JP2006328373A - Manufacturing method of polyester - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for economically and efficiently manufacturing a polyester derived from biomass resources using a dicarboxylic acid and/or a diol derived from the biomass resources as raw materials of the polyester. <P>SOLUTION: In the manufacturing method of the polyester by a reaction of the dicarboxylic acid with the diol, the dicarboxylic acid and/or the diol are those obtained from the biomass resources and the agitation speed during the esterification reaction is caused to be at least 30 rpm. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing polyester. In detail, it is related with the manufacturing method of polyester using the dicarboxylic acid and / or diol induced | guided | derived from biomass resources as a raw material.

  Polyesters such as aromatic polyesters, aliphatic polyesters, wholly aromatic polyesters, semi-aromatic polyesters and polycarbonates are currently one of the typical polymers produced by polycondensation of raw materials derived from fossil fuel resources. However, due to concerns over the recent depletion of fossil fuel resources and global environmental problems such as an increase in atmospheric carbon dioxide, attention has been focused on methods for deriving these polymer raw materials from biomass resources. Yes. 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 1, 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 a biomass resource-derived diol, are obtained by directly obtaining the corresponding dicarboxylic acid from a biomass resource-derived raw material by fermentation. There is a method of obtaining this by hydrogenation using a reduction catalyst after the production by the fermentation method (see Patent Document 3).
Japanese Patent Laid-Open No. 2005-27533 Appl. Microbiol Biotechnol No. 51 (1999) 545-552 Journal of the American Chemical Society No. 116 (1994) 399-400 Biotechnology and Bioengineering Symp. No. 17 (1986) 355-363

  The dicarboxylic acids and diols derived from biomass resources containing a large amount of impurities as described above are usually used after further reducing the amount of impurities by a purification process, but the present inventors have undergone such a purification process. Dicarboxylic acids and diols also contain nitrogen elements contained in biomass resources, nitrogen elements derived from microorganisms and enzymes, nitrogen elements such as ammonia used in purification processes, inorganic acids, organic acids, metal cations, etc. Therefore, it is easier to color than raw materials synthesized from ordinary fossil fuel resources. For example, when 1,4-butanediol is used as the diol, a diol oxidation reaction such as 2- (4-hydroxybutyloxy) tetrahydrofuran is generated. Acquired knowledge that there is a problem that the polyester produced as a result is easy to color because of the characteristics such as easy to produce It was.

An object of the present invention is to provide a method for producing a practical polyester having a high molecular weight and little coloring when a dicarboxylic acid and / or diol derived from a biomass resource is used as a raw material.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have used a biomass resource-derived dicarboxylic acid and / or diol as a raw material because of the high impurity content in the raw material as described above. The present inventors have found that more tetrahydrofuran and 2- (4-hydroxybutyloxy) tetrahydrofuran are by-produced during the esterification reaction and / or transesterification reaction. Furthermore, if the efficiency of distilling off water during the esterification reaction is poor, by-products of tetrahydrofuran during the esterification reaction become significant. As a result, the ratio of diol and dicarboxylic acid in the reaction solution collapses, and the subsequent polymerization reaction The knowledge that the speed decreases was obtained. On the other hand, if the stirring speed during esterification is controlled to a specific value or higher to improve the efficiency of distilling water, the by-production of tetrahydrofuran during esterification is suppressed, and polyester derived from biomass resources is economical and efficient. As a result, the present invention has been completed.

  That is, the first gist of the present invention is a method for producing a polyester by reaction of a didicarboxylic acid and a diol, wherein the dicarboxylic acid and / or diol is obtained from a biomass resource, and the esterification reaction is performed. The polyester production method is characterized in that the reaction is carried out while the stirring speed of the stirring blade in the reaction apparatus is controlled to 30 rpm or more.

  According to the production method of the present invention, when a dicarboxylic acid and / or diol derived from a biomass resource is used as a raw material for the polyester, a polyester derived from the biomass resource can be produced economically and efficiently.

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, 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. 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, and 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 and stored in the form of starch, cellulose, etc. by the photosynthesis of plants, the bodies of animals that grow by eating plants, plants and animals Includes products made by processing the body. Among these, a more preferable biomass resource is a plant resource. , 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, most preferably corn, sugar cane, cassava, sago palm. These biomass resources generally contain a large amount of alkali metals and alkaline earth metals such as nitrogen element, 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 not particularly limited. If necessary, a grinding process is further included with a grinder or a mill. The refined biomass resources are further guided to a carbon source through pretreatment and saccharification processes. Specific methods include acid treatment and alkali treatment with strong acids such as sulfuric acid, nitric acid, hydrochloric acid, and phosphoric acid. , By chemical methods such as ammonia freezing steam explosion method, solvent extraction, supercritical fluid treatment, oxidizing agent treatment, 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 a dicarboxylic acid-producing ability. For example, Anaerobiospirillum (US Pat. No. 5,143,833) is used.
Anaerobic bacteria such as Actinobacillus (US Pat. No. 5,504,004) and Escherichia (US Pat. No. 5,770,435) (E. coli (J. Bacteriol.,). 57: 147-158) or mutants of E. coli strains (Japanese translations of PCT publication No. 2000-500333, US Pat. No. 6,159,738), Corynebacterium genus (JP-A-11-113588), etc. Bacteria, aerobic bacteria belonging to the genus Bacillus, Rizobium, Brevibacterium, Arthrobacter (Japanese Patent Laid-Open No. 2003-235593), Bacteroides ruminicola, Bacteroides amylophilus Anaerobic rumen bacteria such as 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, preferably among 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 parent strains of the above bacteria 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), in the present invention, Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 strain are Corynebacterium glutamicum MJ-233 strain and MJ-233 AB, respectively. It shall be the same stock as -41 stock.

The Brevibacterium flavum MJ-233 was founded 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 as FERM P-3068 in Tsukuba City, Ibaraki Prefecture, Japan, 1st Central, 6), and transferred to an international deposit based on the Budapest Treaty on May 1, 1981, with the accession 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 other purification methods, 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, weakly basic ion A method of treating with an exchange resin or an oxalate salt produced by fermentation as described in JP-A-2-283289 is electrodialyzed and then treated with a strongly acidic ion exchange resin or a weakly basic ion exchange resin. The method of doing is illustrated. Furthermore, USP 6284904 and the method of Unexamined-Japanese-Patent No. 2004-196768 are also used suitably. 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 elemental nitrogen as an impurity due to the purification process including the biomass resource origin, fermentation treatment and neutralization step with acid. Specifically, nitrogen elements 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 1000 wtppm or less, preferably 100 wtppm or less, more preferably the upper limit is 5 wtppm or less, 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 the dicarboxylic acid derived from biomass resources obtained by the above-described method is used 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. Is preferred. Thereby, coloring by oxidation reaction, such as a 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 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 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. If the temperature is too low, storage costs tend to increase, and if it is too high,
There is a tendency for the dehydration reaction of carboxylic acid 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.
The pressure in the dicarboxylic acid storage tank is usually atmospheric pressure (normal pressure).

(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 /
Examples include KOH and Cu / Cr / Zn. 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, in terms of atoms, based on the diol mass, the upper limit is usually 100 wtppm or less, preferably 10 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 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 mentioned above, Most preferably, it is 0.01 vol% or more, and an upper limit is 10 vol% or less normally, Preferably it is 5 vol% or less, More preferably, it is 1 vol% or less, Most preferably, it is 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, a copolymer component may be added 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, and 3 Examples include at least one polyfunctional compound selected from the group consisting of functional or higher oxycarboxylic acids. Among these copolymer components, a bifunctional and / or trifunctional or higher oxycarboxylic acid is particularly preferably used because a polyester having a high polymerization degree 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, the lower limit of the amount used to achieve the effect is usually 0. It is 02 mol% or more, preferably 0.5 mol% or more, more preferably 1.0 mol% or more. 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.

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.
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.

  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.

Specifically, as the diisocyanate compound, 2,4-tolylene diisocyanate,
Known mixtures of 2,4-tolylene diisocyanate and 2,6-tolylene diisocyanate, diphenylmethane diisocyanate, 1,5-naphthylene diisocyanate, xylylene diisocyanate, hydrogenated xylylene diisocyanate, hexamethylene diisocyanate, isophorone diisocyanate, etc. Examples of the diisocyanate are as follows.

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.

In the present invention, a polyester containing substantially no chain extender is most preferable. 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.

<Production method of polyester>
The production of the polyester mainly composed of a diol unit and a dicarboxylic acid unit can be performed by a known technique for producing a polyester, except that the stirring speed of a stirring blade in a reaction apparatus during an esterification reaction is controlled to a specific value or more. 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.
The esterification reaction referred to in the present invention refers to the dicarboxylic acid component and the diol component described above, and in the case of introducing an oxycarboxylic acid unit or a trifunctional or higher component, the dicarboxylic acid component and the diol component including those components. Is defined as a reaction from when raw materials are charged into a reaction vessel and the temperature rise is started until the reaction vessel is depressurized. The
Regarding the stirring speed of the stirring blade in the reaction apparatus during the esterification reaction, it is necessary to control the average stirring speed of the stirring blade of the reaction apparatus in the esterification reaction to 30 ppm or more.

In the present invention, the biomass resource-derived dicarboxylic acid and / or diol obtained by the above-described method is required to control the stirring speed during the esterification reaction during the polyester production reaction when used as a polyester raw material. .
Thereby, the by-product of tetrahydrofuran is reduced and the subsequent polymerization rate can be improved.

The lower limit of the stirring speed during the esterification reaction is 30 rpm or more, preferably 50 rpm or more, more preferably 80 rpm or more, and the upper limit is 1000 rpm or less, preferably 5
00 rpm or less. When the stirring speed is too slow, the distillation efficiency is poor and the esterification reaction tends to be slow, and for example, the diol dehydration reaction or dehydration cyclization tends to be caused. As a result, the ratio of the diol / dicarboxylic acid collapses and the polymerization rate is reduced, and it is necessary to charge an excessive amount of diol, and if it is too fast, it consumes extra power and is economical. Disadvantageous.

  In the present invention, the dicarboxylic acid and / or diol derived from biomass resources obtained by the above-described method is used as a polyester raw material in a reaction tank in which the oxygen concentration during the polyester production reaction is controlled to a specific value or less. It is preferable to produce polyester. As a result, the polyester is colored by an oxidation reaction of the nitrogen compound as an impurity, or 2- (4-hydroxybutyloxy) formed by the oxidation reaction of 1,4-butanediol when, for example, 1,4-butanediol is used as the diol. ) Since the coloring of the polyester by the diol oxidation reaction product such as tetrahydrofuran can be suppressed, a polyester having a good hue can be produced.

The production reaction referred to here is the preparation of a polymer having a desired viscosity under reduced pressure in a polycondensation reaction tank from the time when the raw material is charged into the esterification reaction tank, and the temperature rise is started. It is defined as the period until the pressure is restored.
The lower limit of the oxygen concentration in the reaction tank during the production reaction is not particularly limited, but is usually 1.0 × 10 ⁻⁹ vol% or more, preferably 1.0 × 10 ⁻⁷ vol% or more, and the upper limit is usually 10 vol. % Or less, preferably 1 vol% or less, more preferably 0.1 vol% or less, and most preferably 0.01 vol% or less. When the oxygen concentration is too low, the management process tends to be complicated, and when it is too high, the polyester obtained for the above reason tends to become colored.

Moreover, you may control the stirring speed at the time of the polymerization reaction under reduced pressure. Thereby, especially thermal decomposition and coloring at the time of manufacture of polyester derived from biomass resources which are easy to thermally decompose with many impurities can be controlled.
The lower limit of the stirring speed at the start of polymerization under reduced pressure is usually 10 rpm or more, preferably 20 rpm or more, more preferably 30 rpm or more, most preferably 50 rpm or more, and the upper limit is 200 rpm or less. Preferably it is 100 rpm or less, More preferably, it is 50 rpm or less, Most preferably, it is 30 rpm or less. When the stirring speed is too slow, the polymerization speed tends to be slow or the viscosity of the produced polymer tends to be uneven, and when it is too fast, during the production of a polymer derived from biomass resources having many impurities due to shearing heat generation. Tends to degrade the polymer in particular.

The lower limit of the final stirring speed during the polymerization reaction under reduced pressure is usually 0.1 rpm or higher, preferably 0.5 rpm or higher, more preferably 1 rpm or higher, and the upper limit is 20 rpm or lower, preferably 10 rpm or lower. More preferably, it is 7 rpm or less, and most preferably 5 rpm or less. If the stirring speed is too slow, the polymerization measure tends to be slow or the viscosity of the produced polymer tends to be uneven, and if it is too fast, in the production of polymers derived from biomass resources that have many impurities due to shearing heat generation. In particular, the polymer tends to decompose easily. In the present invention, it is usually preferable to produce the desired polyester by stirring at a rotational speed of at least 10 rpm for at least 5 minutes, preferably at least 10 minutes, more preferably at least 30 minutes.
Here, the stirring speed during the polymerization reaction under reduced pressure is preferably a method in which the stirring speed is reduced continuously or in multiple stages in consideration of an increase in the viscosity of the polyester. More preferably, the average stirring speed for 10 minutes before stopping the polycondensation reaction under reduced pressure is lower than the average stirring speed for 30 minutes after starting the polycondensation reaction under reduced pressure. By performing this adjustment, the thermal decomposition during the production of the biomass resource-derived polyester, which contains many impurities and is easily pyrolyzed, is suppressed, and the polymer can be produced stably.

  Moreover, when using dicarboxylic acid derived from biomass resources as a polyester raw material, the oxygen concentration and humidity when transferring the dicarboxylic acid from the storage tank to the reactor may be controlled. As a result, corrosion in the transfer pipe due to the sulfur component as an impurity can be prevented, and further, coloring due to the oxidation reaction of the nitrogen source can be suppressed, and a polyester having a good hue can be produced.

  Specifically, the transfer pipe is made of a commonly known metal, or an inner surface of which a lining such as glass or resin is applied, or a glass or resin container. 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 oxygen concentration in the transfer pipe is not particularly limited with respect to the total volume of the transfer pipe, 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 capital investment 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 humidity in the transfer pipe 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. Hereinafter, more preferably, 40% R.I. H. It is as follows. If the humidity is too low, the management process tends to be cumbersome and economically disadvantageous, and if it is too high, corrosion of the storage tank and piping tends to be a problem. Furthermore, when the humidity is too high, problems such as adhesion of dicarboxylic acid to storage tanks and piping, blocking of dicarboxylic acid, and the like occur, and corrosion of piping tends to be promoted by these adhesion phenomena.

The lower limit of the temperature in the transfer tube 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, more preferably 50 ° C. or lower. 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 pressure in the transfer pipe is usually from 0.1 kPa to 1 MPa, but is used at a pressure of about 0.05 MPa to 0.3 MPa from the viewpoint of operability.

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 selected from the group consisting of titanium, zirconium, tin, antimony, cerium, germanium, zinc, cobalt, manganese, iron, aluminum, magnesium, calcium, strontium, sodium and potassium
Compounds containing organic groups such as carboxylate salts, alkoxy salts, organic sulfonate salts or β-diketonate salts containing more than one metal, and inorganic compounds such as metal oxides and halides described above, and mixtures thereof Can be mentioned.

  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
product name manufactured by trial Fibers: C-94) is more preferable, in particular, tetra-n
-Butyl titanate, polyhydroxy titanium stearate, titanium (oxy) acetyl acetonate, titanium tetraacetyl acetonate, titania / silica composite oxide (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, sulphate 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.

In the polycondensation reaction after the esterification reaction and / or transesterification reaction between the dicarboxylic acid component and the diol component, the lower limit is usually 0.01 × 10 ³ Pa or more, preferably 0.05 × 1.
0 ³ Pa or more, and the upper limit is usually 1.4 × 10 ³ Pa or less, preferably 0.4 × 10 ³ Pa
The following vacuum is applied. 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. The method of using the stirring tank type reactor which equipped the exhaust pipe for pressure reduction to tie is 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 chain or cyclic ether derived from the diol and / or the diol may also be removed together with the aliphatic dicarboxylic acid and / or the acid anhydride cyclic. 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 inorganic pigments such as carbon black, titanium oxide, zinc oxide, iron oxide, 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>
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.
The nitrogen atom content other than the functional group covalently bonded in the polyester of the present invention is 1000 wtppm or less with respect to the polyester mass. The nitrogen atom content other than the covalently bonded functional group in the polyester is preferably 500 wtppm or less, more preferably 100 wtppm or less, still more preferably 50 wtppm or less, of which 40 wtppm or less is preferable, and further 30 wtppm or less is preferable. 20 wtppm or less is most preferable. The nitrogen atom content other than the functional group covalently bonded to the polyester in the polymer is mainly derived from the nitrogen element in the raw material, but is included other than the functional group covalently bonded to the polyester. It is preferable that the nitrogen atom content is 1000 wtppm or less with respect to the mass of the polyester, so that coloring and foreign matter are less generated at the time of molding, and deterioration or hydrolysis of the product after molding, such as heat or light, hardly occurs. Further, the content of nitrogen atoms contained other than the functional group covalently bonded in the polyester is 100 wtppm or less with respect to the polyester mass, and it is more preferable depending on the use because the polyester is less colored and foreign matter is generated. The effect becomes remarkable when the content is smaller. On the other hand, the nitrogen atom content contained in the polyester other than the covalently bonded functional group is preferably 0.01 wtppm or more with respect to the polyester mass. More preferably, it is 0.05 wtppm or more, More preferably, it is 0.1 wtppm or more, Most preferably, it is 1 wtppm or more. If the nitrogen atom content is less than 0.01 wtppm, a burden is imposed on the purification of the raw material, which is disadvantageous in terms of energy, and the influence on the environment cannot be ignored. Further, when the nitrogen atom content is 1 wtppm or more, the aliphatic polyester is preferable because the biodegradation rate in the soil is accelerated. By using a raw material having a nitrogen atom content in the above range, usually, the polymerization rate of the polyester can be reduced in the polymerization reaction, and the biodegradability of the resulting polyester can be promoted. The nitrogen atom content can be measured by a chemiluminescence method which is a conventionally known method as described later.
In the present invention, the functional group covalently bonded in the polyester is a urethane functional group derived from the diisocyanate compound or carbodiimide compound, an unreacted isocyanate functional group, a urea functional group, an isourea functional group, and Refers to unreacted carbodiimide functional group. Therefore, in the present invention, the nitrogen atom content other than the functional group covalently bonded in the polyester is the above-mentioned urethane functional group, unreacted isocyanate functional group, from the total nitrogen atom content contained in the polyester, This is a value obtained by subtracting the nitrogen atom content attributed to the urea functional group, the isourea functional group and the unreacted carbodiimide functional group. The content of urethane functional group, unreacted isocyanate functional group, urea functional group, isourea functional group and unreacted carbodiimide functional group is the same as the above ¹³ C NMR and IR spectroscopic measurements and polyester production Calculated from the charged amount.

The ratio of the nitrogen atom 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.
The upper limit of the sulfur atom content in the polyester of the present invention is 500 wtppm or less, preferably 50 wtppm or less, more preferably 3 wtppm or less, and most preferably 0.3 wtppm or less with respect to the polyester mass. is there. On the other hand, the lower limit is not particularly limited, but is 0.0001 wtppm or more, preferably 0.001 wtppm or more, more preferably 0.01 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.

  The reduced viscosity (ηsp / c) value of the polyester produced in the present invention is 1.4 or more, and preferably 1.6 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 groups is usually calculated by a known titration method. In the present invention, the obtained polyester is dissolved in benzyl alcohol to give 0.1N.
It is a value titrated with NaOH, and is a carboxyl group equivalent per 1 × 10 ⁶ g.
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 can be produced by a general-purpose plastic molding method such as an injection molding method, a hollow molding method, and an extrusion molding method, etc. 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) was measured, and a 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).

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. 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) containing lysozyme at a concentration of 10 mg / mL
It was suspended in 0.15 mL of 1 mM EDTA · 2Na solution.

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 taken and sodium acetate was 0
. After adding to 3M, 2 volumes of 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 a 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 × 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 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 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 was dephosphorylated with alkaline phosphatase (Alkaline Phosphatase Calf intestine) and 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 transferred to an LB agar medium containing 34 μg / mL chloramphenicol and 10% 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: 1 ng of template DNA, Pyrobest DNA polymerase (Takara Shuzo) 0
. 1 μL, 1 × concentration buffer, 0.5 μM each primer, and 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. Recovery of the target DNA fragment from the gel is performed using the QIAQuick Gel Extraction Kit (manufactured by QIAGEN)
It was performed using. The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by 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. Further, 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 · 4-5H ₂ O 6 mg, biotin 200 μg, thiamine 100 μg, yeast extract 1 g
1 g of casamino acid, 20 g of glucose, dissolved in 1 L of distilled water] In 10 mL, Brevibacterium flavum MJ-233 strain was cultured until the late logarithmic growth phase, and the obtained cells were transferred to (A) of Reference Example 1 above. Genomic DNA was prepared by the method shown.

(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 mixed with 50 μg / mL ampicillin and 50 μg / mL X-Gal LB.
It was smeared on an agar medium.
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 [
10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 20 g of glucose, and 15 g of agar were dissolved in 1 L of distilled water.

  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 was centrifuged (3,000 × g, 4 ° C., 20 minutes) to recover the bacterial cells, and then sodium-phosphate buffer [composition: 5
0 mM sodium phosphate buffer (pH 7.3)].

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 enzyme activity of lactate dehydrogenase 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 pyruvate as a substrate for both crude enzyme solutions [L. Kanarek and R.K. L. Hill, J.M. Biol. Chem. 239, 4202 (1964)]. The reaction was performed at 37 ° C. in the presence of 50 mM potassium-phosphate buffer (pH 7.2), 10 mM pyruvic acid, 0.4 mM NADH. 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 carried out by PCR using SEQ ID NO: 9 and SEQ ID NO: 10).

Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached 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 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. Recovery of the target DNA fragment from the gel is performed using the QIAQuick Gel Extraction Kit (manufactured by QIAGEN)
It was performed using.

The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated 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 mixed with 50 μg / mL ampicillin and 50 μg / mL X-Gal.
The LB agar medium containing

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 cleaved with the restriction enzyme PstI, blunted with Klenow fragment, and then cleaved with the restriction enzyme KpnI to give 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: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentration attached 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 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,
Furthermore, it was kept 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. Recovery of the target DNA fragment from the gel is performed using the QIAQuick Gel Extraction Kit (manufactured by QIAGEN)
It was performed using.

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 mixed with 50 μg / mL ampicillin and 50 μg / mL X-Gal LB.
It was smeared on an agar medium.

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 to Brevibacterium flavum MJ233 / ΔLDH strain is performed by the electric pulse method (Res. Microbiol., Vol. 144, p. 181-185, 1993).
The obtained transformant was smeared on an LBG agar medium containing 10 μg / mL kanamycin [10 g tryptone, 5 g yeast extract, 5 g NaCl, 20 g glucose, and 15 g agar dissolved in 1 L 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. After collecting the obtained cells, 50 mM potassium phosphate buffer (pH 7.5) 50
It was washed with ml and resuspended in 20 ml of the same composition buffer. 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 is 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 , 20 units / 1
. The reaction was performed at 25 ° C. in a reaction solution containing 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 The mixture was placed in a 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 is 10000
g The cells and the supernatant were separated by centrifugation 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 broth obtained as described above was concentrated in a stirred tank 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, cooled to 15 ° C. and stirred for 1 hour, and then at 20 ° 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.
Dissolve 2.0 kg of this crude succinic acid crystal in 28.5 L of demineralized water, and add 1 L of ion exchange resin (
A column filled with SK1BH manufactured by Mitsubishi Chemical Corporation was passed at SV = 2 to obtain about 33 L of a processing solution. 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.

Example 1
In a reaction vessel equipped with a stirrer, a nitrogen inlet, a heating device, a thermometer, and an exhaust port for decompression, a biomass resource derived from biomass resources having a nitrogen atom content of 30 ppm and a sulfur atom content of 18 ppm obtained by the method described above 100 parts by weight of acid, 88.5 parts by weight of industrial grade 1,4-butanediol manufactured by Mitsubishi Chemical Co., Ltd., 0.37 parts by weight of malic acid, and 0.98% by weight of germanium dioxide as a catalyst were previously dissolved. Then, 5.4 parts by weight of 88% lactic acid aqueous solution was charged, and after the pressure was reduced (attainment pressure reduction degree 0.2 kPa), the pressure in the system was returned to atmospheric pressure with nitrogen gas three times, and the inside of the system was put into a nitrogen atmosphere.

Next, the temperature of the reaction solution was raised to 220 ° C. over 1 hour while stirring the system at 100 rpm. Subsequently, the temperature was raised to 230 ° C. over 30 minutes and at the same time 0.07 × 10 ³ Pa over 1.5 hours.
The reaction was carried out for 3 hours under the same pressure reduction. The polyester obtained had a reduced viscosity (ηsp / c) of 2.4.

Example 2
The same procedure as in Example 1 was carried out except that the stirring speed during the esterification reaction after charging the raw materials was 50 rpm. The polymerization reaction time under a reduced pressure of 0.07 × 10 ³ Pa was almost the same as in Example 1. The polyester obtained had a reduced viscosity (ηsp / c) of 2.4.

Comparative Example 1
The same procedure as in Example 1 was performed except that the stirring speed during the esterification reaction after charging the raw materials was 3 rpm. Although the reduced viscosity (ηsp / c) of the obtained polyester was 2.4, the polymerization reaction time under a reduced pressure of 0.07 × 10 ³ Pa required 4 hours.

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 (5)

  In the method for producing a polyester by reaction of a dicarboxylic acid and a diol, the dicarboxylic acid and / or diol is obtained from biomass resources, and when performing the esterification reaction, the stirring speed of the stirring blade in the reactor is increased. A method for producing a polyester, wherein the reaction is carried out while controlling at 30 rpm or higher.   The method for producing a polyester according to claim 1, wherein the biomass resource is a plant resource.   The method for producing a polyester according to claim 1 or 2, wherein the reduced viscosity (ηsp / c) of the polyester is 1.4 or more.   The polyester contains at least one trifunctional or higher polyfunctional compound unit selected from the group consisting of a trifunctional or higher polyhydric alcohol, a trifunctional or higher polyvalent carboxylic acid, and a trifunctional or higher oxycarboxylic acid. The manufacturing method of the polyester of any one of Claims 1-3 characterized by the above-mentioned.   The content of the trifunctional or higher polyfunctional compound unit is 0.0001 mol% or more and 0.5 mol% or less with respect to 100 mol% of all monomer units constituting the polyester. 4. A process for producing a polyester as described in 4.

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