JP5168072B2 - Polyester manufacturing method - Google Patents

Description

  The present invention relates to a method for producing a polyester comprising a dicarboxylic acid and / or a diol as a constituent component.

  The aliphatic polyester having biodegradability has been applied to fibers, molded articles, films, sheets, and the like as a resin that avoids environmental burdens more than ever because of increasing awareness of environmental problems. For example, polybutylene succinate and / or polybutylene adipate having biodegradability has been developed as an alternative polymer for polyethylene because it has similar mechanical properties to polyethylene.

  However, in the case of production of aliphatic polyester, since a high polymerization degree polyester having low polymerization activity and practically sufficient strength cannot be obtained, for example, when a titanium compound is used as a catalyst, , Diisocyanate (for example, see Patent Document 1) and diphenyl carbonate (for example, see Patent Document 2) have been devised.

  Further, as a polymerization catalyst, Ge compounds (for example, see Patent Document 3), zinc compounds (for example, see Patent Document 4), Fe, Mn, Co, Zr, V, Y, La, Ce, Li, Ca, and other acetyls A production method using acetonate (for example, see Patent Document 5) or an organic alkoxytitanium compound (for example, see Patent Document 6) has also been proposed. In any of these cases, the polymerization activity is low, and the degree of polymerization is low. In the same way, a chain extender was added.

  In addition, a trifunctional group (for example, see Patent Document 7) or a tetrafunctional group (for example, see Patent Document 8) is introduced into an aliphatic polyester, or a branched structure is introduced by introducing an epoxy group, Although attempts have been made to increase the melt tension, in spite of great efforts to improve the melt viscosity and the melt tension as described above, the predetermined viscosity (degree of polymerization) is still not reached. In most cases, diisocyanate is added late in the polymerization. In the only example in which no chain extender is used, a method requiring a very high capital investment for making a very high vacuum has been proposed (see, for example, Patent Document 9), but it is not a practical method.

  On the other hand, when lactic acid is added to the polymerization component to form a ternary system (1,4-butylene glycol, succinic acid, lactic acid) or a quaternary system (1,4-butylene glycol, succinic acid, adipic acid, lactic acid), the catalyst Although it has been disclosed that a polyester having a high degree of polymerization can be produced by using a Ge-based catalyst, the polymerization activity is not sufficient (for example, see Patent Document 10).

  As described above, in the conventional method, the polymerization activity is not sufficient, and it is necessary to maintain a high vacuum for a long time.

  On the other hand, for the purpose of shortening the polymerization time, an increase in the amount of catalyst, etc. can be mentioned. However, an increase in the catalyst content in the polymer causes problems such as degradation in hydrolysis resistance and thermal stability. There wasn't.

  By the way, these polyesters are currently one of the typical polymers produced by polycondensation of raw materials derived from fossil fuel resources. However, there are concerns about recent depletion of fossil fuel resources and atmospheric carbon dioxide. Attention has been focused on methods for deriving raw materials for these polymers from biomass resources due to the increasing global environmental problems. These methods are expected to become a particularly important process from the viewpoint of carbon neutral since this resource is a renewable resource.

  So far, technologies for producing dicarboxylic acids such as succinic acid and adipic acid from glucose, glucose, cellulose, oils and fats derived from biomass resources using fermentation methods have been developed. (See Patent Document 11, Non-Patent Documents 1 and 2).

  However, these processes are processes for producing the target dicarboxylic acid through steps such as neutralization, extraction, and crystallization after once obtaining the dicarboxylic acid as an organic acid salt by fermentation. In addition to the nitrogen element contained in biomass resources, there are features that many impurities such as nitrogen element derived from fermentation bacteria, ammonia and metal cations are mixed.

In addition, 1,4-butanediol, 1,3-propanediol, ethylene glycol, and the like, which are components of biomass resource-derived diols, are obtained by directly obtaining fermentation resources from raw materials derived from biomass resources and the corresponding dicarboxylic acids described above. There is a method of obtaining the product by fermentation using a reduction catalyst and then hydrogenating it with a reduction catalyst (see Non-Patent Document 3).
JP-A-4-189822 JP-A-8-301999 JP-A-5-39350 JP-A-5-39352 JP-A-5-39353 JP-A-5-70566 JP-A-5-295099 JP-A-5-295098 JP-A-5-310898 JP-A-8-239461 Japanese Patent Laid-Open No. 2005-27533 Appl. Microbiol Biotechnol No. 5 1 (1999) 545-552 Journal of the American Chemical Society No. 116 (1994) 399-400 Biotechnology and Bioengineering Symp. No. 17 (1986) 355-363

  This invention is made | formed in view of the said situation, The objective is to provide the manufacturing method of polyester which can obtain a target molecular weight with an industrially advantageous and efficient manufacturing method.

  As a result of intensive studies to solve the above problems, the present inventors have improved the polymerization rate when a sulfur atom having a content within a specific range is present during polyester production, but the acid concentration in the reaction system is high. As a result, it was found that there was a problem that corrosion of the reactor occurred. Therefore, as a result of further earnest research, it was found that by performing the reaction in the coexistence of sulfur and nitrogen atoms, the polymerization rate was maintained and corrosion of the reaction vessel was prevented, and the present invention was completed. It came.

  That is, the gist of the present invention is that in the method for producing a polyester by the reaction of a diol and a dicarboxylic acid, sulfur atoms and nitrogen atoms are contained in the dicarboxylic acid in a ratio of sulfur atom weight / nitrogen atom weight greater than 0 in terms of atoms. It exists in the manufacturing method of polyester characterized by making it exist at 10 or less.

The present invention contributes to solving environmental problems, fossil resource problems, etc., and when a resin is produced using the production method of the present invention, a resin having practical physical properties can be provided. The utility value is extremely large. In addition, the polyester of the present invention produced from biomass resources is surprisingly better biodegradable than the polyester produced from petroleum-derived raw materials having the same structure. Further, it is possible to provide a high molecular weight polyester that is excellent in biodegradability, has little coloration, and has excellent mechanical properties and can be used in various applications.

  In addition, since a so-called diol unit or dicarboxylic acid unit obtained from a natural material vegetated under the present global environment in the atmosphere by a technique such as fermentation is used as a polyester monomer, the raw material can be obtained at a very low cost. Since plant raw material production can be dispersed and diversified in various places, the supply of raw materials is very stable, and the difference in the mass balance of carbon dioxide absorption and emission is made because it is done in the global environment of the atmosphere. It is relatively balanced. Moreover, it can be recognized as an environmentally friendly and safe polyester.

  Such a polyester of the present invention not only can be evaluated in the physical properties, structure and function of the material, but also potentially has the feasibility of a recycling society including recycling that cannot be expected at all from polyester derived from fossil fuels. Have advantages. This is to provide a new polyester manufacturing process that is different from conventional fossil fuel-dependent orientation, so the use of plastic materials from a completely new perspective, a new second stage plastic. And contribute significantly to development. The polyester of the present invention is less likely to generate harmful substances and offensive odors even if the soil dumping is stopped and incinerated. You can overcome such problems.

  Hereinafter, the present invention will be described in detail.

<Polyester>
The polyester which is the object of the present invention contains a dicarboxylic acid unit and a diol unit as essential components. In the present invention, it is preferable that at least one of the dicarboxylic acid and diol constituting the dicarboxylic acid unit and the diol unit is derived from biomass resources.

  Of the polyesters derived from these biomass resources, aliphatic polyesters composed of aliphatic dicarboxylic acid units and aliphatic diol units are preferred.

(1) Dicarboxylic acid unit
Examples of the dicarboxylic acid constituting the dicarboxylic acid unit include aromatic dicarboxylic acids, aliphatic dicarboxylic acids, and mixtures thereof. Examples of the aromatic dicarboxylic acid include terephthalic acid and isophthalic acid, and examples of the aromatic dicarboxylic acid derivative include lower alkyl esters of aromatic dicarboxylic acid, specifically methyl ester, ethyl ester, propyl ester, and butyl. Examples include esters. Among these, terephthalic acid is preferable as the aromatic dicarboxylic acid, and dimethyl terephthalate is preferable as the derivative of the aromatic dicarboxylic acid. Even when the aromatic dicarboxylic acid disclosed in the present specification is used, a desired aromatic polyester can be produced by using any aromatic dicarboxylic acid such as polyester of dimethyl terephthalate and 1,4-butanediol. it can.

As the aliphatic dicarboxylic acid component, an aliphatic dicarboxylic acid or a derivative thereof is used. Specific examples of the aliphatic dicarboxylic acid include oxalic acid, succinic acid, glutaric acid, adipic acid, sebacic acid, dodecanedioic acid, dimer acid, and cyclohexanedicarboxylic acid, which usually have 2 to 40 carbon atoms. Examples include chain or alicyclic dicarboxylic acids. Examples of the aliphatic dicarboxylic acid derivative include lower alkyl esters such as methyl ester, ethyl ester, propyl ester, and butyl ester of the aliphatic dicarboxylic acid, and cyclic acid anhydrides of the aliphatic dicarboxylic acid such as succinic anhydride. Can be used. Among these, the aliphatic dicarboxylic acid is preferably adipic acid, succinic acid, dimer acid or a mixture thereof from the viewpoint of the physical properties of the resulting polymer. The aliphatic dicarboxylic acid derivatives are adipic acid and succinic acid. The methyl esters of these, or mixtures thereof, are preferred.

  These dicarboxylic acids can be used alone or in combination of two or more.

  In the present invention, these dicarboxylic acids are preferably derived from biomass resources.

  Biomass resources as used in the present invention are those in which solar light energy is converted into a form such as starch or cellulose by the photosynthetic action of plants, animals that grow by eating plants, plants or animals Includes products made by processing the body. Among these, more preferable biomass resources are plant resources, such as wood, rice straw, rice husk, rice bran, old rice, corn, sugar cane, cassava, sago palm, okara, corn cob, tapioca cass, bagasse, vegetable oil cass, straw , Buckwheat, soybeans, fats and oils, waste paper, papermaking residue, marine product residue, livestock excrement, sewage sludge, food waste and the like. Among them, plant resources such as wood, rice straw, rice husk, rice bran, old rice, corn, sugar cane, cassava, sago palm, okara, corn cob, tapioca cass, bagasse, vegetable oil residue, buckwheat, soy, oil, waste paper, paper residue Wood, rice straw, rice husk, old rice, corn, sugar cane, cassava, sago palm, straw, oil and fat, waste paper, papermaking residue, and most preferably corn, sugar cane, cassava, sago palm. These biomass resources generally contain many alkali metals and alkaline earth metals such as nitrogen atoms, Na, K, Mg, and Ca.

  These biomass resources are not particularly limited, but are induced to carbon sources through known pretreatment and saccharification processes such as chemical treatment with acids and alkalis, biological treatment with microorganisms, physical treatment, and the like. Is done. The process usually includes, for example, a micronization process by pretreatment such as chipping, scraping, or crushing biomass resources, although it is not particularly limited. If necessary, a grinding process is further included with a grinder or a mill. The biomass resources refined in this way are further guided to a carbon source through pretreatment and saccharification processes, and specific methods include acid treatment and alkali treatment with strong acids such as sulfuric acid, nitric acid, hydrochloric acid, and phosphoric acid. , Chemical methods such as ammonia freezing steam explosion method, solvent extraction, supercritical fluid treatment, oxidant treatment, etc., physical methods such as fine grinding, steam explosion method, microwave treatment, electron beam irradiation, microorganisms and enzyme treatment Biological treatment such as hydrolysis may be mentioned.

  Carbon sources derived from the above biomass resources usually include hexoses such as glucose, mannose, galactose, fructose, sorbose, tagatose, pentoses such as arabinose, xylose, ribose, xylulose, ribulose, pentose, saccharose, starch, cellulose Disaccharides and polysaccharides such as butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, monoctinic acid, arachidic acid, eicosene Fermentation of fats and oils such as acids, arachidonic acid, behenic acid, erucic acid, docosapentaenoic acid, docosahexaenoic acid, lignoceric acid, ceracolonic acid, polyalcohols such as glycerin, mannitol, xylitol, ribitol Carbohydrate are used, these glucose, fructose, xylose are preferred, and glucose is particularly preferred. As a carbon source derived from a broader plant resource, cellulose which is a main component of paper is preferable.

  Using these carbon sources, dicarboxylic acids can be produced by microbial conversion fermentation methods, chemical conversion methods including hydrolysis, dehydration reaction, hydration reaction, oxidation reaction, etc., and combinations of these fermentation methods and chemical conversion methods. Synthesized. Among these, the fermentation method by microbial conversion is preferable.

The microorganism used for microbial conversion is not particularly limited as long as it has the ability to produce dicarboxylic acid. For example, anaerobic bacteria such as Anaerobiospirillum (US Pat. No. 5,143,833), Actinobacillus (US Pat. No. 5,504,004) ), A facultative anaerobic bacterium (E. coli (J. Bacteriol., 57: 147-158) such as the genus Escherichia (US Pat. No. 5,770,435) or a mutant of a strain of E. coli (Special Table 2000) Aerobic bacteria such as the genus Corynebacterium (Japanese Patent Laid-Open No. 11-113588), the genus Bacillus, the genus Rizobium, the genus Brevibacterium, the Arthro Aerobic bacteria belonging to the genus Bacter (Japanese Patent Application Laid-Open No. 2003-235593), Bacteroides ruminicola, Bacteroides amylophilus, and the like can be used.

  More specifically, the parent strain of bacteria that can be used in the present invention is preferably a coryneform bacterium, a Bacillus bacterium, or a Rhizobium bacterium, and more preferably a coryneform bacterium. These bacteria have the ability to produce succinic acid by microbial conversion.

  Examples of coryneform bacteria include microorganisms belonging to the genus Corynebacterium, microorganisms belonging to the genus Brevibacterium, or microorganisms belonging to the genus Arthrobacter, and among these, those belonging to the genus Corynebacterium or Brevibacterium And more preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium amniagenes or Brementac fermentum bacterium. And microorganisms belonging to

  Particularly preferred specific examples of the above-mentioned bacterial parent strains include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872, Coryne And bacteria glutamicum ATCC 31831 and Brevibacterium lactofermentum ATCC 13869. Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. and Schleiffer, K. H., International Journal). of Systemic Bacteriology, 1991, vol. 41, p255-260), Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 strain are respectively corynebacterium glutamicum MJ. -233 strain and MJ-233 AB-41 strain.

  Brevibacterium flavum MJ-233 was released on April 28, 1975 at the Institute of Biotechnology, National Institute of Technology, Ministry of International Trade and Industry (currently the National Institute of Advanced Industrial Science and Technology, Patent Deposit Center). Deposited under the deposit number FERM P-3068 in Tsukuba City, 1-1-1 Higashi 1-chome, Ibaraki, Japan, and transferred to an international deposit under the Budapest Treaty on May 1, 1981, with the deposit number FERM BP-1497 Has been granted.

  Reaction conditions such as reaction temperature and pressure in microbial conversion will depend on the activity of microorganisms such as selected cells and molds, but suitable conditions for obtaining dicarboxylic acids should be selected in each case. That's fine.

  In microbial conversion, neutralizing agents are usually used because the metabolic activity of microorganisms decreases or the activity of microorganisms stops when pH decreases, and the production yield deteriorates or the microorganisms die. To do. Usually, the pH in the reaction system is measured by a pH sensor, and the pH is adjusted by adding a neutralizing agent so as to be in a predetermined pH range. There is no restriction | limiting in particular about the addition method of a neutralizing agent, Continuous addition or intermittent addition may be sufficient.

Examples of the neutralizing agent include ammonia, ammonium carbonate, urea, alkali metal hydroxide, alkaline earth metal hydroxide, alkali metal carbonate, and alkaline earth metal carbonate. Ammonia, ammonium carbonate and urea are preferred. Examples of the alkali (earth) metal hydroxide include NaOH, KOH, Ca (OH) ₂ , Mg (OH) ₂ , or a mixture thereof. Examples of the alkali (earth) metal carbonate include _{is, Na 2 CO 3, K 2} CO 3, CaCO 3, MgCO 3, NaKCO 3 etc., or the like and mixtures thereof.

  The pH value is adjusted to a range in which the activity is most effectively exhibited depending on the type of microorganisms such as fungus bodies and molds to be used. In general, the pH value is about 4 to 10, preferably about 6 to 9. It is a range.

  As a method for purifying a dicarboxylic acid obtained by a production method including a fermentation method, a method using electrodialysis, a method using an ion exchange resin, a salt exchange method, and the like are known. For example, it can be produced by using a combination of electrodialysis and water splitting steps to separate the dicarboxylate salt to produce pure acid, and further purification can be achieved by passing the product stream through a series of ion exchange columns. Alternatively, hydrolytic electrodialysis for conversion to a supersaturated solution of dicarboxylic acid may be used (US Pat. No. 5,034,105). Further, as a salt exchange method, for example, dicarboxylic acid and ammonium sulfate may be mixed and reacted with ammonium hydrogen sulfate and / or sulfuric acid at a sufficiently low pH to produce dicarboxylic acid and ammonium sulfate (Japanese Patent Publication No. 2001-514900). Publication). As a specific method using an ion exchange resin, after removing solids such as cells from a dicarboxylic acid solution by centrifugation, filtration, etc., desalting with an ion exchange resin and crystallization or column chromatography from the solution. Can be used to separate and purify the dicarboxylic acid. As another purification method, as described in JP-A-3-30685, fermented with calcium hydroxide as a neutralizing agent, precipitated and removed calcium sulfate with sulfuric acid, then strongly acidic ion exchange resin, weak base After the electrodialysis of the succinate produced by the fermentation method as described in JP-A-2-283289 or the method of treating with a basic ion exchange resin, a strongly acidic ion exchange resin, a weakly basic ion exchange resin The method of processing using is illustrated. Furthermore, the methods described in US Pat. No. 6,284,904 and JP-A No. 2004-196768 are also preferably used. That is, in the present invention, any purification method may be used, such as the method using electrodialysis, the method using ion exchange resin, the method of treating with an acid such as sulfuric acid, water, alcohol, carvone. By repeating the crystallization using an acid or a mixture thereof and any unit operations described in the above-mentioned known documents such as washing, filtration, drying, etc. and the reference examples of the present invention in any combination, and repeatedly as necessary. A purified monomer raw material suitable for the present invention can be produced. Among these, the ion exchange method or the salt exchange method is particularly preferable in terms of cost and efficiency, and the salt exchange method is particularly preferable in terms of industrial productivity.

  It is usually necessary to reduce the amount of impurity nitrogen compounds and metal cations contained in the dicarboxylic acid by purification in order to obtain a practical polymer.

  The dicarboxylic acid derived from the biomass resource by the above-mentioned method contains nitrogen atoms as impurities due to the purification process including the biomass resource origin, fermentation treatment and neutralization step with acid. Specifically, nitrogen atoms such as amino acids, proteins, ammonium salts, urea, and fermenting bacteria are included.

  The nitrogen atom content contained in the dicarboxylic acid derived from the biomass resource by the above-mentioned method is usually 2000 wtppm or less, preferably 1000 wtppm or less, more preferably, in terms of atoms with respect to the dicarboxylic acid mass. 100 wtppm or less, most preferably 50 wtppm or less. The lower limit is usually 0.01 wtppm or more, preferably 0.1 wtppm or more for reasons of economic efficiency of the purification process.

  In the present invention, wtppm means mass ppm.

  The nitrogen atom content is measured by a known method such as elemental analysis or an amino acid analyzer and a method of separating amino acids and ammonia in a sample under biological amino acid separation conditions and detecting them by ninhydrin color development. Value.

  Use of a dicarboxylic acid having a nitrogen atom content in the above range is advantageous in reducing coloring of the resulting polyester. It also has the effect of suppressing the delay of the polyester polymerization reaction.

  As a specific method for efficiently reducing the amount of impurity ammonia contained in the dicarboxylic acid, there is a reaction crystallization method using a weakly acidic organic acid having a pH higher than that of the target dicarboxylic acid.

  Moreover, when using the dicarboxylic acid manufactured by the fermentation method, a sulfur atom may be contained by the refinement | purification process including the neutralization process by an acid. Specifically, examples of the impurities containing sulfur atoms include sulfuric acid, sulfate, sulfurous acid, organic sulfonic acid, and organic sulfonate.

  The sulfur atom content contained in the dicarboxylic acid is, in terms of atoms, based on the dicarboxylic acid mass, the upper limit is usually 100 wtppm or less, preferably 20 wtppm or less, more preferably the upper limit is 5 wtppm or less, and most preferably the upper limit. Is 0.5 wtppm or less. On the other hand, the lower limit is usually 0.001 wtppm or more, preferably 0.01 wtppm or more, more preferably 0.1 wtppm or more. When the amount is too large, the polymerization reaction tends to be delayed or the stability of the polymer tends to be lowered. There is also a problem of corrosion of the storage container. On the other hand, an excessively small system is a preferred form, but the purification process becomes complicated and economically disadvantageous. The sulfur atom content is a value measured by a known elemental analysis method.

  Specifically, the sulfur atom content in the present invention can be measured by causing a hydrogen peroxide solution to absorb a sulfur content generated by burning a sample and using an ion chromatograph for the sulfate ion.

  In the present invention, when using the dicarboxylic acid derived from biomass resources obtained by the above-mentioned method as a polyester raw material, the oxygen concentration in the tank for storing the dicarboxylic acid connected to the polymerization system is controlled to a certain value or less. Also good. Thereby, coloring by the oxidation reaction of the nitrogen source which is an impurity of polyester can be prevented.

  A tank is usually used to control the oxygen concentration and store the raw material. However, the apparatus is not particularly limited as long as the apparatus can control the oxygen concentration other than the tank. The type of the storage tank is not specifically limited, and a known metal or those whose inner surfaces are lined with glass, resin, or the like, or a glass or resin container is used. From the viewpoint of strength, etc., those made of metal or those lining them are preferably used. As the metal tank material, known materials are used. Specifically, carbon steel, ferritic stainless steel, martensitic stainless steel such as SUS410, austenitic stainless steel such as SUS310, SUS304, and SUS316, clad Steel, cast iron, copper, copper alloy, aluminum, inconel, hastelloy, titanium and the like can be mentioned.

  The lower limit of the oxygen concentration in the dicarboxylic acid storage tank is not particularly limited with respect to the total volume of the storage tank, but is usually 0.00001 vol% or more, preferably 0.01 vol% or more. On the other hand, the upper limit is 16 vol% or less, preferably 14 vol% or less, and more preferably 12 vol% or less. When the oxygen concentration is too low, the equipment and the management process become complicated, which is economically disadvantageous. On the other hand, when the oxygen concentration is too high, coloring of the produced polymer tends to increase.

  The lower limit of the temperature in the dicarboxylic acid storage tank is usually −50 ° C. or higher, preferably 0 ° C. or higher. On the other hand, the upper limit is usually 200 ° C. or lower, preferably 100 ° C. or lower, and more preferably 50 ° C. or lower. However, the method of storing at room temperature is most preferable because there is no need for temperature control. When the temperature is too low, the storage cost tends to increase. When the temperature is too high, the dehydration reaction of carboxylic acid tends to occur simultaneously.

  The lower limit of the humidity in the dicarboxylic acid storage tank is not particularly limited, but is usually 0.0001% R.D. H. Or more, preferably 0.001% R.V. H. More preferably, 0.01% R.D. H. Or more, most preferably 0.1% R.I. H. The upper limit is 80% R.I. H. Below, preferably 60% R.I. H. Or less, more preferably 40% R.I. H. Hereinafter, more preferably 30% R.I. H. Hereinafter, most preferably 10% R.I. H. It is as follows. When the humidity is too low, the management process tends to be complicated and economically disadvantageous. When it is too high, the dicarboxylic acid adheres to the storage tank and piping, the dicarboxylic acid is blocked, and the storage tank. When the metal is made of metal, tank corrosion tends to be a problem. In particular, the tendency tends to increase as the impurity sulfur atom content increases.

  In general, the dicarboxylic acid used in the present invention is preferably less colored. The upper limit of the yellowness (YI value) of the dicarboxylic acid used in the present invention is usually 50 or less, preferably 20 or less, more preferably 10 or less, still more preferably 6 or less, and particularly preferably 4 or less. On the other hand, the lower limit thereof is not particularly limited, but is usually −20 or more, preferably −10 or more, more preferably −5 or more, particularly preferably −3 or more, and most preferably −1 or more. The use of dicarboxylic acids exhibiting a high YI value has a significant drawback in the coloration of the polymer produced. On the other hand, a dicarboxylic acid exhibiting a low YI value is a more preferable form, but it is economically disadvantageous in that it requires a very large capital investment for its production and requires a large production time. In the present invention, the YI value is a value measured by a method based on JIS K7105.

(2) Diol unit
In the present invention, the diol unit is derived from an aromatic diol and / or an aliphatic diol, and a known compound can be used, but an aliphatic diol is preferably used.

  The aliphatic diol is not particularly limited as long as it is an aliphatic and alicyclic compound having two OH groups, but the lower limit of the carbon number is 2 or more, and the upper limit is usually 10 or less, preferably 6 The following aliphatic diols are mentioned.

  Specific examples of the aliphatic diol include, for example, ethylene glycol, 1,3-propylene glycol, neopentyl glycol, 1,6-hexamethylene glycol, decamethylene glycol, 1,4-butanediol, and 1,4- And cyclohexanedimethanol. These may be used alone or as a mixture of two or more.

  Among these, ethylene glycol, 1,4-butanediol, 1,3-propylene glycol and 1,4-cyclohexanedimethanol are preferable, and among them, ethylene glycol and 1,4-butanediol are preferable. Furthermore, 1,4-butanediol is particularly preferable.

The aromatic diol is not particularly limited as long as it is an aromatic compound having two OH groups, and examples thereof include aromatic diols having a lower limit of 6 or more carbon atoms and an upper limit of usually 15 or less. . Specific examples of the aromatic diol include, for example, hydroquinone, 1,5-dihydroxynaphthalene, 4,4′-dihydroxydiphenyl, bis (p-hydroxyphenyl) methane and bis (p-hydroxyphenyl) -2,2-propane. Etc. In the present invention, the content of the aromatic diol in the total amount of diol is usually 30 mol% or less, preferably 20 mol% or less, more preferably 10 mol% or less.

  Further, both terminal hydroxy polyethers may be used by mixing with the above aliphatic diol. As both-end hydroxy polyether, the lower limit is usually 4 or more, preferably 10 or more, and the upper limit is usually 1000 or less, preferably 200 or less, more preferably 100 or less.

  Specific examples of both terminal hydroxy polyethers include diethylene glycol, triethylene glycol, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, poly 1,3-propanediol, and poly 1,6-hexamethylene glycol. . In addition, a copolymerized polyether of polyethylene glycol and polypropylene glycol can be used. The use amount of these both terminal hydroxy polyethers is an amount calculated as a content in the polyester of usually 90% by weight or less, preferably 50% by weight or less, more preferably 30% by weight or less.

  In the present invention, these diols may be derived from biomass resources. Specifically, the diol compound may be produced directly from a carbon source such as glucose by a fermentation method, or a dicarboxylic acid, dicarboxylic acid anhydride, or cyclic ether obtained by the fermentation method is converted into a diol compound by a chemical reaction. May be.

  For example, 1,4-butanediol is chemically synthesized from succinic acid, succinic anhydride, succinic ester, maleic acid, maleic anhydride, maleic ester, tetrahydrofuran, γ-butyrolactone, etc. to produce 1,4-butanediol. Alternatively, 1,4-butanediol may be produced from 1,3-butadiene obtained by a fermentation method. Among these, a method of hydrogenating succinic acid with a reduction catalyst to obtain 1,4-butanediol is efficient and preferable.

Examples of catalysts for hydrogenating succinic acid include Pd, Ru, Re, Rh, Ni, Cu, Co and compounds thereof, and more specifically, Pd / Ag / Re, Ru / Ni / Co / ZnO. Cu / Zn oxide, Cu / Zn / Cr oxide, Ru / Re, Re / C, Ru / Sn, Ru / Pt / Sn, Pt / Re / alkali, Pt / Re, Pd / Co / Re, Cu / Si, Cu / Cr / Mn, ReO / CuO / ZnO, CuO / CrO, Pd / Re, Ni / Co, Pd / CuO / CrO ₃ , phosphoric acid Ru, Ni / Co, Co / Ru / Mn, Cu / Pd / KOH and Cu / Cr / Zn are mentioned. Among these, Ru / Sn or Ru / Pt / Sn is preferable in terms of catalytic activity.

  Furthermore, a method of producing a diol compound from biomass resources by a combination of known organic chemical catalytic reactions is also actively used. For example, when pentose is used as a biomass resource, a diol such as butanediol can be easily produced by a combination of a known dehydration reaction and catalytic reaction.

  The diol derived from the biomass resource may contain nitrogen atoms as impurities due to the purification process including the biomass resource origin, fermentation treatment, and neutralization step with acid. In this case, specifically, nitrogen atoms derived from amino acids, proteins, ammonia, urea, and fermenting bacteria are included.

  The nitrogen atom content contained in the diol produced by the above-mentioned method is 2,000 wtppm or less, preferably 1000 wtppm or less, more preferably 100 wtppm or less, and most preferably 50 wtppm in terms of atoms with respect to the diol mass. It is as follows. The lower limit is not particularly limited, but is usually 0.01 wtppm or more, preferably 0.1 wtppm or more for reasons of economic efficiency of the purification process.

When using the diol manufactured by the fermentation method, a sulfur atom may be contained by the refinement | purification process including the neutralization process by an acid. In this case, specific examples of impurities containing sulfur atoms include sulfuric acid, sulfurous acid, and organic sulfonates.

  The sulfur atom content contained in the diol is usually 100 wtppm or less, preferably 10 wtppm or less, more preferably 5 wtppm or less, and most preferably 0 upper limit in terms of atoms relative to the diol mass. .5 wtppm or less. On the other hand, the lower limit is not particularly limited, but is usually 0.001 wtppm or more, preferably 0.01 wtppm or more, more preferably 0.1 wtppm or more. When the amount is too large, the polymerization reaction tends to be delayed, or the stability of the polymer to be produced tends to decrease. On the other hand, the smaller the sulfur atom content, the more preferable it is. However, the purification process becomes complicated and economically disadvantageous. The sulfur atom content is a value measured by a known elemental analysis method.

  In the present invention, when using the biomass resource-derived diol obtained by the above-mentioned method as a polyester raw material, in order to suppress the coloration of the polyester due to the impurities, in the tank for storing the diol linked to the polymerization system The oxygen concentration and temperature may be controlled. By this control, the oxidation reaction of the diol promoted by the impurities is suppressed, and the coloring of the impurities themselves and the coloring of the polyester by the diol oxidation product can be prevented.

  A tank is usually used to control the oxygen concentration and store the raw material. However, the apparatus is not particularly limited as long as the apparatus can control the oxygen concentration other than the tank. The type of the storage tank is not specifically limited, and a known metal or those whose inner surfaces are lined with glass, resin, or the like, or a glass or resin container is used. From the viewpoint of strength, etc., those made of metal or those lining them are preferably used. As the material for the metal tank, known materials are used. Specifically, carbon steel, ferritic stainless steel, martensitic stainless steel such as SUS410, austenitic stainless steel such as SUS310, SUS304, and SUS316, clad Steel, cast iron, copper, copper alloy, aluminum, inconel, hastelloy, titanium and the like can be mentioned.

  The lower limit of the diol concentration in the storage tank is not particularly limited, but is usually 0.00001 vol% or more, preferably 0.0001 vol% or more, and more preferably 0.001 vol%. As described above, it is most preferably 0.01 vol% or more, and the upper limit is usually 10 vol% or less, preferably 5 vol% or less, more preferably 1 vol% or less, and most preferably 0.1 vol% or less. If the oxygen concentration is too low, the management process becomes complicated and tends to be economically disadvantageous. If it is too high, the coloring of the polymer by the oxidation reaction product of the diol tends to increase.

  The lower limit of the diol storage temperature in the storage tank is usually 15 ° C. or higher, preferably 30 ° C. or higher, more preferably 50 ° C. or higher, most preferably 100 ° C. or higher, and the upper limit is 230 ° C. or lower. Is 200 ° C. or lower, more preferably 180 ° C. or lower, and most preferably 160 ° C. or lower. If the temperature is too low, it takes time to raise the temperature during the production of the polyester, and the polyester production tends to be economically disadvantageous, and may solidify depending on the type of diol. On the other hand, if it is too high, the diol vaporization requires a high-pressure storage facility, which is not only economically disadvantageous but also tends to increase the degradation of the diol.

  The pressure in the diol storage tank is usually atmospheric pressure (normal pressure). If the pressure is too low or too high, the management facilities become complicated and economically disadvantageous.

  In the present invention, the upper limit of the content of the oxidation product of the diol used for producing a polymer having a good hue is usually 10,000 ppm or less, preferably 5000 ppm or less, more preferably 3000 ppm or less, most preferably 2000 ppm or less in the diol. is there. On the other hand, the lower limit is not particularly limited, but is usually 1 ppm or more, preferably 10 ppm or more, more preferably 100 ppm or more for reasons of economic efficiency of the purification step.

  In the present invention, the diol is usually used as a polyester raw material after a purification step by distillation.

  The polyester of the present invention includes all polyesters produced by the reaction of components mainly composed of various compounds belonging to the categories of dicarboxylic acid units and diol units listed above, but the polyesters of the present invention are typical ones. As specific examples, the following polyesters can be exemplified.

  Polyesters using succinic acid include succinic acid and ethylene glycol polyester, succinic acid and 1,3-propylene glycol polyester, succinic acid and neopentyl glycol polyester, succinic acid and 1,6-hexamethylene glycol. And polyesters of succinic acid and 1,4-butanediol, polyesters of succinic acid and 1,4-cyclohexanedimethanol, and the like.

  As polyesters using oxalic acid, oxalic acid and ethylene glycol polyester, oxalic acid and 1,3-propylene glycol polyester, oxalic acid and neopentyl glycol polyester, oxalic acid and 1,6-hexamethylene glycol And polyester of oxalic acid and 1,4-butanediol, polyester of oxalic acid and 1,4-cyclohexanedimethanol, and the like.

  Examples of polyesters using adipic acid include adipic acid and ethylene glycol polyester, adipic acid and 1,3-propylene glycol polyester, adipic acid and neopentyl glycol polyester, adipic acid and 1,6-hexamethylene glycol And polyester of adipic acid and 1,4-butanediol and 1,4-cyclohexanedimethanol of adipic acid.

  In addition, a polyester in which the above dicarboxylic acid is combined is also a preferable combination, a polyester of succinic acid, adipic acid and ethylene glycol, a polyester of succinic acid, adipic acid and 1,4-butanediol, a terephthalic acid, adipic acid and 1, Examples include 4-butanediol polyester and terephthalic acid, succinic acid, and 1,4-butanediol polyester.

  The present invention is also directed to a copolymer polyester in which a copolymer component is added as a third component in addition to the diol component and the dicarboxylic acid component. Specific examples of the copolymer component include a bifunctional oxycarboxylic acid, a trifunctional or higher polyhydric alcohol, a trifunctional or higher polyvalent carboxylic acid and / or an anhydride thereof to form a crosslinked structure, and Examples thereof include at least one polyfunctional compound selected from the group consisting of trifunctional or higher functional oxycarboxylic acids. Among these copolymer components, a bifunctional and / or trifunctional or higher functional oxycarboxylic acid is particularly preferably used because a copolymer polyester having a high degree of polymerization tends to be easily produced. Among them, the use of a trifunctional or higher functional oxycarboxylic acid is the most preferable method because a polyester having a high degree of polymerization can be easily produced in a very small amount without using a chain extender described later.

Specific examples of the bifunctional oxycarboxylic acid include lactic acid, glycolic acid, hydroxybutyric acid, hydroxycaproic acid, 2-hydroxy3,3-dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid, and 2-hydroxyisocaprone. Examples of the acid include esters of oxycarboxylic acids, lactones, and derivatives of oxycarboxylic acid polymers. These oxycarboxylic acids can be used alone or as a mixture of two or more. When optical isomers exist in these, any of D-form, L-form, and racemic form may be sufficient, and a form may be a solid, a liquid, or aqueous solution. Of these, readily available lactic acid or glycolic acid is particularly preferable. The form is preferably 30 to 95% because an aqueous solution can be easily obtained. When a bifunctional oxycarboxylic acid is used as a copolymerization component for the purpose of easily producing a polyester having a high degree of polymerization, a desired copolymerized polyester can be produced by adding any bifunctional oxycarboxylic acid during polymerization. Specifically, the lower limit of the amount used to exhibit the effect is usually 0.02 mol% or more, preferably 0.5 mol% or more, more preferably 1.0 mol%, based on the raw material monomer. That's it. On the other hand, the upper limit of the amount used is usually 30 mol% or less, preferably 20 mol% or less, more preferably 10 mol% or less.

  Specifically, when the polyester is used, when lactic acid is used as the bifunctional oxycarboxylic acid, for example, a copolymer polyester of succinic acid-1,4-butanediol-lactic acid or succinic acid-adipic acid-1,4 is used. -Copolymerized polyester of butanediol-lactic acid. When glycolic acid is used, for example, it is a copolymer polyester of succinic acid-1,4-butanediol-glycolic acid.

  Specific examples of the trifunctional or higher polyhydric alcohol include glycerin, trimethylolpropane, pentaerythritol and the like, and they can be used alone or as a mixture of two or more.

  When pentaerythritol is used as a trifunctional or higher functional polyhydric alcohol as a copolymerization component, for example, succinic acid-1,4-butanediol-pentaerythritol copolymerized polyester or succinic acid-adipic acid-1,4-butanediol- It becomes a copolyester of pentaerythritol. A desired copolyester can be produced by arbitrarily changing a trihydric or higher polyhydric alcohol. High molecular weight polyesters obtained by chain extending (coupling) these copolymerized polyesters also belong to the category of the polyester of the present invention.

  Specific examples of the tri- or higher functional polycarboxylic acid or anhydride thereof include propanetricarboxylic acid, pyromellitic anhydride, benzophenonetetracarboxylic anhydride, cyclopentatetracarboxylic anhydride, and the like. It can also be used as a mixture of two or more.

  Specific examples of the tri- or higher functional oxycarboxylic acid include malic acid, hydroxyglutaric acid, hydroxymethylglutaric acid, tartaric acid, citric acid, hydroxyisophthalic acid, hydroxyterephthalic acid, and the like. It can also be used as a mixture of In particular, malic acid, tartaric acid, citric acid and a mixture thereof are preferable because of easy availability.

  Specifically, when the embodiment of the polyester is shown, when malic acid is used as the trifunctional oxycarboxylic acid of the copolymer component, for example, a copolymer polyester of succinic acid-1,4-butanediol-malic acid, succinic acid- Adipic acid-1,4-butanediol-malic acid copolyester, succinic acid-1,4-butanediol-malic acid-tartaric acid copolyester, succinic acid-adipic acid-1,4-butanediol-apple Acid-tartaric acid copolyester, succinic acid-1,4-butanediol-malic acid-citric acid copolyester, succinic acid-adipic acid-1,4-butanediol-malic acid-citric acid copolyester It becomes. A desired copolyester can be produced by arbitrarily changing the trifunctional oxycarboxylic acid.

Of course, in combination with a bifunctional oxycarboxylic acid, for example, succinic acid-1,4-butanediol-malic acid-lactic acid copolymer polyester, succinic acid-adipic acid-1,4-butanediol-malic acid -Copolymerized polyester of lactic acid, succinic acid-1,4-butanediol-malic acid-tartaric acid-lactic acid copolymerized polyester, succinic acid-adipic acid-1,4-butanediol-malic acid-tartaric acid-lactic acid copolymerized Polyester, succinic acid-1,4-
A copolymer polyester of butanediol-malic acid-citric acid-lactic acid, and a copolymer polyester of succinic acid-adipic acid-1,4-butanediol-malic acid-citric acid-lactic acid.

  Since the amount of the above-mentioned trifunctional or higher polyfunctional compound unit causes generation of gel, the upper limit is usually 5 mol% or less, preferably 100 mol% of all monomer units constituting the polyester. Is 1 mol% or less, more preferably 0.50 mol% or less, and particularly preferably 0.3 mol% or less. On the other hand, when a tri- or higher functional compound is used as a copolymerization component for the purpose of easily producing a polyester having a high degree of polymerization, the lower limit of the amount used to achieve its effect is usually 0.0001 mol% or more, Preferably, it is 0.001 mol% or more, more preferably 0.005 mol% or more, and particularly preferably 0.01 mol% or more.

  Moreover, the minimum of melting | fusing point of polyester is 50 degreeC or more normally, Preferably, it is 60 degreeC or more, More preferably, it is 80 degreeC or more. In the case of aliphatic polyester, the upper limit is 140 ° C. or lower, preferably 130 ° C. or lower.

  The polyester of the present invention may use a chain extender such as a carbonate compound or a diisocyanate compound, but the amount thereof is usually 10 mol of carbonate bond and urethane bond with respect to all monomer units constituting the polyester. % Or less. However, when the polyester of the present invention is used as a biodegradable resin, the presence of diisocyanate or carbonate bond may inhibit biodegradability. The carbonate bond is less than 1 mol%, preferably 0.5 mol% or less, more preferably 0.1 mol% or less, and the urethane bond is less than 0.06 mol%, preferably less than 0.06 mol%, based on the body unit. It is at most 01 mol%, more preferably at most 0.001 mol%.

  Specific examples of the carbonate compound include diphenyl carbonate, ditolyl carbonate, bis (chlorophenyl) carbonate, m-cresyl carbonate, dinaphthyl carbonate, dimethyl carbonate, diethyl carbonate, dibutyl carbonate, ethylene carbonate, diamyl carbonate, and dicyclohexyl. Examples include carbonate. In addition, carbonate compounds composed of the same or different hydroxy compounds derived from hydroxy compounds such as phenols and alcohols can be used.

  Specific examples of the diisocyanate compound include 2,4-tolylene diisocyanate, a mixture of 2,4-tolylene diisocyanate and 2,6-tolylene diisocyanate, diphenylmethane diisocyanate, and 1,5-naphthylene diisocyanate. And known diisocyanates such as xylylene diisocyanate, hydrogenated xylylene diisocyanate, hexamethylene diisocyanate, and isophorone diisocyanate.

  Moreover, you may use a dioxazoline, a silicate ester, etc. as another chain extender. Specific examples of the silicate ester include tetramethoxysilane, dimethoxydiphenylsilane, dimethoxydimethylsilane, and diphenyldihydroxylane.

Silicate esters are not particularly limited in terms of use for reasons of environmental protection and safety, but they are used in small quantities because they can be cumbersome and affect the polymerization rate. Sometimes it is better. Therefore, this content is preferably 0.1 mol% or less, more preferably 10 ⁻⁵ mol% or less, based on all monomer units constituting the polyester.

  Thus, the polyester of the present invention is a generic term for polyesters, polyesters, copolymer polyesters, chain-extended (coupled) high molecular weight polyesters, and modified polyesters.

  In the present invention, a polyester containing substantially no chain extender is preferred. However, in order to increase the melt tension, a small amount of peroxide may be added as long as a low toxicity compound is added.

  In the present invention, the polyester terminal group may be sealed with carbodiimide, an epoxy compound, a monofunctional alcohol or carboxylic acid.

  Examples of the carbodiimide compound include compounds having one or more carbodiimide groups in the molecule (including polycarbodiimide compounds). Specifically, as the monocarbodiimide compound, dicyclohexylcarbodiimide, diisopropylcarbodiimide, dimethylcarbodiimide, diisobutylcarbodiimide, Examples include dioctylcarbodiimide, t-butylisopropylcarbodiimide, diphenylcarbodiimide, di-t-butylcarbodiimide, di-β-naphthylcarbodiimide, N, N′-di-2,6-diisopropylphenylcarbodiimide and the like. As the polycarbodiimide compound, those having a degree of polymerization of usually 2 or more, preferably 4 or more and an upper limit of usually 40 or less, preferably 30 or less, are used, U.S. Pat. No. 2,941,956, Japanese Patent Publication No. 47-33279, J. Pat. Org. Chem. 28, p2069-2075 (1963), and Chemical Review 1981, 81, No. 4, p. And those produced by the method described in 619-621 and the like.

  Examples of the organic diisocyanate that is a raw material for producing the polycarbodiimide compound include aromatic diisocyanates, aliphatic diisocyanates, alicyclic diisocyanates, and mixtures thereof. Specifically, 1,5-naphthalene diisocyanate, 4 , 4'-diphenylmethane diisocyanate, 4,4'-diphenyldimethylmethane diisocyanate, 1,3-phenylene diisocyanate, 1,4-phenylene diisocyanate, 2,4-tolylene diisocyanate, 2,6-tolylene diisocyanate, 2,4 A mixture of tolylene diisocyanate and 2,6-tolylene diisocyanate, hexamethylene diisocyanate, cyclohexane-1,4-diisocyanate, xylylene diisocyanate, isophorone diisocyanate And 4,4'-dicyclohexylmethane diisocyanate, methylcyclohexane diisocyanate, tetramethylxylylene diisocyanate, 2,6-diisopropylphenyl isocyanate, 1,3,5-triisopropylbenzene-2,4-diisocyanate, etc. .

  Specific examples of industrially available polycarbodiimides include Carbodilite HMV-8CA (Nisshinbo), Carbodilite LA-1 (Nisshinbo), Starbazole P (Rhein Chemie), Starbazole P100 (Rhein Chemie) and the like. Is done.

  The carbodiimide compound can be used alone, but a plurality of compounds can also be mixed and used.

<Production method of polyester>
The production of a polyester mainly composed of a diol unit and a dicarboxylic acid unit can be carried out by a known technique except that the reaction is carried out in the presence of a sulfur atom and a nitrogen atom at a specific ratio. The polymerization reaction at the time of producing this polyester can set appropriate conditions conventionally employed and is not particularly limited. Specifically, in the case of introducing the dicarboxylic acid component and the diol component, and further an oxycarboxylic acid unit or a trifunctional or higher component, an esterification reaction between the dicarboxylic acid component including these components and the diol component, and It can be produced by a general method of melt polymerization, such as a polycondensation reaction under reduced pressure after transesterification, or a known solution heating dehydration condensation method using an organic solvent. From the viewpoints of performance and simplicity of the production process, a melt polymerization method performed in the absence of a solvent is preferable.

In the present invention, it is essential to carry out the reaction under such conditions that a sulfur atom and a nitrogen atom are present in the polyester at the following specific ratio.

  In the present invention, the upper limit of the atomic weight ratio (sulfur atom weight / nitrogen atom weight) in terms of atoms of sulfur atom and nitrogen atom is usually 100 or less, preferably 10 or less, more preferably 1 or less, and the lower limit is Usually, it is larger than 0, preferably 0.000001 or more, more preferably 0.00001 or more, and further preferably 0.0001 or more. When this ratio is too high, there is a tendency for the polymerization reaction to be delayed due to corrosion of the reaction vessel or diol modification, and when it is too low, the coloring of the polyester or the delay of the polymerization reaction tends to be a problem. .

  Specifically, the amount of each atom present in the reaction system is a sulfur atom content in the reaction system in a mass ratio to the polyester, and the upper limit is usually 100 wtppm or less, preferably 20 wtppm or less, more preferably. The upper limit is 5 wtppm or less, and most preferably the upper limit is 0.5 ppm or less. On the other hand, the lower limit is not particularly limited, but is usually greater than 0 wtppm, preferably 0.001 wtppm or more, more preferably 0.01 wtppm or more, and further preferably 0.1 wtppm or more. When the amount is too large, there is a tendency that the reaction of the reaction vessel is delayed, the polymerization reaction is delayed due to the modification of the diol, and the stability of the polymer to be produced is lowered. On the other hand, if the amount is too small, the process of refining and managing the copolymer component becomes complicated, which is economically disadvantageous.

  The nitrogen atoms present in the reaction system are in a mass ratio to the polyester, and the upper limit is usually 2000 wtppm or less, preferably 1000 wtppm or less, more preferably 100 wtppm or less, and most preferably 50 wtppm or less. The lower limit is not particularly limited, but is usually greater than 0 wtppm, preferably 0.01 wtppm or more, more preferably 0.1 wtppm or more for reasons of economic efficiency of the purification process. When there are too many nitrogen atoms, there is a tendency that the coloring of the polyester and the delay of the polymerization reaction become prominent, and when it is too small, there are problems such as corrosion of the reaction tank, the delay of the polymerization reaction, and a decrease in the stability of the polymer to be produced. Tend to occur.

  In the present invention, in order to carry out the reaction in the presence of sulfur atoms and nitrogen atoms at the above-mentioned ratio, the following methods are mentioned, but there is no particular limitation, and there is a specific ratio of sulfur atoms and nitrogen atoms in the reaction system. As long as it exists.

  For example, as a method for causing nitrogen atoms and sulfur atoms to exist in the reaction system within the above range, (i) a method of adding a compound containing sulfur atoms and / or nitrogen atoms during polymerization, and (ii) a raw material monomer. And a method using a dicarboxylic acid and / or diol containing a nitrogen atom and / or a sulfur atom. However, when biomass resources are used as the monomer, the latter method is preferred for the reason of simplifying the production process.

(i) When using the method of adding a compound containing a sulfur atom and a nitrogen atom during the polymerization, the following compounds may be appropriately combined to give a predetermined ratio. The timing for adding the compound is not particularly limited as long as it is before the polyester is extracted from the polymerization reaction tank.
Examples of the compound containing a nitrogen atom include amino acids, proteins, ammonia, ammonium salts, urea, and mixtures thereof. Among these, ammonium salts are preferable because the resulting polyester has relatively little coloration. A representative compound is an ammonium salt of a dicarboxylic acid. Two or more of these compounds may be used.

  Examples of the compound containing a sulfur atom include sulfuric acid, sulfate, sulfurous acid, organic sulfonic acid, organic sulfonate, and mixtures thereof. Among these, sulfuric acid, Preference is given to sulfates such as sodium sulfate, magnesium sulfate and calcium sulfate, organic sulfonic acids such as tolsulfonic acid, and organic acid salts such as sodium tolsulfonic acid. Two or more of these compounds may be used.

A compound having both a nitrogen atom and a sulfur atom is also preferably used, and a typical example thereof is ammonium sulfate. (ii) Examples of the method using dicarboxylic acid and / or diol containing nitrogen atoms and / or sulfur atoms as raw material monomers include the above-described methods using dicarboxylic acid and / or diol derived from biomass resources.

  In order to control a predetermined ratio of sulfur atoms and nitrogen atoms in the reaction system, (i) a method of adding a compound containing sulfur atoms and / or nitrogen atoms during the polymerization, and (ii) a raw material You may combine suitably the method using the dicarboxylic acid and / or diol containing a nitrogen atom and / or a sulfur atom as a monomer.

  The amount of diol used in the production of the polyester is substantially equimolar with respect to 100 mol of the dicarboxylic acid or derivative thereof, but in general during the esterification and / or transesterification and / or polycondensation reactions. Is used in excess of 0.1 to 20 mol%.

  The polycondensation reaction is preferably performed in the presence of a polymerization catalyst. The addition timing of the polymerization catalyst is not particularly limited as long as it is before the polycondensation reaction, and it may be added when the raw materials are charged, or may be added at the start of pressure reduction.

  As the polymerization catalyst, in general, a compound containing a group 1 to group 14 metal element excluding hydrogen and carbon in the periodic table can be used. Specifically, at least one metal selected from the group consisting of titanium, zirconium, tin, antimony, cerium, germanium, zinc, cobalt, manganese, iron, aluminum, magnesium, calcium, strontium, sodium, and potassium. And compounds containing an organic group such as carboxylate, alkoxy salt, organic sulfonate, or β-diketonate salt, and inorganic compounds such as metal oxides and halides described above, and mixtures thereof.

  Among these, metal compounds containing titanium, zirconium, germanium, zinc, aluminum, magnesium and calcium, and mixtures thereof are preferable, and among these, titanium compounds and germanium compounds are particularly preferable. In addition, the catalyst is preferably a compound that is liquid at the time of polymerization or that dissolves in an ester low polymer or polyester because the polymerization rate increases when it is melted or dissolved at the time of polymerization.

The titanium compound is preferably a tetraalkyl titanate, specifically, tetra-n-propyl titanate, tetraisopropyl titanate, tetra-n-butyl titanate, tetra-t-butyl titanate, tetraphenyl titanate, tetracyclohexyl titanate, tetra Examples include benzyl titanate and mixed titanates thereof. In addition, titanium (oxy) acetylacetonate, titanium tetraacetylacetonate, titanium (diisoproxide) acetylacetonate, titanium bis (ammonium lactate) dihydroxide, titanium bis (ethylacetoacetate) diisopropoxide, titanium (triethanolaminate) ) Isopropoxide, polyhydroxytitanium stearate, titanium lactate, titanium triethanolamate, butyl titanate dimer and the like are also preferably used. Furthermore, titanium oxide and composite oxides containing titanium and silicon (for example, titania / silica composite oxide (product name: C-94) manufactured by Acordis Industrial Fibers) are preferably used. Among these, tetra-n-propyl titanate, tetraisopropyl titanate and tetra-n-butyl titanate, titanium (oxy) acetylacetonate, titanium tetraacetylacetonate, titanium bis (ammonium lactate) dihydroxide, polyhydroxytitanium stearate Rate, titanium lactate, butyl titanate dimer, titanium oxide, titania / silica composite oxide (for example, product name: C-94 manufactured by Acordis Industrial Fibers), tetra-n-butyl titanate, titanium (oxy) acetylacetate Nate, titanium tetraacetylacetonate, polyhydroxytitanium stearate, titanium lactate, butyl titanate dimer, titania / silica composite oxide (for example, Aco dis Indus
Trial Fibers product name: C-94) is more preferred, especially tetra-n-butyl titanate, polyhydroxy titanium stearate, titanium (oxy) acetyl acetonate, titanium tetraacetyl acetonate, titania / silica composite oxidation. (For example, product name: C-94 manufactured by Acordis Industrial Fibers) is preferable.

  Specific examples of the zirconium compound include zirconium tetraacetate, zirconium acetylate hydroxide, zirconium tris (butoxy) stearate, zirconyl diacetate, zirconium oxalate, zirconyl oxalate, potassium potassium oxalate, and polyhydroxyzirconium. Examples include stearate, zirconium ethoxide, zirconium tetra-n-propoxide, zirconium tetraisopropoxide, zirconium tetra-n-butoxide, zirconium tetra-t-butoxide, zirconium tributoxyacetylacetonate and mixtures thereof. Furthermore, zirconium oxide and composite oxides containing, for example, zirconium and silicon are also preferably used. Among these, zirconyl diacetate, zirconium tris (butoxy) stearate, zirconium tetraacetate, zirconium acetate acetate, zirconium ammonium oxalate, zirconium potassium oxalate, polyhydroxyzirconium stearate, zirconium tetra-n- Propoxide, zirconium tetraisopropoxide, zirconium tetra-n-butoxide, zirconium tetra-t-butoxide are preferred, zirconyl diacetate, zirconium tetraacetate, zirconium acetate acetate hydroxide, zirconium tris (butoxy) stearate, sulphur Zirconium ammonium acid, zirconium tetra-n-propoxide, zirconium tetra-n-butoxide are more preferred, Preferred for reasons of zirconium tris (butoxy) stearate is easily obtained polyester having a high degree of polymerization without colored.

  Specific examples of the germanium compound include inorganic germanium compounds such as germanium oxide and germanium chloride, and organic germanium compounds such as tetraalkoxygermanium. In view of price and availability, germanium oxide, tetraethoxygermanium, tetrabutoxygermanium, and the like are preferable, and germanium oxide is particularly preferable.

  The amount of catalyst added in the case of using a metal compound as the polymerization catalyst, the lower limit value is usually 5 wtppm or more, preferably 10 wtppm or more, and the upper limit value is usually 30000 wtppm or less, preferably 1000 wtppm or less as the metal atom mass with respect to the produced polyester. More preferably, it is 250 wtppm or less, and particularly preferably 130 wtppm or less. If too much catalyst is used, it is not only economically disadvantageous but also lowers the thermal stability of the polymer. Conversely, if it is too little, the polymerization activity is lowered, and as a result, the polymer decomposes during polymer production. Is more likely to be triggered.

  The lower limit of the esterification reaction and / or transesterification reaction temperature between the dicarboxylic acid component and the diol component is usually 150 ° C or higher, preferably 180 ° C or higher, and the upper limit is usually 260 ° C or lower, preferably 250 ° C or lower. The reaction atmosphere is usually an inert gas atmosphere such as nitrogen or argon. The reaction pressure is usually normal pressure to 10 kPa, but normal pressure is preferred.

  The lower limit of the reaction time is usually 1 hour or longer, and the upper limit is usually 10 hours or shorter, preferably 4 hours or shorter.

Polycondensation reaction after the esterification reaction and / or ester exchange reaction of a dicarboxylic acid component and a diol component, the pressure, the lower limit is usually 0.01 × 10 ³ Pa or more, preferably 0.05 × 1 0 ³ Pa or more The upper limit is usually 1.4 × 10 ³ Pa or less, preferably 0.4 × 10 ³ Pa or less. At this time, the lower limit of the reaction temperature is usually 150 ° C. or higher, preferably 180 ° C. or higher, and the upper limit is usually 260 ° C. or lower, preferably 250 ° C. or lower. The lower limit of the reaction time is usually 2 hours or more, and the upper limit is usually 15 hours or less, preferably 10 hours or less.

  In the present invention, a known vertical or horizontal stirred tank reactor can be used as a reaction apparatus for producing polyester. For example, using the same or different reaction apparatus, it is carried out in two stages, that is, a step of esterification and / or transesterification of melt polymerization and a step of polycondensation under reduced pressure. A method of using a stirred tank reactor equipped with a evacuation pipe for decompression to be connected can be mentioned. In addition, a method in which a condenser is connected between the vacuum exhaust pipe connecting the vacuum pump and the reactor, and the volatile components and unreacted dicarboxylic acid generated during the polycondensation reaction in the condenser is preferred. Used.

  In the present invention, as a method for producing a polyester, after performing an esterification reaction and / or transesterification reaction of a conventional dicarboxylic acid component containing an aliphatic dicarboxylic acid and an aliphatic diol component, under reduced pressure, A method of increasing the degree of polymerization of the polyester while distilling off the diol produced by the transesterification reaction at the alcohol end of the polyester, or distilling the aliphatic dicarboxylic acid and / or its acid anhydride ring from the end of the aliphatic carboxylic acid of the polyester. A method of increasing the degree of polymerization of the polyester while leaving is used. In the latter case, the removal of the aliphatic carboxylic acid and / or its anhydride ring is usually carried out during the polycondensation reaction under reduced pressure in the latter stage of the melt polymerization step. Although the method of distilling the body by heating is employed, since the aliphatic dicarboxylic acid easily becomes an acid anhydride ring under the polycondensation reaction conditions, it may be distilled by heating in the form of an acid anhydride ring. Many. At this time, the linear or cyclic ether derived from the diol and / or the diol may also be removed together with the aliphatic dicarboxylic acid and / or the anhydride cyclic product thereof. Furthermore, the method of distilling off both the dicarboxylic acid component and the diol component of the cyclic monomer is a preferred embodiment because the polymerization rate is improved.

  In addition, various additives such as plasticizers, UV stabilizers, anti-coloring agents, matting agents, deodorizers in the course of the polyester production process, or within the range where the properties of the produced polyester are not impaired. If necessary, inorganic fine particles or organic compounds may be added as agents, flame retardants, weathering agents, antistatic agents, yarn friction reducing agents, mold release agents, antioxidants, ion exchange agents, or coloring pigments. Color pigments include carbon black, titanium oxide, zinc oxide, iron oxide, and other inorganic pigments, as well as cyanine, styrene, phthalocyanine, anthraquinone, perinone, isoindolinone, quinophthalone, and quinocridone. Organic pigments such as those based on thioindigo can be used. Moreover, modifiers such as calcium carbonate and silica can also be used.

<Physical properties of polyester>
In addition, polyester is obtained from polyester which shows the following physical property.

The polyester of the present invention not only has many uses to replace general-purpose polymer materials, but also has physical properties such as aliphatic diols and aliphatic dicarboxylic acids such as polybutylene succinate and polybutylene succinate adipate. Taking polyester as an example, the density is 1.2 to 1.3 g / cm ³ , the melting point is 80 to 120 ° C., the tensile strength is 30 to 80 Mpa, the ultimate elongation is 300 to 600%, the tensile elastic modulus is 400 to 700 Mpa, the tensile yield. It possesses characteristics of general-purpose polymers such as a point strength of 30 to 40 MPa, an impact test strength of about 5 to 20 kJ / m ² , and a glass transition point of −45 to −25 ° C. Moreover, when targeting a specific use, it can be set as the polyester which has the characteristics of arbitrary wide ranges beyond the range of the above ranges. Furthermore, it can have the melting point, melt index, and melt viscoelasticity properties so that a molded product can be produced by various molding means. These characteristics can be arbitrarily adjusted by changing the kind of polyester raw material and additives, polymerization conditions, molding conditions and the like according to the purpose of use.

  The melting point of the polyester of the present invention is not particularly limited, but is usually 40 ° C to 270 ° C, preferably 50 ° C to 230 ° C, and more preferably 60 ° C to 130 ° C. These are determined by the above-described components, and it is possible to produce a polyester having a melting point within the above range by appropriately selecting the components.

  The number average molecular weight of the polyester of the present invention is usually at least 5000, preferably 10,000 or more, more preferably 15,000 or more in terms of polystyrene, and the upper limit is usually 500,000 or less, preferably 300,000. It is as follows.

  As for the composition ratio of the polyester copolymer, the molar ratio of the diol unit and the dicarboxylic acid unit needs to be substantially equal.

  The nitrogen content contained in the polyester excluding nitrogen contained in the functional group covalently bonded in the molecule of the polyester of the present invention is a mass ratio with respect to the polyester, and the upper limit is usually 1000 wtppm or less, preferably 500 wtppm or less. More preferably, it is 100 wtppm or less, More preferably, it is 50 wtppm or less, Among these, 40 wtppm or less is preferable, Furthermore, 30 wtppm or less is preferable, and 20 wtppm or less is the most preferable. On the other hand, the lower limit is not particularly limited, but the lower limit is usually 0.0001 wtppm or more, preferably 0.001 wtppm or more, more preferably 0.01 wtppm or more, and particularly preferably 0.05 wtppm or more. Preferably, it is 0.1 wtppm or more. The above-mentioned nitrogen content in the polymer is mainly derived from ammonia in the raw material, but when the above-mentioned nitrogen content is 1000 ppm or less, there is little coloring during molding, and heat or light of the product after molding, etc. Degradation and hydrolysis are not likely to occur, which is preferable. Further, when the above-mentioned nitrogen content of the polyester is 100 ppm or less, the polyester is less colored and more preferable depending on the application. The nitrogen content is a value measured by a chemiluminescence method.

  The ratio of the above-mentioned nitrogen content contained in the polyester of the present invention to the ammonia content contained in the raw material is preferably more than 0 and 0.9 or less, more preferably more than 0 and 0.6 or less, particularly preferably. Is 0.3 or less.

  The sulfur atom content in the polyester of the present invention is, by mass ratio with respect to the polyester, the upper limit is 20 wtppm or less, preferably 5 wtppm or less, more preferably 3 wtppm or less, and most preferably 0.3 wtppm or less. On the other hand, the lower limit is not particularly limited, but is 0.0001 wtppm or more, preferably 0.001 wtwt ppm or more, more preferably 0.01 wtppm or more, particularly preferably 0.05 wtppm or more, and most preferably 0. .1 wtppm or more. If the sulfur content is too high, the thermal stability and hydrolysis resistance of the polyester tend to be reduced, and if the amount is too low, the purification cost becomes remarkably high and the polyester tends to be economically disadvantageous.

  In the polymer of the present invention, in particular, in the case of a polyester using a raw material derived from biomass resources, for example, volatile organic components such as tetothehydrofuran and acetaldehyde tend to be easily contained in the polyester. The upper limit of these contents is usually 10000 wtppm or less, preferably 3000 wtppm or less, more preferably 1000 wtppm or less, and most preferably 500 wtppm or less based on the mass of the polyester. On the other hand, the lower limit is not particularly limited, but is usually 1 wtppb or more, preferably 10 wtppb or more, more preferably 100 wtppb or more. When the volatile content is large, it may cause odor and may cause foaming during melt molding and deterioration of storage stability. On the other hand, an excessively small system is a preferred form, but it is economically disadvantageous to produce such a polymer, such as requiring a very large capital investment and requiring a large production time.

The reduced viscosity (ηsp / c) value of the polyester produced according to the present invention is 0.5 or more, and preferably 1.0 or more, more preferably 1.8, because practically sufficient mechanical properties can be obtained. The above is preferable, and 2.0 or more is particularly preferable. The upper limit of the reduced viscosity (ηsp / c) value is usually 6.0 or less, preferably 5.0 or less, from the viewpoint of operability such as ease of extraction after the polyester polymerization reaction and ease of molding. Preferably it is 4.0 or less.

  The reduced viscosity as used in the present invention is measured under the following measurement conditions.

[Reduced viscosity (ηsp / c) measurement conditions]
Viscosity tube: Ubbelohde viscosity tube
Measurement temperature: 30 ° C
Solvent: phenol / tetrachloroethane (1: 1 weight ratio) solution
Polyester concentration: 0.5 g / dl
The polyester of the present invention is preferably a polyester that dissolves uniformly when a polyester (0.5 g) is dissolved in a phenol / tetrachloroethane (1: 1 weight ratio) solution (volume: 1 dl) at room temperature. In general, the amount of insoluble components in the total polyester is preferably 1% by weight or less, more preferably 0.1% by weight or less, and particularly preferably 0.01% by weight or less.

The terminal carboxyl group concentration of the polyester of the present invention is usually 100 equivalents / ton or less, more preferably, the concentration is 50 equivalents / ton or less, particularly 35 equivalents / ton or less, and more preferably 25 equivalents / ton. Or less, preferably 0.1 equivalent / ton or more, preferably 0.5 equivalent / ton or more, particularly preferably 1 equivalent / ton or more. When this amount increases, the thermal stability during molding of the polymer and the hydrolysis resistance during relatively long-term use / storage tend to decrease, and a polymer having too few carboxyl groups is a more preferable form, In order to produce such a polymer, it is economically disadvantageous in that it requires a very large amount of capital investment and requires a lot of production time. The amount of terminal carboxyl group is usually calculated by a known titration method. In the present invention, the obtained polyester is a value obtained by dissolving the obtained polyester in benzyl alcohol and titrating with 0.1 N NaOH, and per 1 × 10 ⁶ g. The carboxyl group equivalent.

  The polyester produced in the present invention is usually preferably a polyester with little coloring. The upper limit of the yellowness (YI value) of the polyester of the present invention is usually 50 or less, preferably 30 or less, more preferably 20 or less, still more preferably 15 or less, particularly preferably 10 or less, The lower limit is not particularly limited, but is usually −20 or more, preferably −10 or more, more preferably −5 or more, particularly preferably −3 or more, and most preferably −1 or more. Polyesters exhibiting high YI values have the disadvantage that their use applications such as films and sheets are limited. On the other hand, polyesters exhibiting a low YI value are a more preferred form, but there are economical disadvantages in producing such polymers, such as complicated manufacturing processes and very high capital investment. In the present invention, the YI value is a value measured by a method based on JIS K7105.

<Use of polyester composition>
The polyester according to the present invention is formed by a general-purpose plastic molding method such as an injection molding method, a hollow molding method and an extrusion molding method, etc., a film, a laminate film, a sheet, a plate, a stretched sheet, a monofilament, a multifilament, a nonwoven fabric, a flat yarn, a staple. It can be used for molded products such as crimped fibers, striped tapes, split yarns, composite fibers, blow bottles and foams. At that time, a crystal nucleating agent, an antioxidant, a lubricant, a colorant, a release agent, a filler, other polymers, and the like can be added as necessary.

Furthermore, since the polyester according to the present invention has less burden on the environment than conventional petroleum-derived polymers, in the future, shopping bags, garbage bags, agricultural films, cosmetic containers, detergent containers, bleach containers, fishing lines, fishing nets, Expected to be used in applications such as ropes, binding materials, surgical threads, sanitary cover stock materials, cold insulation boxes, cushion materials, medical materials, electrical equipment materials, home appliance housings, and automotive materials.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples, unless the summary is exceeded. The characteristic values in the following examples were measured by the following method.

Dilute solution viscosity (reduced viscosity): Polyester is dissolved in phenol / tetrachloroethane (1/1 (mass ratio) mixed solution) to a concentration of 0.5 g / dL, and the solution is a viscosity tube in a thermostatic bath at 30 ° C. The time t (sec) for dropping was measured. In addition, the time t ₀ (s ec) when the solvent alone falls was measured, and the reduced viscosity η _sp / C (= (t−t ₀ ) / t ₀ · C) at 30 ° C. was calculated (C is the concentration of the solution). .

  Nitrogen atom content: A sample of 10 mg was taken into a quartz boat, the sample was burned using a total nitrogen analyzer (TN-10 manufactured by Mitsubishi Chemical Corporation), and determined by a chemiluminescence method.

  Sulfur atom content: About 0.1 g of sample was collected in a platinum boat and burned in a quartz tube tubular furnace (AQF-100 (concentration system) manufactured by Mitsubishi Chemical Corporation), and the sulfur content in the combustion gas was 0.1%. -Absorbed with hydrogen peroxide. Thereafter, sulfate ions in the absorbing solution were measured using an ion chromatograph (ICS-1000 type, manufactured by Dionex).

Terminal carboxyl group amount: The value obtained by dissolving the obtained polyester in benzyl alcohol and titrating with 0.1N NaOH, which is the carboxyl group equivalent per 1 × 10 ⁶ g.
YI value: Measured based on the method of JIS K7105.

Reference example 1
<Construction of vector for gene disruption>
(A) Extraction of Bacillus subtilis genomic DNA
Bacillus subtilis (Bacillus subtilis ISW1214) was cultured in 10 mL of LB medium [composition: tryptone 10 g, yeast extract 5 g, NaCl 5 g dissolved in 1 L of distilled water], and the cells were collected. The obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) / 1 mM EDTA · 2Na solution containing lysozyme at a concentration of 10 mg / mL.

Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. To this lysate, add an equal amount of phenol / chloroform solution, shake gently at room temperature for 10 minutes, and then centrifuge (5,000 × g, 20 minutes, 10-12 ° C.) to obtain a supernatant. The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice as much ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol and then air-dried. To the obtained DNA, 5 mL of 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution was added and left overnight at 4 ° C., and used as template DNA for subsequent PCR.

(B) Amplification and cloning of SacB gene by PCR
The Bacillus subtilis SacB gene was obtained by using the DNA prepared in (A) as a template and a synthetic DNA (SEQ ID NO: 1 and sequence) designed based on the already reported base sequence of the gene (GenBank Database Accession No. X02730). Performed by PCR using number 2).

Composition of reaction solution: template DNA 1 μL, Pfx DNA polymerase (manufactured by Invitrogen) 0.2 μL, 1-fold concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄
0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 2 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 68 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplified product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 2 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated with a ligation kit ver. 2 (manufactured by Takara Shuzo Co., Ltd.) was used to bind to the EcoRV site of the E. coli vector (pBluescript II: manufactured by STRATEGENE), and E. coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium [10 g tryptone, 5 g yeast extract, 5 g NaCl, and 15 g agar dissolved in 1 L distilled water] containing 50 μg / mL ampicillin and 50 μg / mL X-Gal. .

  Clones that formed white colonies on this medium were then transferred to LB agar medium containing 50 μg / mL ampicillin and 10% sucrose and cultured at 37 ° C. for 24 hours. Among these clones, those that could not grow on a medium containing sucrose were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. Strains in which the SacB gene is functionally expressed in E. coli should be unable to grow on sucrose-containing media. The obtained plasmid DNA was cleaved with restriction enzymes SalI and PstI, whereby an inserted fragment of about 2 kb was observed, and the plasmid was named pBS / SacB.

(C) Construction of chloramphenicol resistant SacB vector
The restriction enzyme PshBI10 unit was reacted with 500 ng of E. coli plasmid vector pHSG396 (Takara Shuzo: Chloramphenicol resistance marker) at 37 ° C. for 1 hour, and then recovered by phenol / chloroform extraction and ethanol precipitation. After smoothing both ends with Klenow Fragment (manufactured by Takara Shuzo), ligation kit ver. MluI linker (Takara Shuzo) was ligated and circularized using 2 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. Plasmid DNA was prepared from the obtained clones by a conventional method, and a clone having a restriction enzyme MluI cleavage site was selected and named pHSG396Mlu.

On the other hand, pBS / SacB constructed in (B) above was cleaved with restriction enzymes SalI and PstI, and the ends were blunted with Klenow fragment. Ligation kit ver. After linking the MluI linker using 2 (Takara Shuzo), a DNA fragment of about 2.0 kb containing the SacB gene was separated and recovered by 0.75% agarose gel electrophoresis. This SacB gene fragment was cleaved with the restriction enzyme MluI, and then the pHSG396Mlu fragment and the ligation kit ver. 2 (manufactured by Takara Shuzo) was used to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 34 μg / mL chloramphenicol. The colonies thus obtained were then obtained with 34 μg / mL chloramphenicol and 10%
The mixture was transferred to an LB agar medium containing sucrose and cultured at 37 ° C. for 24 hours. Among these clones, plasmid DNA was purified by a conventional method for those that could not grow on a medium containing sucrose. As a result of analyzing the plasmid DNA thus obtained by MluI digestion, it was confirmed that it had an inserted fragment of about 2.0 kb, and this was named pCMB1.

(D) Acquisition of kanamycin resistance gene
The kanamycin resistance gene was obtained by PCR using the DNA of E. coli plasmid vector pHSG299 (Takara Shuzo: kanamycin resistance marker) as a template and the synthetic DNAs shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers.

  Composition of reaction solution: Template DNA 1 ng, Pyrobest DNA polymerase (Takara Shuzo) 0.1 μL, 1-fold concentration buffer, 0.5 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 62 ° C. for 15 seconds, and 72 ° C. for 1 minute 20 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 1.1 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN). The recovered DNA fragment was phosphorylated at the 5 'end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: Takara Shuzo).

(E) Construction of kanamycin resistant SacB vector
The approximately 3.5 kb DNA fragment obtained by cleaving pCMB1 constructed in (C) above with restriction enzymes Van91I and ScaI was separated and recovered by 0.75% agarose gel electrophoresis. This was mixed with the kanamycin resistance gene prepared in (D) above, and ligation kit ver. 2 (manufactured by Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

  It was confirmed that the strain grown on this kanamycin-containing medium was unable to grow on the sucrose-containing medium. Moreover, the plasmid DNA prepared from the same strain produced fragments of 354, 473, 1807, and 1997 bp by digestion with the restriction enzyme HindIII. Therefore, it was determined that the structure shown in FIG. 1 was correct, and the plasmid was designated as pKMB1. Named.

Reference example 2
<Preparation of LDH gene disruption strain>
(A) Extraction of Brevibacterium flavum MJ233-ES strain genomic DNA
A medium [urea 2 g, (NH ₄ ) ₂ SO ₄ 7 g, KH ₂ PO ₄ 0.5 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ .7H ₂ O 0.5 g, FeSO ₄ · 7H ₂ O 6 mg, MnSO _4. 4-5H2O 6 mg, biotin 200 μg, thiamine 100 μg, yeast extract 1 g, casamino acid 1 g, glucose 20 g, dissolved in 1 L of distilled water] In 10 mL, the Brevibacterium flavum MJ-233 strain was cultured until the late logarithmic growth phase. Genomic DNA was prepared from the obtained cells by the method shown in (A) of Reference Example 1 above.

(B) Cloning of lactate dehydrogenase gene
The MJ233 strain lactate dehydrogenase gene was obtained by using the DNA prepared in the above (A) as a template and a synthetic DNA (SEQ ID NO: 5 and SEQ ID NO: 6) designed based on the base sequence of the gene described in JP-A-11-206385. By PCR using

  Composition of reaction solution: Template DNA 1 μL, Taq DNA polymerase (Takara Shuzo) 0.2 μL, 1 × concentration attached buffer, 0.2 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 55 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 0.95 kb. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.

  Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, an insert fragment of about 1.0 kb was recognized, and this was named pGEMT / CgLDH.

(C) Construction of plasmid for disrupting lactate dehydrogenase gene
The coding region of lactate dehydrogenase consisting of about 0.25 kb was cut out by cutting the pGEMT / CgLDH prepared in (B) above with restriction enzymes EcoRV and XbaI. The ends of the remaining approximately 3.7 kb DNA fragment were blunted with Klenow fragment, and ligated kit ver. 2 (manufactured by Takara Shuzo) was cyclized to transform E. coli (DH5α strain). The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin. The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, a clone in which an insert fragment of about 0.75 kb was recognized was selected and named pGEMT / ΔLDH.

  Next, a DNA fragment of about 0.75 kb generated by cleaving the above pGEMT / ΔLDH with restriction enzymes SacI and SphI is separated and recovered by 0.75% agarose gel electrophoresis, and a lactate dehydrogenase gene fragment containing a defective region Was prepared. This DNA fragment was mixed with pKMB1 constructed in Reference Example 1 cut with restriction enzymes SacI and SphI, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin and 50 μg / mL X-Gal.

  Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes SacI and SphI, and a plasmid with an inserted fragment of about 0.75 kb was selected and named pKMB1 / ΔLDH (FIG. 2).

(D) Preparation of lactate dehydrogenase gene disruption strain derived from Brevibacterium flavum MJ233-ES strain
Plasmid DNA used for transformation of Brevibacterium flavum MJ-233 strain was prepared from Escherichia coli JM110 strain transformed by pKMB1 / ΔLDH by the calcium chloride method (Journal of Molecular Biology, 53, 159, 1970).

  The Brevibacterium flavum MJ233-ES strain was transformed by the electric pulse method (Res. Microbiol., Vol. 144, p. 181-185, 1993), and the obtained transformant was treated with 50 μg / mL of kanamycin. LBG agar medium containing [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water] was smeared.

  Since the strain grown on this medium is a plasmid in which pKMB1 / ΔLDH cannot be replicated in Brevibacterium flavum MJ233-ES, the lactate dehydrogenase gene of the plasmid and Brevibacterium flavum MJ-233 strain As a result of homologous recombination with the same gene on the genome, the kanamycin resistance gene and SacB gene derived from the plasmid should be inserted on the same genome.

  Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing kanamycin 50 μg / mL. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, about 10 strains that were considered to be sucrose-insensitive due to elimination of the SacB gene by the second homologous recombination were obtained.

  Among the strains thus obtained, those in which the lactate dehydrogenase gene is replaced with a mutant derived from pKMB1 / ΔLDH and those in which the wild type is restored are included. Confirmation of whether the lactate dehydrogenase gene is a mutant type or a wild type is easily confirmed by subjecting the cells obtained by liquid culture in LBG medium to direct PCR reaction and detecting the lactate dehydrogenase gene. it can. When the lactate dehydrogenase gene is analyzed using primers (SEQ ID NO: 7 and SEQ ID NO: 8) for PCR amplification, a wild-type DNA fragment of 720 bp and a mutant type having a deletion region should have a DNA fragment of 471 bp.

  As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔLDH.

(E) Confirmation of lactate dehydrogenase activity
The Brevibacterium flavum MJ233 / ΔLDH strain prepared in (D) above was inoculated into the A medium, and cultured under aerobic shaking at 30 ° C. for 15 hours. The obtained culture is centrifuged (3,000 × g, 4 ° C., 20 minutes) to recover the bacterial cells, and then sodium-phosphate buffer [composition: 50 mM sodium phosphate buffer (pH 7.3)]. Washed with.

  Next, 0.5 g (wet weight) of washed cells was suspended in 2 mL of the sodium-phosphate buffer, and the cells were crushed by applying an ultrasonic crusher (manufactured by Branson) under ice cooling. The crushed material was centrifuged (10,000 × g, 4 ° C., 30 minutes), and the supernatant was obtained as a crude enzyme solution. As a control, a crude enzyme solution of Brevibacterium flavum MJ233-ES strain was similarly prepared and subjected to the following activity measurement.

Confirmation of the lactate dehydrogenase enzyme activity was carried out by measuring, as an absorbance change at 340 nm, that the coenzyme NADH was oxidized to NAD + with the production of lactic acid using pyruvic acid as a substrate for both crude enzyme solutions [L. Kanarek and R.K. L. Hill, J.M. Biol. Chem. 239, 4202 (1964)]. Reaction is 50 mM
Potassium-phosphate buffer (pH 7.2), 10 mM pyruvate, 0.4 mM NADH
Performed at 37 ° C. in the presence. As a result, the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233 / ΔLDH was 10 minutes compared to the lactate dehydrogenase activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233-ES strain. 1 or less.

Reference example 3
<Construction of Coryneform Bacterial Expression Vector>
(A) Preparation of promoter fragment for coryneform bacteria
It was decided to use a DNA fragment (hereinafter referred to as TZ4 promoter) described in SEQ ID NO: 4 of JP-A-7-95891, which was reported to have a strong promoter activity in coryneform bacteria. This promoter fragment was obtained by using the Brevibacterium flavum MJ233 genomic DNA prepared in (A) of Reference Example 2 as a template and a synthetic DNA designed based on the sequence described in SEQ ID NO: 4 of JP-A-7-95891 ( It was performed by PCR using SEQ ID NO: 9 and SEQ ID NO: 10).

Reaction solution composition: template DNA 1 μL, Pfx DNA polymerase (Invitrogen)
0.2 μL, 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄ and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.

  The amplification product was confirmed by separation by 2.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.25 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated with a ligation kit ver. 2 (Takara Shuzo) was used to bind to the SmaI site of E. coli vector pUC19 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.

  Six clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified and the nucleotide sequence was determined. Among them, a clone in which the TZ4 promoter was inserted so as to have a transcriptional activity in the reverse direction to the lac promoter of pUC19 was selected and named pUC / TZ4.

  Next, a DNA fragment prepared by cleaving pUC / TZ4 with restriction enzymes BamHI and PstI consists of synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) phosphorylated at the 5 ′ end, and BamHI and A DNA linker having a sticky end against PstI was mixed, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. This DNA linker includes a ribosome binding sequence (AGGAGG) and a cloning site (PacI, NotI, ApaI in this order from the upstream).

Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected and named pUC / TZ4-SD.

  The thus constructed pUC / TZ4-SD was digested with the restriction enzyme PstI, blunted with Klenow fragment, and then cleaved with the restriction enzyme KpnI to obtain a promoter fragment of about 0.3 kb. Separated and collected by 0% agarose gel electrophoresis.

(B) Construction of coryneform bacterial expression vector
As a plasmid that can stably replicate independently in coryneform bacteria, pHSG298par-rep described in JP-A-12-93183 is used. This plasmid comprises a replication region and a region having a stabilizing function of the natural plasmid pBY503 possessed by Brevibacterium stachynis IFO12144, a kanamycin resistance gene derived from E. coli vector pHSG298 (Takara Shuzo), and a replication region of E. coli. The DNA prepared by cleaving pHSG298par-rep with the restriction enzyme SseI, blunting the ends with Klenow fragment, and then cleaving with the restriction enzyme KpnI is mixed with the TZ4 promoter fragment prepared in (A) above and ligated. Kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

  The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected, and the plasmid was named pTZ4 (construction procedure is shown in FIG. 3).

Reference example 4
<Preparation of a strain with enhanced pyruvate carboxylase activity>
(A) Acquisition of pyruvate carboxylase gene
The pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) of Reference Example 2 as a template, and the gene of Corynebacterium glutamicum ATCC13032 strain for which the entire genome sequence was reported. PCR was performed using synthetic DNA (SEQ ID NO: 13 and SEQ ID NO: 14) designed based on the sequence (GenBank Database Accession No. AP005276).

Composition of reaction solution: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.2 μL, 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

  Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 4 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 68 ° C. in the final cycle was 10 minutes. After completion of the PCR reaction, 0.1 μL of Takara Ex Taq (Takara Shuzo) was added, and the mixture was further incubated at 72 ° C. for 30 minutes.

  Confirmation of the amplification product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining, and a fragment of about 3.7 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).

  The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega) and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.

  Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes PacI and ApaI, an insertion fragment of about 3.7 kb was observed, which was designated as pGEM / MJPC.

  The base sequence of the insert fragment of pGEM / MJPC was determined using a base sequencing device (Model 377XL) manufactured by Applied Biosystems and a Big Dye Terminator cycle sequence kit ver3. The resulting DNA base sequence and deduced amino acid sequence are set forth in SEQ ID NO: 15. Only the amino acid sequence is shown in SEQ ID NO: 16. Since this amino acid sequence shows extremely high homology (99.4%) with that derived from Corynebacterium glutamicum ATCC13032, the insert fragment of pGEM / MJPC is a pyruvate carboxylase gene derived from Brevibacterium flavum MJ233. I determined that there was.

(B) Construction of pyruvate carboxylase activity enhancing plasmid
The approximately 3.7 kb pyruvate carboxylase gene fragment produced by cleaving pGEM / MJPC prepared in (A) above with restriction enzymes PacI and ApaI was separated and recovered by 0.75% agarose gel electrophoresis.

  This DNA fragment was mixed with pTZ4 constructed in Reference Example 3 cleaved with restriction enzymes PacI and ApaI, and ligation kit ver. After ligation using 2 (Takara Shuzo), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

  The strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes PacI and ApaI, and a plasmid in which an insertion fragment of about 3.7 kb was recognized was selected and named pMJPC1 (FIG. 4).

(C) Transformation to Brevibacterium flavum MJ233 / ΔLDH strain
Plasmid DNA for transformation with pMJPC1 capable of replicating in Brevibacterium flavum MJ233 strain was prepared from E. coli (DH5α strain) transformed in (B) above.

  Transformation into the Brevibacterium flavum MJ233 / ΔLDH strain was performed by the electric pulse method (Res. Microbiol., Vol. 144, p. 181-185, 1993), and the obtained transformant was kanamycin 50 μg / mL. In an LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water].

  From the strain grown on this medium, liquid culture was performed by a conventional method, and then plasmid DNA was extracted and analyzed by restriction enzyme digestion. As a result, it was confirmed that the strain retained pMJPC1. The strain was named as bacterial flavum MJ233 / PC / ΔLDH strain.

(D) Pyruvate carboxylase enzyme activity
The transformant Brevibacterium flavum MJ233 / PC / ΔLDH obtained in (C) above was cultured overnight in 100 ml of A medium containing 2% glucose and 25 mg / L kanamycin. The obtained cells were collected, washed with 50 ml of 50 mM potassium phosphate buffer (pH 7.5), and resuspended in 20 ml of the same composition. The suspension was crushed with SONIFIER350 (manufactured by BRANSON), and the centrifuged supernatant was used as a cell-free extract. The pyruvate carboxylase activity was measured using the obtained cell-free extract. Measurement of enzyme activity was 100 mM Tris / HCl buffer (pH 7.5), 0.1 mg / 10 ml biotin, 5 mM magnesium chloride, 50 mM sodium bicarbonate, 5 mM sodium pyruvate, 5 mM sodium adenosine triphosphate, 0.32 mM NADH. The reaction was carried out at 25 ° C. in a reaction solution containing 20 units / 1.5 ml malate dehydrogenase (manufactured by WAKO, derived from yeast) and the enzyme. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol NADH per minute. The specific activity in the cell-free extract in which pyruvate carboxylase was expressed was 0.2 U / mg protein. It should be noted that pyruvate carboxylase activity was not detected by this activity measurement method in cells obtained by similarly culturing the parent strain MJ233 / ΔLDH strain using A medium.

Reference Example 5
<Preparation of fermentation broth>
Urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, sulfuric acid Manganese hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, and distilled water: 1000 mL of a medium of 100 mL is placed in a 500 mL Erlenmeyer flask at 120 ° C. for 20 minutes. Heat sterilized. This was cooled to room temperature, 4 mL of 50% glucose aqueous solution sterilized in advance and 50 μL of sterile 5% kanamycin aqueous solution were added, and inoculated with Brevibacterium flavum MJ233 / PC / ΔLDH prepared in Reference Example 4 (C). The seed culture was performed at 30 ° C. for 24 hours.

  Urea: 12 g, ammonium sulfate: 42 g, monopotassium phosphate: 1.5 g, dipotassium phosphate 1.5 g, magnesium sulfate heptahydrate: 1.5 g, ferrous sulfate heptahydrate: 60 mg, sulfuric acid Manganese hydrate: 60 mg, D-biotin: 600 μg, thiamine hydrochloride: 600 μg, yeast extract 3 g, casamino acid 3 g, antifoaming agent (Adecanol LG294: manufactured by Asahi Denka): 1 mL and distilled water: 2500 mL of 5 mL of medium It put into the fermenter and sterilized by heating at 120 ° C. for 20 minutes. After cooling this to room temperature, 500 mL of a 12% aqueous glucose solution sterilized in advance was added, and the whole amount of the above-mentioned seed culture solution was added thereto, and the mixture was kept at 30 ° C. Main culture was performed at aeration of 500 mL / min and stirring at 500 rpm. Glucose was almost consumed after 12 hours.

  Magnesium sulfate heptahydrate: 1.5 g, Ferrous sulfate heptahydrate: 60 mg, Manganese sulfate / hydrate: 60 mg, D-biotin: 600 μg, Thiamine hydrochloride: 600 μg, Antifoam (Adecanol LG294 : Manufactured by Asahi Denka): 5 ml and distilled water: 1.5 L of a medium was placed in a 3 L Erlenmeyer flask and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, microbial cells collected by centrifugation at 10,000 g for 5 minutes were added to the culture solution obtained by the above main culture. D. It was resuspended so that (660 nm) was 60. 1.5 L of this suspension and 1.5 L of a 20% glucose solution sterilized in advance were mixed in a 5 L jar fermenter, and kept at 35 ° C. The pH was kept at 7.6 using 2M ammonium carbonate, and the reaction was carried out with aeration at 500 mL / min and stirring at 300 rpm. In about 50 hours after the start of the reaction, glucose was almost consumed. 57 g / L of succinic acid was accumulated. This fermentation broth was separated into cells and supernatant by centrifugation at 10,000 g for 5 minutes and ultrafiltration (NTU-3000-C1R manufactured by Nitto Denko Corporation). By performing the above operation 30 times, 103 L of a succinic acid fermentation broth supernatant could be obtained.

<Purification of succinic acid from succinic acid fermentation broth>
103 L (succinic acid content 5.87 kg) of the succinic acid fermentation supernatant obtained as described above was concentrated in a stirred tank equipped with a jacket while reducing the pressure. The concentration of succinic acid was 32.9% and ammonia 11.9% concentrate: 17.8 kg (calculated value) was obtained. To this, 8.58 kg of acetic acid (manufactured by Daicel Chemical Industries) was added and cooled to 30 ° C., 4.0 kg of methanol (manufactured by Kishida Chemical Co., Ltd.) was added and cooled to 15 ° C. Stirring was continued for 4 hours.

  Crystals were precipitated, and this was filtered with a centrifugal filter to obtain 4.95 kg of crystals containing 74.6% oxalic acid, 3.5% acetic acid, and 12.2% ammonia.

  4.9 kg of crystals obtained in 11.3 kg of acetic acid were added, dissolved at 85 ° C., and immediately cooled to 20 ° C. Crystals were already precipitated, but the mixture was further stirred for 3 hours, and then filtered with a centrifugal filter. Crystals containing oxalic acid 87.9%, acetic acid 8.4%, and ammonia 0.6% 2.44 kg was obtained.

  The obtained crystals were washed with 3.5 L of demineralized water cooled to 5 ° C., and filtered through a centrifugal filter. As a result, 90% oxalic acid, 1.7% acetic acid, 0.05% ammonia (approximately 500 ppm) ) Containing 2.08 kg of crystals was obtained.

  This crude oxalic acid crystal (2.0 kg) was dissolved in 28.5 L of demineralized water, and passed through a tower filled with 1 L of ion exchange resin (SK1BH, manufactured by Mitsubishi Chemical Corporation) at SV = 2, and approximately 33 L of processing solution. Got. While continuously feeding this to a rotary evaporator under reduced pressure, the solution was concentrated to approximately 5.2 L. Crystals had already precipitated at this stage. Further, after cooling to 5 ° C. and continuing stirring for 2 hours, this was filtered to obtain 1.76 kg of crystals of 96.7% oxalic acid. When this was dried with a vacuum dryer, 1.68 kg of oxalic acid could be obtained.

<Production of polyester and pellets using biomass resource-derived succinic acid (sulfur atom weight / nitrogen atom weight = 0.04) having a nitrogen atom content of 5 ppm and a sulfur atom content of 0.2 ppm>
Example 1
In a reaction vessel equipped with a stirrer, a nitrogen inlet, a heating device, a thermometer and an exhaust port for decompression, 100 parts by weight of a biomass resource-derived succinic acid having a nitrogen atom content of 5 ppm and a sulfur atom content of 0.2 ppm (YI = 2) 5), 88.5 parts by weight of industrial grade 1,4-butanediol manufactured by Mitsubishi Chemical Corporation, 0.37 parts by weight of malic acid, and 88% lactic acid aqueous solution 5 in which 0.98% by weight of germanium dioxide was previously dissolved as a catalyst 5 .4 parts by weight were charged, and after reducing pressure (attainment pressure reduction degree 0.2 kPa), the system was put under nitrogen atmosphere by repeating the operation of returning to atmospheric pressure with nitrogen gas three times.

Next, the temperature in the system was increased to 220 ° C. while stirring, and the reaction was performed at this temperature for 1 hour. Next, the temperature was raised to 230 ° C. over 30 minutes, and at the same time, the pressure was reduced to 0.07 × 10 ³ Pa over 1.5 hours, and the reaction was performed at the same degree of pressure for 1.8 hours. The obtained polyester was extracted as a strand from the bottom of the reaction vessel at 220 ° C., submerged in 10 ° C. water, and then cut with a cutter to obtain white pellets (yellowness YI of 11). The pellet was heat-dried at 80 ° C. for 8 hours under vacuum to obtain a pellet having a water content of 358 ppm. The nitrogen element content and the sulfur element content in the polyester after drying were 2 ppm and 0.1 ppm, respectively, the reduced viscosity (ηsp / c) of the polyester was 2.5, and the terminal carboxyl group content was 26 equivalents / ton. It was. The obtained polyester (0.5 g) was uniformly dissolved in 1 dL of phenol / tetrachloroethane (1/1 (mass ratio) mixed solution) at room temperature.

<Polyester using biomass resource-derived succinic acid (sulfur atom weight / nitrogen atom weight = 0.41) having a nitrogen atom content of 12 ppm and a sulfur atom content of 5 ppm>
Example 2
As a raw material, derived from a biomass resource containing 12 ppm nitrogen element and 5 ppm sulfur atom content instead of 100 parts by weight of succinic acid derived from biomass resource containing 5 ppm nitrogen element and 0.2 ppm sulfur atom content of Example 1 The same white polyester pellets as in Example 1 were produced under the same conditions as in Example 1 except that 100 parts by weight of succinic acid (yellowness YI was 7) was used. The polymerization reaction time under reduced pressure of 0.07 × 10 ³ Pa was 2 hours.

  The obtained polyester (yellowness YI is 22) has a nitrogen element content of 3.6 ppm, a sulfur atom content of 2.6 ppm, a reduced viscosity (ηsp / c) of the polyester of 2.3, and a terminal carboxyl group content. Was 19 equivalents / ton. The obtained polyester (0.5 g) was uniformly dissolved in 1 dL of phenol / tetrachloroethane (1/1 (mass ratio) mixed solution) at room temperature.

<Polyester using biomass resource-derived succinic acid having a nitrogen atom content of 16 ppm and a sulfur atom content of 2 ppm (sulfur atom weight / nitrogen atom weight = 0.13)>
Example 3
As raw material, instead of 100 parts by weight of succinic acid derived from biomass resource containing 5 ppm of nitrogen element and 0.2 ppm sulfur atom content of Example 1, derived from biomass resource containing 16 ppm nitrogen element and 2 ppm sulfur atom content The same white polyester pellets as in Example 1 were produced under the same conditions as in Example 1 except that 100 parts by weight of succinic acid (yellowness YI was 3) was used. The polymerization reaction time under a reduced pressure of 0.07 × 10 ³ Pa was 2.1 hours.

  The obtained polyester (yellowness YI is 19) has a nitrogen element content of 3.4 ppm, a sulfur atom content of 1.4 ppm, a reduced viscosity (ηsp / c) of the polyester of 2.4, and a terminal carboxyl group content. Was 15 equivalents / ton. The obtained polyester (0.5 g) was uniformly dissolved in 1 dL of phenol / tetrachloroethane (1/1 (mass ratio) mixed solution) at room temperature.

<Polyester using petroleum-derived succinic acid containing no nitrogen and sulfur atoms>
Comparative Example 1
In place of the succinic acid produced by the fermentation method in Example 1, a polyester was produced using a petroleum-derived commercially available raw material that does not contain elemental nitrogen and sulfur. The same method as in Example 1 except that succinic acid uses an industrial grade manufactured by Kawasaki Kasei Co., Ltd., and 1,4-butanediol uses an industrial grade manufactured by Mitsubishi Chemical Corporation. A polyester was produced. The polymerization reaction time under a reduced pressure of 0.07 × 103 Pa was 2.5 hours. Neither elemental nitrogen nor elemental sulfur was detected in the produced polyester.

<Polyester using biomass resource-derived succinic acid having a nitrogen atom content of 3 ppm and a sulfur atom content of 34 ppm (sulfur atom weight / nitrogen atom weight = 11)>
Comparative Example 2
As raw materials, derived from biomass resources containing 3 ppm nitrogen element and 34 ppm sulfur atom content instead of 100 parts by weight succinic acid derived from biomass resources containing 5 ppm nitrogen element and 0.2 ppm sulfur atom content of Example 1 The same polyester pellets as in Example 1 were produced under the same conditions as in Example 1 except that 100 parts by weight of succinic acid was used. The polymerization reaction time under a reduced pressure of 0.07 × 10 ³ Pa was 7 hours.

  The resulting polyester (yellowness YI was 38) had a reduced viscosity (ηsp / c) of 2.4 and an amount of terminal carboxyl groups of 30 equivalents / ton. The obtained polyester (0.5 g) was dissolved in 1 dL of phenol / tetrachloroethane (1/1 (mass ratio) mixed solution) at room temperature, but a small amount of insoluble matter was observed.

  From this example and the comparative example, it can be seen that the polymerization rate is improved when a specific amount of sulfur atom is contained in the polyester. However, it can be seen that when the ratio of sulfur atom weight / nitrogen atom weight exceeds 10, not only the polymerization reaction is delayed, but also the polyester is colored and the generation of foreign matters is caused.

Physical property evaluation example
<Evaluation of biodegradability>
The polyester produced in Example 1 and Comparative Example 1 was formed into a film using an inflation molding machine at a molding temperature of 160 ° C., a blow ratio of 2.5, and a thickness of 20 μm. The formed film was cut to a size of 5 cm × 18 cm and embedded in soil. The weight loss rate of the film was measured for 1 month, 2 months, 3 months, and 6 months, and a biodegradation test was performed. The results are shown in Table 1. From Table 1, it was confirmed that the polyester using fermented succinic acid has a high biodegradation rate in soil.

<Biodegradability test in soil>
The film produced by the above method was cut into a size of 5 cm × 18 cm and embedded in soil. The weight loss rate of the film at 1 month, 2 months, 3 months, and 6 months was measured. The results are shown in the table.

An outline of the construction of pKMB1 is shown. An outline of the construction of pKMB1 / ΔLDH is shown. An outline of the construction of the pTZ vector is shown. An outline of the construction of pMJPC1 is shown.

Claims (8)

A succinic acid derived from a biomass resource, comprising a sulfur atom and a nitrogen atom in a ratio of sulfur atom weight / nitrogen atom weight in an atomic conversion of more than 0 and 10 or less. Succinic acid according to claim 1, characterized in that the biomass resource is a plant resource. The succinic acid according to claim 1 or 2, wherein the succinic acid contains 100 mass ppm or less and 0.001 mass ppm or more of sulfur atoms in terms of atoms with respect to the mass of the dicarboxylic acid. The succinic acid according to any one of claims 1 to 3, wherein the succinic acid contains 50 mass ppm or less and 0.01 mass ppm or more of nitrogen atoms in terms of atoms relative to the mass of succinic acid . The succinic acid according to any one of claims 1 to 4, wherein the yellowness degree (YI) of the dicarboxylic acid is 50 or less. A polyester obtained by reacting the succinic acid according to any one of claims 1 to 5. By purifying succinic acid derived from biomass resources by a purification method including a crystallization step, sulfur atoms and nitrogen atoms in the succinic acid are converted into atomic weight ratios of sulfur atomic weight / nitrogen atomic weight. A method for producing succinic acid , characterized by being made greater than 0 and less than or equal to 10. A polyester obtained by reacting succinic acid obtained by the method for producing succinic acid according to claim 7 .

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