of Rhizopus oryzae and Rhizopus microsporus var. Chinensis, and the microorganism of the genus Mucor is a Mucor plumbeus, A species selected from the group consisting of Mucor circinelloides (Mucor circinelloides f. Circinelloides), Mucor racemosus, Mucor subtilissimus and Mucor ramannianus The production method according to any one of claims 12 to 15, which is a microorganism.   The Rhizopus genus microorganism is a Rhizopus oryzae identified by receipt number: NITE ABP-945, and the Mucor genus microorganism is a Mucor sarcineroides varieties Sarcineroides NBRC4570 or a receipt number: Mucor racemosas identified by NITE ABP-946. The manufacturing method as described in any one of Claims 12-15 which exists.   A cellulose decomposition method, wherein a cellulose material is treated with cellulase in the presence of the cellulose decomposition accelerator according to any one of claims 1 to 11.   The method for decomposing cellulose according to claim 18, wherein the cellulase is a cellulase derived from the genus Trichoderma, Aspergillus or Acremonium.   The manufacturing method of ethanol including the step which carries out ethanol conversion of the saccharide | sugar produced | generated by the cellulose decomposition method of Claim 18 or 19.   The method for producing ethanol according to claim 20, wherein the treatment with cellulase and the ethanol conversion are performed in parallel.   A microorganism producing a polypeptide exhibiting a cellulose degradation-promoting activity, which is Rhizopus oryzae identified by NITE ABP-945 or Mucor racemosus identified by NITE ABP-946.

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