WO2000043489A1 - Method and additive for reducing solid distillation residue - Google Patents

Abstract

A method for reducing a solid distillation residue characterized by involving the step of effecting distillation in the presence of a polymerase which exerts its activity under the distillation conditions.

Description

 Description Method for reducing solid content of distillation residue and additives Technical field

 The present invention relates to a method for reducing the solid content of a distillation residue, which is useful for improving the efficiency of a distillation step of a fermentation product, and an additive for reducing the solid content for use in the method. Background art

 The phenomenon in which carbohydrates are anoxically decomposed by microorganisms is called fermentation. In this process, useful substances (fermentation products) are produced as metabolites, and fermentation involves alcohol (ethanol). It has been used as a means of producing various useful substances.

 Distillation is used to recover volatile fermentation products, and many distillation residues are formed after distillation. The same applies to alcohol fermentation. Shochu, for example, is a distilled liquor produced by performing alcohol fermentation using rice, buckwheat, wheat, brown sugar, etc. as raw materials, and distilling the obtained moromi. A large amount of distillation residue is produced even during the industrial production of shochu. Most of this distillation residue is disposed of by ocean dumping, but the disposal of the distillation residue by ocean is being abolished by the London Convention on the Parties of the Treaty of 1993.

 As typical treatment means for solving these problems, (1) a method in which a distillation residue is subjected to a methane fermentation treatment to reduce the concentration of organic substances, followed by an activated sludge treatment, (2) a solid-liquid treatment of the waste liquid Separation is performed, solid components are incinerated, and liquid components are concentrated and then used as food materials, feed, fertilizer, or activated sludge treatment.

However, even if either of the above methods is used, it takes a longer time to process the distillation residue after distillation. In particular, the methane fermentation method (1) is a method for treating solid organic matter. Performance is low and the processing equipment must be large. In the method (2), solid-liquid separation is difficult, or a large amount of solid components are contained. There is a problem that the active ingredient is incinerated without being used.

 Japanese Patent No. 2,916,666 discloses a technique for reducing the solid content in the residue after distillation by allowing an enzyme to act on mash during fermentation. However, in this method, changes in components in the mash may affect the growth and fermentation ability of microorganisms contributing to fermentation, so fermentation conditions are adjusted according to the type and amount of enzyme to be added. There is a need. Purpose of the invention

 Accordingly, an object of the present invention is to provide a means for previously reducing the amount of solid components in a distillation residue generated in a distillation process of a fermentation product by allowing a polymer degrading enzyme to act during distillation. As described above, by reducing the amount of solids in the distillation residue in advance, it is possible to process the distillation residue in a shorter time and more efficiently, and further, to increase the liquid content of the active ingredient. The components can be effectively used as feed and medium. Summary of the Invention

 According to an outline of the present invention, the first invention of the present invention comprises a step of performing distillation in the presence of a polymerase having activity under distillation conditions, wherein the distillation residue is reduced in solid content. The present invention relates to a method, wherein the second invention comprises a polymerase that is active under distillation conditions and a microorganism that produces Z or a polymerase that is active under distillation conditions, and is added to the fermentation mash before the start of distillation. The present invention relates to an additive for reducing the amount of solids in a distillation residue, which is characterized in that:

 By causing a polymer degrading enzyme having an activity under distillation conditions to act on the fermentation mash during distillation, the solid content in the distillation residue is solubilized, and the weight of the solid content can be reduced.

The present inventors have conducted intensive studies to solve the above-mentioned problems of the prior art.As a result, in the distillation step, a polymer degrading enzyme having an activity under distillation conditions in fermentation mash, for example, a heat-resistant protease was used. It was found that the solidification in the distillation residue was solubilized by the action, and the weight of the solids was reduced. In addition, the above-mentioned polymer degrading enzyme A microorganism that produces the desired fermentation product is co-cultured during fermentation, and a microorganism that produces the desired fermentation product is given the ability to express the above-mentioned high-molecular-weight degrading enzyme and used for fermentation. The fermentation mash obtained by such a method as above is used for fermentation using the recombinant organism into which the polymer degrading enzyme gene has been introduced, and the solid content is efficiently solubilized in the distillation process. However, it was found that the weight of the solid content in the residue was reduced. BRIEF DESCRIPTION OF THE FIGURES

 Fig. 1: A diagram showing the decrease in the amount of solids in shochu distillation residue by protease treatment. The horizontal axis is the enzyme used and the processing temperature (° C), and the vertical axis is the decrease rate of the solid content.

(%). In the bar graph, black (solid) shows the results of the reaction with ethanol added, and white (mouth) shows the results of the reaction without ethanol added.

 FIG. 2 is a graph showing the decrease in the amount of solids in the residue when protease is acted on at various temperatures. The horizontal axis shows the incubation temperature (° C), and the vertical axis shows the solid content reduction rate (%).

 Figure 3: A graph showing the decrease in the solid content in the residue when cellulase and protease were allowed to act. The horizontal axis indicates the added enzyme, and the vertical axis indicates the rate of decrease (%) in the solid content. Figure 4: A graph showing the amount of γ-polyglutamic acid obtained by culturing Bacillus nut using the distillation residue as a medium. The horizontal axis indicates the used residue, and the vertical axis indicates the γ-polyglutamic acid concentration (mg Zm 1) in the culture supernatant.

 FIG. 5 is a view showing a decrease in the amount of solid content in the residue when a microorganism into which a protease gene is introduced is added and the microorganism is allowed to act. The horizontal axis indicates the amount of microbial suspension added (μ 1), and the vertical axis indicates the rate of decrease in solid content (%).

FIG. 6 is a diagram showing the efficiency of filtration of the residue from the combustion distillation by protease treatment. The horizontal axis shows the time (minutes) of centrifugation, and the vertical axis shows the filtration rate (%). In the figure, open squares (◊) represent the control, and closed triangles (▲), open circles (〇), and closed circles (黒) represent the results obtained using the 2 U, 4 U, and 8 U proteases, respectively. Detailed description of the invention Hereinafter, the present invention will be described specifically.

 The “fermentation” referred to in the present specification is not particularly limited, and examples thereof include alcohol (ethanol) fermentation, acetone butanol fermentation, lactic acid fermentation, propionic acid fermentation, and glycerin fermentation. Fermentation is a phenomenon in which various microorganisms such as acetic acid bacteria and lactic acid bacteria, such as yeasts and koji molds, are used to decompose sugar and starch to produce alcohol and other organic acids and carbon dioxide.

 The term “fermentation product” as used herein refers to a metabolite produced by fermentation. INDUSTRIAL APPLICATION This invention can be utilized for manufacture of the fermentation product which can be collect | recovered by distillation, and can be suitably used especially for manufacture of volatile fermentation products, such as ethanol, acetone, and butanol. For example, the following are produced using alcohol fermentation and distillation. In other words, shochu, awamori, and fruits obtained by distilling a fermented liquid (fermented moromi) prepared from rice, wheat, sweet potato, potato potato, buckwheat, and the like as raw materials, and starch in the raw materials through sugar cane dipping are used as raw materials. These include brandy obtained by distilling the obtained fruit wine, whiskey obtained by distilling the malt fermented liquid, molasses obtained from sugarcane and sugar beet, and alcohol for fuel using corn and the like as raw materials.

 As used herein, the term "distillation conditions" refers to the distillation conditions used to obtain the desired fermentation product. Distillation conditions vary depending on the desired fermentation product. For example, distillation of shochu is generally carried out at 94 to 100 ° C for about 8 hours in normal pressure distillation and about 5 hours at 48 ° C in vacuum distillation.

 As used herein, the term "microorganism that produces a target fermentation product" refers to a microorganism used for fermentation to produce a target product. Microorganisms used for alcohol fermentation include yeast of the genus Saccharomyces, yeast of the genus Zygosaccharomyces, and Clostridium.

 Applied Biochemistry and Biotechnology, Vol. 57, 599-9-60 (Applied Biochemistry and Biotechnology)

P. 4 (1 9 9 6), Journal of Ob 'Batatellology 1 (Journal of

Bacteriology), Volume 170, 280-9-28 15 (1988)], genus Zymomonas (Critical Review in Biotechnology) , Vol. 13, Vol. 57-98 (1 9 9 3), Biochemical Society Symposia, 48, 53-86 (1983)], genus Thermoanaerobacter [Archives of Microbiology] Microbiology), Vol. 168, pp. 114-119 (1997)], bacteria of the genus Mucor, genus Fusarium, Rhizopus

 (Rhizopus) genus and the like. In addition, recombinant microorganisms having alcohol fermentation ability (Applied and Environmental Microbiology, Vol. 57, pp. 893-900 (1991), current Microbiology (Current

Microbiology), Volume 33, pages 256-260 (1996), Applied 'And. Environmental' Microbiology, Volume 57, 2810-2815 (1991), U.S. Pat. No. 5482846, Applied 'and' Environmental 'Microbiology, Vol. 62, 4648-4651 (1996), Applied' and 'Environmental Microbiology, Vol. 64, 1852-1859 The present invention can also be applied to fermentation using page (1998)].

 In the present specification, “fermentation moromi” means a mixture obtained by fermenting a raw material in a fermentation step, and also means the contents of a distillation apparatus during distillation. The mixture of solids and liquid remaining inside the distillation apparatus after the fermentation mash has been distilled is called "distillation residue". Accordingly, the term "distillation residue solids" as used herein refers to solids in the distillation residue.

 The present invention is characterized in that a polymer degrading enzyme having activity under distillation conditions is allowed to act on fermentation mash during distillation to reduce the amount of solids in the distillation residue. It should be noted that the scope of the present invention is not limited by whether or not the polymer degrading enzyme acts on the distillation residue after the distillation step. In general, the distillation residue maintains the same temperature and pH conditions for a considerable time after the distillation as during the distillation, and in practicing the present invention, the polymer degrading enzyme may act on the distillation residue after the distillation is completed. .

The type of the polymer degrading enzyme that can be used in the present invention is not particularly limited as long as it is substantially active under distillation conditions and can reduce the solid content of the distillation residue. Absent. For example, in view of the temperature during and after distillation, a heat-resistant polymer-degrading enzyme having activity under the temperature conditions at which distillation is performed can be suitably used in the present invention. In addition, it is desirable that the enzyme used in the present invention has resistance to the target fermentation product. For example, in the case of alcohol fermentation, an enzyme that does not easily deactivate at a high temperature of 50 ° C. or higher and coexistence of ethanol and can exhibit enzymatic activity can be suitably used.

 As used herein, the term “polymer” refers to components contained in fermentation raw materials and microorganisms used for fermentation, for example, proteins, cellulose, hemicellulose, pectin, cell wall components (mucopeptide, chitin, | 3-glucan), nucleic acids and the like.

 The polymer degrading enzyme used in the present invention can be selected according to the components contained in the fermentation raw material and the microorganism used in the fermentation, for example, protease, cellulase, hemicellulase, pectinase, cell wall degrading enzyme, Examples include nucleases. These enzymes may be used alone or in combination of a plurality of enzymes.

 Protease is a general term for enzymes that cleave peptide bonds such as proteins and polypeptides. The enzyme is used for solubilizing protein components derived from fermentation raw materials, for example, rice, wheat, sweet potato, potato, soba, and the like, and microorganisms used for fermentation, for example, yeast and Escherichia coli. However, it can act on fermented moromi.

The protease that can be used in the present invention is not particularly limited, but from the viewpoint of the temperature during and after distillation, a protease that is active at a high temperature (for example, 55 ° C. or higher) and has heat resistance is preferable. Enzymes derived from microorganisms can be used. As proteases derived from microorganisms, for example, subtilisin and thermolysin derived from Bacillus genus bacteria can be used. It can be suitably used for the invention. The protease PFUS (International Publication No. WO97 / 21823) used in the following examples is an enzyme having extremely high heat resistance and can be used particularly preferably in the present invention. Cellulase is a general term for enzymes that degrade cellulose, that is, a polymer of dulcose consisting of j3-1,4-darcoside bonds, and can be broadly classified into end-type and exo-type. Endo-type cellulases, such as carboxymethyl cellulase (CMCase), mainly hydrolyze j3-1,4-darcoside bonds in amorphous cellulose randomly to produce reducing sugars. Exo-type cellulases, such as Avicelase, have a high activity of degrading crystalline cellulose, and cleave cellulose from the non-reducing end to produce mainly cellobiose. Examples of cellulase-producing microorganisms include filamentous fungi belonging to the genus Trichoderma (Aspergillus), such as the genus Clostridium, the anaerobic bacteria belonging to the genus Ruminococcus, and the genus Bacteroides. Can be mentioned. Cellulases are known to degrade crystalline cellulose through the synergistic action of several different cellulase properties.

 Hemicellulase is a general term for hemicellulose, an enzyme that degrades insoluble polysaccharides other than cellulose and pectin, which are components of the cell wall of land plants. For example, xylan, mannan, araban, xyloglucan contained in cell walls of many dicotyledonous plants, and arapinoglucuronoxylan contained in cell walls of monocotyledonous plants, mainly grasses, are hemicellulose. Are known. Enzymes that hydrolyze these include, for example, xylanase, mannase, and arabinase. Microorganisms that produce hemicellulase include, for example, Aspergillus niger, which produces cell-mouth synths.

 Pectinase is a generic name for enzymes that degrade pectin in cell walls of higher plants. Pectin is a mixture of protopectin, pectinic acid, pectinic acid, etc., but the main component is an acidic polysaccharide consisting of α-1,4-linked D-galataturonic acid. Pectinase is known to hydrolyze the α-1,4 bond of pectin.

Examples of microorganisms that produce actinase include Aspergillus niger, Cibuithyrium diplodiella, Fusarium monilifu and Fusarium moniliforme. For example, when fermentation is performed using rice, wheat, sweet potatoes, potatoes, buckwheat, etc., or cellulosic biomass such as rice straw, straw, bagasse, etc. And polysaccharides such as hemicellulose and pectin. Therefore, by allowing cellulase, hemicellulase, and actinase to act on the fermentation mash, these polysaccharides can be decomposed and the solid content in the distillation residue can be reduced.

 When fermentation is performed using woody biomass such as wood as a raw material, lignin-degrading enzymes can act to decompose lignin contained in fermentation mash and distillation residues. The enzyme is a general term for lignin, an enzyme that reduces the molecular weight of a hydroxyphenylpropane compound, and is known to exhibit activities such as laccase, peroxidase, and oxygenase. Microorganisms that produce lignin-degrading enzymes include basidiomycetes, such as Coriolus versicolor, Kinia mushrooms (Poria subacida), and filamentous fungi belonging to ascomycetes, such as Fusarium * solani (Fusarium solani). be able to.

 Cell wall degrading enzymes are used to degrade the cell walls of microorganisms used in fermentation, such as yeast and E. coli. For example, lysozyme, chitinase,] 3-dalcanase and the like are used as cell wall degrading enzymes. '

 Lysozyme is widely distributed in the animal and plant kingdoms such as chicken egg whites, human tears and saliva, and papaya, and β-1,1, which exists between mucopeptides such as mucopeptides in bacterial cell walls, between N-acetylmuramic acid and N-acetyldarcosamine. 4 It is an enzyme that hydrolyzes one bond. Microorganisms that produce lysozyme include Streptomyces erythraeus, which belongs to the genus Streptomyces.

 Chitinase hydrolyzes the i3-1,4-single bond of chitin, the main polysaccharide that constitutes arthropods, molluscs, exo-animals, fungal cell walls, etc., to produce N-acetyldarcosamine and its oligosaccharides. A generic term for enzymes. Examples of microorganisms that produce chitinase include Streptomyces antibiotics (btreptomyces antibiotics) belonging to the genus Streptomyces.

β-glucanase is a major component of the yeast cell wall. 1,3—The enzyme that breaks bonds. Microorganisms that produce j3-glucanase include, for example, Bacillus circulans.

 Nuclease is a general term for enzymes that degrade nucleic acids, that is, polynucleotides.It refers to ribonucleases that specifically degrade RNA, deoxyribonucleases that specifically degrade DNA, and specific base sequences of DNA. Recognition restriction enzymes, enzymes that act on both DNA and RNA, such as Micrococcus endonuclease, are included. Nucleases can be broadly classified into endo- and exo-types. End-type nucleases, for example, DNase I derived from the skeletal knee, restriction enzymes, etc., bind to the 3'5, monophosphodiester bond inside the polynucleotide chain. To yield fragmented polynucleotide-oligonucleotides.

 An exo-type nuclease is one that degrades a polynucleotide chain sequentially from one end to produce a mononucleotide, and includes, for example, the 3 ′ → 5 ′ exonuclease activity of eukaryotic DNA polymerase δ. Examples of the nuclease-producing microorganism include Escherichia coli, bacteria belonging to the genus Bacillus and Streptomyces, and filamentous fungi such as Aspergillus oryzae.

The above-mentioned high-molecular-weight degrading enzyme may be one obtained by purifying from its original source or one produced by genetic engineering using a gene encoding the enzyme. . When the enzyme is an enzyme derived from a thermophilic bacterium, the gene encoding the enzyme is introduced into a host microorganism that grows at room temperature, such as Escherichia coli or Bacillus bacterium, to express the enzyme. Purification becomes easy. For example, the protease PFUS can be secreted and expressed outside the host at 37 ° C. by using a bacterium belonging to the genus Bacillus (International Publication WO97 / 21823). If the amount of solids in the distillation residue can be reduced, these high-molecular-weight degrading enzymes need not necessarily be purified. Further, the enzyme may be modified by a known method. Examples of such enzymes include those in which the original amino acid sequence has been modified by substitution, deletion, insertion, addition, or the like by genetic engineering techniques, or in which the amino acid residue has been chemically modified. One.

 There is no limitation on the method of treatment with the above-mentioned polymer degrading enzyme, and the treatment may be carried out under conditions where the polymer degrading enzyme exhibits activity. If the pH of the fermentation mash after fermentation is significantly different from the optimal pH of the polymer-degrading enzyme, the pH needs to be adjusted so that the enzyme acts.

 Not only known high-molecular-weight degrading enzymes can be used, but also those more suitable for achieving the object of the present invention can be screened and used. For example, a thermostable high molecular weight degrading enzyme suitable for the present invention can be screened and used from microorganisms, preferably thermophilic microorganisms.

 The novel polymer-degrading enzyme can be obtained, for example, by using a gene encoding a known high-molecular-weight enzyme. For example, a DNA fragment consisting of a nucleotide sequence encoding an amino acid sequence of a region showing high homology to an amino acid sequence such as subtilisin in an acid sequence encoding a protease PFUL described in WO 95/34645, or By using an oligonucleotide designed based on the base sequence as a probe or a primer, another Pyrococcus furiosus-derived protease gene different from protease PFUL, that is, a protease PFUS gene, can be obtained. It has been isolated and its amino acid sequence has been elucidated (WO 97Z21 823).

 In addition, in order to obtain a macromolecule-degrading enzyme that can act more efficiently on fermentation mash during distillation, a heat-resistant enzyme can be produced from a known enzyme using evolutionary molecular engineering. For example, the kanamycin resistant enzyme of Escherichia coli is unstable at 60 ° C or higher, but can be grown at 71 ° C in the presence of kanamycin by incorporating the gene of the enzyme into moderate thermophiles. Procedurals of the National 'Academy' of the University of the United States

 (Proceedings of National Academy of Sciences USA) Vol. 83, Vol. 576-

580 (1986)].

The method of adding these high-molecular-weight degrading enzymes is not particularly limited. For example, they are used by adding to fermentation mash before distillation or during distillation. On the other hand, by adding a microorganism producing the polymer degrading enzyme to the fermentation raw material at the start of fermentation and during Z or the fermentation process, The enzyme can be produced in the fermentation mash during the fermentation process. When this fermented mash is subjected to distillation, the expressed polymer-degrading enzyme solubilizes solid components during distillation. In this case, as the microorganism that produces the polymer-degrading enzyme, a microorganism that naturally has the ability to produce the polymer-degrading enzyme may be used, or a suitable gene into which a foreign gene encoding the enzyme is introduced may be used. Microorganisms can also be used. Furthermore, by introducing a gene encoding the enzyme into a microorganism that produces the fermentation product of interest, for example, in alcohol fermentation, a single microorganism can perform fermentation and produce a polymer-degrading enzyme. . In addition, by introducing the above-mentioned polymer-degrading enzyme gene into an organism that becomes a fermentation raw material or that produces the same, a fermentation material containing the polymer-degrading enzyme is prepared and used for fermentation and distillation. It is also possible to carry out the present invention.

 When performing distillation using the method of the present invention, the operation is not particularly limited, and may be performed according to a known distillation method. In addition, at the time of distillation, by setting the inside of the distillation vessel under reduced pressure, the target fermentation product can be distilled at a temperature lower than its boiling point. If the target fermentation product has a high boiling point and the polymer-degrading enzyme to be used may not work sufficiently, take the above measures to make it suitable for the enzyme that uses the distillation temperature. Can reduce the amount of solids.

As used herein, the term “weight loss” refers to a reduction in the weight of the distillation residue solids as compared to the case where a high-molecular-weight enzyme is not used. Whether or not the above-mentioned polymer-degrading enzyme can reduce the amount of solids in the distillation residue can be confirmed, for example, by the following method. That is, the enzyme to be used is added to the fermentation mash and distilled, and the solid content in the resulting distillation residue is collected by filtration, centrifugation, etc., and the dry weight is measured. It can be checked by comparing with those without enzyme addition. The effects of the polymer-degrading enzyme are as follows: 1) the presence of a component that is solubilized by the enzyme in the distillation residue; and 2) the enzyme can use the component of 1) under the conditions where distillation is performed. It can be known by examining that it can be solubilized. Therefore, in the above method, if the fermentation mash is incubated at the same temperature as the distillation time using a device capable of distilling out the desired fermentation product, the fermentation product does not always need to be recovered. More conveniently, for example, by normal distillation W

 12 Add the fermentation product equivalent to fermentation moromi to the distillation residue obtained, and conduct the incubation as described above to check the decrease in solid content in the residue to confirm the effect of adding the polymer-degrading enzyme. be able to. According to the method of the present invention, the solid content of the distillation residue is preferably reduced by 10% or more, more preferably 20% or more, and most preferably 30% or more.

 The present invention will be specifically described below by taking protease as an example.

 A thermostable protease that is stable at 55 ° C or higher, for example, a protease produced by a Bacillus bacterium W ai 21a strain having a half-life of 4.5 to 9.5 hours at 60 ° C and pH 3.0. [International Journal of Biochemistry 'and' Cell Biology

 (International Journal of Biochemistry and Cell Biology, Vol. 27, pp. 729-739 (1955)] Furthermore, hyperthermostable proteases that exhibit activity even at 80 ° C. or higher, for example, Pyrococcus belonging to the genus Pyrococcus Pyrocoscus (Fyrococcus furiosus) プ ロ テ ア ー ゼ Thermococcus belonging to the genus Thermococcus' Protease produced by the cello (Thermococcus celer), etc. [Applied and Environmental Microbiology, No. 60 Vol., Pp. 4559-4566 (1994), International Publication WO 97/21823].

Examples of proteases produced by Pyrococcus furiosus include protease PFUL and protease PFUS derived from Pyrococcus furiosus D SM3638. Protease PFUL has the activity of decomposing proteins such as casein and gelatin at 95 ° C, and its optimum pH is around pH 9.0 to 10.0. The protease has high thermostability and retains almost 100% activity after heat treatment at 95 ° C for 4 hours. This thermal stability is similar even in the presence of 0.1% SDS. The optimal temperature of protease PFUS is 80-95 ° C, and the optimal pH is around pH 6-8. The enzyme also has high thermostability, and retains about 80% of the activity after heat treatment at 95 ° C for 3 hours when treated in a buffer solution at pH 7.5. . Furthermore, the enzyme is stable in the presence of organic solvents, for example at 95 ° C in the presence of 50% (V / V) acetonitrile. Even after 1 hour of treatment, it has an activity of 80% or more before the treatment.

 The method of using the protease is not particularly limited. For example, by directly adding protease to fermentation mash and performing distillation, the amount of solids in the distillation residue remaining after distillation can be reduced. During fermentation, a protease-producing microorganism, such as Bacillus thermoproteolyticus, which is a thermolysin-producing bacterium, or a foreign protease gene, such as a protease PFUL gene derived from Pyrococcus furiosus, or a protease PFUS gene. The protease may be produced during fermentation by co-culturing the incorporated recombinant with a microorganism producing the desired fermentation product, so that the protease may be allowed to act during the distillation. Alternatively, an exogenous protease gene may be introduced into a microorganism that produces the desired fermentation product, the protease may be expressed during fermentation and after or after fermentation, and the resulting fermented mash may be subjected to distillation. Furthermore, a protease gene, such as a protease PFUL gene or a protease PFUS gene, is introduced into an organism (eg, a plant) from which the fermentation raw material is derived, and is expressed at an appropriate time. ) Is not particularly limited as far as the pH at which the protease is allowed to act on the starting material is within the range of pH at which the enzyme exhibits activity. If the pH of the fermentation mash after fermentation is significantly different from the optimal pH of the protease, it is necessary to adjust the pH so that the protease acts efficiently. For example, when a protease PFUS is allowed to act, ^^ should be adjusted to around 6 to 8. There is no particular limitation on the action temperature and time in the enzymatic reaction, but it can be set at 5 to 120 ° C. within a range of several minutes to several days. However, for the purpose of this patent to improve the energy efficiency in biomass utilization, the protease is preferably allowed to act during and after distillation at a high temperature, so that the working temperature is 50 ° C or higher, preferably 6 ° C or higher. The temperature is preferably at least 0 ° C, more preferably at least 80 ° C. Similarly, the action time of the enzyme may be any time required to reach the target reduction in solids content, but from the viewpoint of efficient distillation, a shorter time, for example, 1 to 48 hours Is preferred.

The gene for the protease is introduced into another host different from the original strain producing the enzyme. It is also possible to produce enzymes by using For example, plasmid pSNP1 into which the gene for protease PFUS has been integrated or DN encoding the promoter signal peptide derived from the subtilisin gene upstream of protease PFUS

The protease PFUS can be purified from a culture of Bacillus subtilis DB104 transformed with the plasmid p NAPS 1 into which the A fragment has been introduced (International Publication WO 97/2 1 8 2 3). The host into which the thermostable enzyme gene is introduced may be a microorganism that is not directly involved in fermentation or biomass production as described above, or may be a microorganism that produces the desired fermentation product. It may be a producing organism.

 Transformation of the alcohol-producing recombinant Bacillus bacterium disclosed in U.S. Pat.No. 5,482,846 with, for example, the above-described plasmid pSNP1 to confer protease PFUS-producing ability. Can be. By performing alcohol fermentation and distillation using this recombinant, it is possible to reduce the solid content of the distillation residue as compared with, for example, a recombinant obtained without transforming with plasmid pSNP1. it can. Similarly, when alcohol fermentation is performed using yeast Saccharomyces cerevisiae into which the protease PFUS gene has been introduced and then distillation is performed, the distillation residue is lower than when fermentation is performed using yeast not having the gene. Solid content can be reduced.

 The additive for reducing the solid content in the distillation residue of the present invention (hereinafter, referred to as the additive of the present invention) may contain a polymer-degrading enzyme and Z or a microorganism that produces a polymer-degrading enzyme. It can be prepared and prepared by a known method similar to the enzyme preparation. By adding the additive to the fermentation mash, the amount of solids in the distillation residue can be reduced.

The polymer-degrading enzyme contained in the additive of the present invention may be a purified polymer-degrading enzyme or an unpurified enzyme as long as it has an action of reducing the solid content in the distillation residue. There may be. Examples of the unpurified enzyme include a culture supernatant of an enzyme-producing microorganism in the case of an extracellular enzyme, a crude cell extract in the case of an intracellular enzyme, and a concentrated or dried product thereof. Examples of the additive of the present invention containing a microorganism that produces a polymer degrading enzyme include a liquid culture, a solid culture, and a dry culture of the microorganism. Those containing dried cells and the like can be mentioned. Further, the additive of the present invention may contain a mixture of two or more substances selected from the above enzymes and microorganisms.

 There is no particular limitation on the timing of addition of the additive of the present invention as long as it is added to the fermentation mash during the distillation, before the fermentation, during the fermentation, after the fermentation (before the distillation), and after the distillation is started. You. Preferably, it is added to the fermentation mash before the start of distillation, from the viewpoint of making the above-mentioned polymer-degrading enzyme act efficiently.

 The additive of the present invention may contain various components other than the enzyme as long as the activity of the polymer degrading enzyme is not impaired and the collection of fermentation products by distillation is not hindered. . Such components include, for example, components for stabilizing enzymes (glycerol, polyethylene glycol, saccharides, etc.), components for adjusting pH, and additives for facilitating removal of the additives. Excipients and the like. Example

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the scope of these Examples. Example 1

 (1) Distilled residue of B2 rice shochu was added to 700 μl, 1 μm sodium carbonate buffer (ρΗ9.0) 100 μl, and 8 U each of Esperase (Bacillus licheniformis). ) -Derived protease; 100 μl of an enzyme solution containing 100 μl of an enzyme solution containing Novo Noredisk Industries, Inc.) or PFUS (Pfu protease, a product of Takara Shuzo), and 100 μl of ethanol. Incubated at 70, 80 or 95 ° C for 15 hours. As a control, a reaction in which no protease was added and a reaction in which ethanol was not added were simultaneously performed. After completion of the reaction, the reaction solution was centrifuged to remove the supernatant, the precipitate was kept at 95 ° C for 18 hours, dried, weighed, and the obtained value was taken as the weight of the solid content. did. From the measured values, the decrease in solid content due to the addition of each protease was compared, and the results are shown in FIG.

As shown in FIG. 1, the weight of the solid content of the distillation residue was reduced by the addition of the protease. When protease PFUS is added, the decrease rate increases with increasing temperature However, the solid content in the distillation residue was reduced even in the presence of ethanol. When esperase was added, the decrease rate decreased with increasing temperature, and the decrease in solid content was significantly suppressed in the presence of ethanol.

 From the above results, it was shown that shochu distillation residue contains components solubilized by protease. In addition, protease PFUS, in particular, reduces the weight of solids in the residue under high temperature conditions in the presence of ethanol, suggesting that the enzyme can be used in the distillation step.

 (2) 1M sodium carbonate buffer (ρΗ9.0) 100μ1, 91.5πιυ / μ1 protease PFUS solution 87.4μ1 is added to fermentation moromi of rice shochu (80μμ1), 70 ° C For 4 hours, 80 ° C for 2 hours or 95 ° C for 2 hours in an open system. As a control, a sample to which no protease was added was prepared and incubated at the same time. After completion of the reaction, the reaction solution was centrifuged to collect the supernatant and the precipitate, respectively, and the ethanol concentration of the supernatant was measured using F-kit ethanol (manufactured by Roche Diagnostics). The precipitate was dried at 95 ° C. for 18 hours, weighed, and the reduction rate of the solid content in the residue was calculated from the obtained results. The results are shown in FIG.

 Since ethanol was not detected in the supernatant of the reaction solution, it was confirmed that heating was performed under conditions similar to actual distillation under the experimental conditions in this experiment. Further, as shown in FIG. 2, the weight of the solid content in the residue was reduced by about 20% or more by adding the protease PFUS. This indicates that, when the protease was added to the fermentation mash during distillation and distillation was performed, the weight of solids in the residue after distillation was reduced.

 (3) The operation in (2) was scaled up and performed using a distillation apparatus. That is, 4 Oml of fermented moromi of the second kind of rice shochu was added to 5 ml of 1 M sodium carbonate buffer (pH 9.0), 91.5 mU / μ1 protease PFUS solution 4 · 37 m

1 was added and distillation was performed at 80 ° C for 2 hours or at 95 ° C for 6 hours. As a control, distillation of mash without added protease was also performed. After distillation, the residue remaining in the device was centrifuged to collect the supernatant and the precipitate, respectively.The absence of ethanol in the supernatant was confirmed by the same method as above, and the precipitate was collected at 95 ° C. 18:00 at C After drying, the weight was measured. Also in this case, it was confirmed that the solid content of the distillation residue from the mash to which the protease PFUS was added was reduced by about 20% as compared with the control. Example 2

 (1) Fermentation mash of B-type rice shochu adjusted to pH 7 with 0.1 N sodium hydroxide solution 800 μl, 91.5πυ / μ1 protease PFUS solution 8 7.4 Add 1 μl, incubate in an open system at 95 ° C for 2 hours, and apply 100 μl of 100 mg / ml cellulase “On ozuka R—10” (Yakult) solution [1]. The cells were further incubated at 50 ° C for 4 hours. As controls, those without cellulase, those without protease, and those without both enzymes were also incubated under the same conditions. After completion of the reaction, centrifugation was performed, and the collected precipitate was dried at 95 ° C. for 18 hours, and its weight was measured. From the obtained measured values, the rate of weight reduction relative to a control in which neither of the above enzymes was added was determined and is shown in FIG.

 The solid content in the residue could be reduced by about 30% when only cellulase was added, and about 20% when only protease was added. Furthermore, when both enzymes were added stepwise, the solid content could be reduced by 50% or more. This suggests that not only protease but also high molecular weight degrading enzyme such as cellulase has an effect on reducing the solid content of distillation residue, and more effective solidification can be achieved by using multiple polymer degrading enzymes in combination. It was shown that the amount could be reduced.

 (2) In the same operation as in Example 2 (1), the fermentation mash of the second class rice shochu to which protease and cellulase were added was incubated, and then the supernatant was recovered from the reaction solution.

The amount of reducing sugars contained in this supernatant was measured by the Park and Johnson method. That is, 10 μl of the reaction solution supernatant was added to 90 μl of distilled water, and a cyanide carbonate solution (5.3 g of sodium carbonate and 0.65 g of potassium cyanide dissolved in 1 liter of water) 1 The mixture was mixed with 100 μl and a 0.05% aqueous solution of potassium ferricified solution (100 μl) and incubated in a boiling water bath for 15 minutes. After completion of the incubation, add 500 μl of iron alum to the reaction mixture (1.5 g of iron alum and 1 g of alum). Sodium laurino sulfate (SDS) dissolved in 1 liter of 0.15 N sulfuric acid) was mixed, left at room temperature for 15 minutes, and the absorbance at 690 nm was measured. The amount of reduced terminal was determined as a glucose conversion amount based on a calibration curve prepared using glucose of known concentration.

 Further, the amount of glucose contained in the supernatant of the above reaction solution was measured using Glucose CII Test Co. (Wako Pure Chemical Industries, Ltd.).

 Furthermore, 2 μl of the above reaction solution supernatant was concentrated to dryness, azeotropic hydrochloric acid was added, and the mixture was heated at 135 ° C for 3 hours to hydrolyze peptides contained in the supernatant to amino acid. . After dissolving and diluting the dried hydrolyzate in distilled water, add 200 μl of 0.2 diboric acid / hydroxy sodium hydroxide buffer (ρ Η 9.0) to diluent 20 β1 and 50 μl of 0.3 mg / ml Fluram (acetonitrile) solution (manufactured by Fluka) was added, and amino groups were quantified from the increase in fluorescence intensity. It was considered that all the obtained amino groups were derived from amino acids, and the average molecular weight of amino acids was 110, and the amount of amino acids was calculated.

 Table 1 shows the above measurement results. The values in Table 1 are the values obtained by subtracting the values measured with fermentation mash that was incubated without adding the enzyme and those measured with only the enzyme from the above measurement values. , Reducing sugars and darcos as increase (mg). table 1

Thus, by distilling the fermented mash in the presence of hydrolases such as protease and cellulase, the amount of glucose, reducing sugar, and amino acid Z-peptide contained in the soluble fraction in the residue after distillation is significantly increased. Increased. Example 3

 To 40 ml of the fermentation mash of the varieties of rice shochu adjusted to pH 7 with 0.1 N sodium hydroxide solution, 3.87 ml of a 91.5πιυ / μ1 protease PFUS solution was added, and 95. C, distillation for 6 hours was performed. After completion of the distillation, 5 ml of a 10 OmgZm 1 cellulase “Onozuka R-10” solution was added, and the mixture was further incubated at 50 ° C for 4 hours. As a control, an operation of distilling and incubating under the same conditions without adding both enzymes was performed.

 A portion of the obtained residue was taken, and the precipitate was collected by centrifugation, dried at 95 ° C for 18 hours, and weighed. As a result, when the enzyme was added and distillation and incubation were performed, the solid content in the residue was reduced by about 30% compared to the control.

 Bacillus natto IFO 3335 (purchased from the Fermentation Research Institute), a polyglutamic acid-producing Bacillus subtilis, was treated with 5 ml of LB medium (1% tryptone, 0.5% yeast extract, 0.5% Incubation was carried out at 37 ° C for 18 hours in a sodium chloride solution (pH 7.2). 50 μl of the obtained preculture was inoculated into 5 ml of the above-mentioned residue, and was incubated at 37 ° C for 48 hours. To the culture supernatant collected by centrifugation, 1/10 volume of a saturated sodium chloride solution and 2.2 volumes of 90% ethanol were added and stirred. The resulting aggregates of γ-polyglutamic acid were collected with a pipet and further washed three times with ethanol. After the obtained γ-polyglutamic acid was air-dried, the dry weight was measured to determine the amount of γ-polyglutamic acid produced.

 Figure 4 shows the production of γ-polyglutamic acid. When Bacillus nut was cultured using the residue obtained by adding protease and cellulase as a medium, the production of γ-polyglutamic acid was significantly increased as compared with the control. Example 4

(1) Escherichia coli JM109 / pTPR12 into which the protease PFUL gene has been introduced as described in WO 95/34645 (this strain was FERM BP— 5 1 03 W

 Was deposited in LB medium at 37 ° C for 24 hours, and the cells were recovered by centrifugation. 3 g of the cells are suspended in 0.1 M potassium phosphate buffer (pH 7.0) to make a total volume of 2.6 ml. In addition to μ1, 50 μl of 1% sodium carbonate buffer (ρΗ9) was added, and then distilled water was added to adjust the total amount to 500 1. After incubating these reaction solutions at 95 ° C for 15 hours, the precipitate obtained by centrifugation was dried at 95 ° C for 18 hours, and the weight was measured. The results are shown in FIG. The solid content was reduced by adding genetically modified microorganisms that express hyperthermostable proteases to the shochu distillation residue. Therefore, it was shown that microorganisms that produce high-molecular-weight degrading enzymes can be used as additives for reducing the solid content in distillation residues.

 (2) Prepare a microbial suspension similar to the above, add 4 ml of the suspension to 5 ml of fermentation mash of B-type rice shochu, and add 1 ml of 1 M sodium carbonate buffer (pH 9) to a total volume of 1 ml. A sample of Oml was prepared. The sample was placed in a distillation apparatus, distilled at 95 ° C for 6 hours, and the precipitate was collected from the residue after distillation by centrifugation. The obtained precipitate was dried at 95 ° C for 18 hours, weighed, and added with a suspension of Escherichia coli JM109 that did not retain plasmid to perform distillation. And the dry weight of the precipitate. As a result, it was shown that the amount of solids in the distillation residue was reduced by about 10% by adding a suspension of microorganisms producing protease P FUL and performing distillation. Example 5

 The residue obtained by distillation performed in the presence of the protease PFUS described in Example 11 (2) was examined for its filterability. As a control, a residue obtained by distillation without adding protease was used.

 Take 400 μl of the above reaction solution on a spin column (Ultra Free MC, manufactured by Millipore) with a pore size of 5 μπι, centrifuge at 3000 X g for 2, 4, 8, or 16 minutes and pass through the filter. The weight of the liquid thus obtained was measured, and the ratio of the filtrate to the whole reaction solution was calculated to obtain the filtration rate. The results are shown in FIG.

The residue obtained by incubation with the protease is added to the protease. W

 21 The efficiency of filtration was higher than that in which no filtration was performed, and the maximum filtration rate was about 50% higher than that of the control. In other words, it has been clarified that solid-liquid separation during the treatment of the residue is facilitated by adding a polymer-degrading enzyme such as a protease during distillation. Industrial applicability

 As described above, the present invention provides a method for reducing the solid content in a distillation residue. By implementing the method of the present invention, the load in the distillation waste liquid treatment step is reduced, for example, the solid-liquid separation step is facilitated, and the energy efficiency throughout the entire process from fermentation to distillation and distillation waste liquid treatment can be improved. .

 In the method of the present invention, the fermentation step can be carried out in the same manner as in the prior art, since there is no change in the mash component during the fermentation step because the enzyme acts after the fermentation. In addition, since the enzyme acts at high temperature, the solid content in the residue is reduced extremely efficiently by the denaturation of the polymer to be degraded by heating, solubilization, and the synergistic action of the degradation and the degradation by the enzyme. be able to.

 The present invention also provides an additive for reducing the amount of solids in a distillation residue, and the additive can be suitably used in the above method.

 Further, by treating the fermentation mash by the method of the present invention, amino acids, oligopeptides, carbohydrates, and the like can be more efficiently solubilized than the solid content of the distillation residue conventionally treated as waste. Distillation residues can be used as feed, fertilizer, and microbial media, but their amino acids, oligopeptides, carbohydrates, etc. are soluble and their nutritional value is improved. In addition, by using a distillation residue supernatant containing more solubilized amino acids, oligopeptides and carbohydrates, biological treatment such as methane fermentation can be performed efficiently, and the utilization efficiency as biomass can be improved. Is improved.

Claims

 The scope of the claims

I. A method for reducing the solid content of a distillation residue, comprising a step of performing distillation in the presence of a polymer degrading enzyme having activity under distillation conditions.

2. Distillation 5 0 ^D distillation residue solids method of weight loss according to claim 1, characterized in that it is implemented in C or more.

 3. The method for reducing solids content of a distillation residue according to claim 1 or 2, wherein fermentation mash obtained by co-culturing a microorganism that produces a polymer degrading enzyme and a microorganism that produces a target fermentation product is used for distillation. .

 4. The reduction of distillation residue solid content according to claim 1, wherein an exogenous polymer degrading enzyme gene is incorporated into a microorganism that produces the target fermentation product, and fermentation mash obtained using the microorganism is used for distillation. Method.

 5. The method for reducing the solid content of distillation residue according to claim 1, wherein a fermentation mash obtained by using an organism-derived material into which an exogenous polymer-degrading enzyme gene has been introduced as a fermentation raw material is used for distillation.

 6. The method for reducing the solid content of a distillation residue according to any one of claims 1 to 5, wherein the fermentation is alcoholic fermentation.

 7. The method according to any one of claims 1 to 6, wherein at least one kind of high-molecular-weight degrading enzyme selected from protease, cellulase, hemicellulase, actinase, cell wall degrading enzyme, and nuclease is used. Method for reducing solid content of distillation residue.

 8. The method according to claim 7, wherein the protease is a thermostable protease.

 9. The method according to claim 8, wherein the heat-resistant protease is a hyperthermostable protease.

 10. It contains a polymerase that is active under distillation conditions and / or a microorganism that produces a polymerase that is active under distillation conditions, and is added to the fermentation mash before the start of distillation. For reducing the amount of solids in the distillation residue.

II. The additive according to claim 10, wherein the high-molecular-weight degrading enzyme is one or more types of high-molecular-weight degrading enzymes selected from proteases, cellulases, hemicellulases, actinases, cell wall degrading enzymes, and nucleases. .

12. The additive according to claim 1, wherein the protease is a thermostable protease.

 13. The additive according to claim 12, wherein the thermostable protease is a hyperthermostable protease.