JPH0731489A - Separation of bio-polyester from bio-polyester-containing microorganism - Google Patents
Separation of bio-polyester from bio-polyester-containing microorganismInfo
- Publication number
- JPH0731489A JPH0731489A JP5196671A JP19667193A JPH0731489A JP H0731489 A JPH0731489 A JP H0731489A JP 5196671 A JP5196671 A JP 5196671A JP 19667193 A JP19667193 A JP 19667193A JP H0731489 A JPH0731489 A JP H0731489A
- Authority
- JP
- Japan
- Prior art keywords
- suspension
- polyester
- bio
- biopolyester
- pressure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 244000005700 microbiome Species 0.000 title claims abstract description 18
- 229920000728 polyester Polymers 0.000 title abstract description 13
- 238000000926 separation method Methods 0.000 title description 8
- 239000000725 suspension Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000007900 aqueous suspension Substances 0.000 claims abstract description 9
- 238000010008 shearing Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 230000000813 microbial effect Effects 0.000 abstract description 12
- 238000010438 heat treatment Methods 0.000 abstract description 10
- 239000003513 alkali Substances 0.000 abstract description 8
- 239000012530 fluid Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 39
- 230000001580 bacterial effect Effects 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 229920000642 polymer Polymers 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000000638 solvent extraction Methods 0.000 description 3
- SJZRECIVHVDYJC-UHFFFAOYSA-M 4-hydroxybutyrate Chemical compound OCCCC([O-])=O SJZRECIVHVDYJC-UHFFFAOYSA-M 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WHBMMWSBFZVSSR-GSVOUGTGSA-M (R)-3-hydroxybutyrate Chemical compound C[C@@H](O)CC([O-])=O WHBMMWSBFZVSSR-GSVOUGTGSA-M 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000793839 Aquaticitalea lipolytica Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 101100264195 Caenorhabditis elegans app-1 gene Proteins 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 150000008044 alkali metal hydroxides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 238000004177 carbon cycle Methods 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002079 cooperative effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000006140 methanolysis reaction Methods 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 239000013502 plastic waste Substances 0.000 description 1
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 1
- 229920000903 polyhydroxyalkanoate Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、生分解性を有するバイ
オポリエステルの菌体からの分離方法に関する。TECHNICAL FIELD The present invention relates to a method for separating biodegradable biopolyester from bacterial cells.
【０００２】[0002]
【従来の技術】現在、プラスチック廃棄物は焼却、埋立
などによって処理されているが、これらの処理方法に
は、それぞれ地球の温暖化、埋め立て地の地盤弛緩等の
問題がある。そのため、プラスチックリサイクルへの社
会意識の高まりとともに、リサイクルシステム化が進み
つつある。しかし、リサイクル可能な用途には限りがあ
り、実際問題としてプラスチック廃棄物処理方法として
は、焼却、埋立、リサイクルだけでは対応しきれず、自
然環境中に放置されたままになるものも多い。そこで、
廃棄後は自然界の物質循環に取り込まれ、分解生成物が
有害物質とならないような生分解性プラスチックが注目
されており、その開発が進められている。このようなプ
ラスチックとして、特に、微生物が菌体内で生成するポ
リエステルは、自然界の炭素循環プロセスに組み込まれ
て生態系の安定化がなされると予想されている。また、
医療分野においても、回収不要のインプラント材料、薬
物担体としての利用が可能である。2. Description of the Related Art Currently, plastic wastes are treated by incineration, landfill, etc., but these treatment methods have problems such as global warming and land relaxation of landfill sites. For this reason, as the social awareness of plastic recycling increases, a recycling system is being developed. However, recyclable uses are limited, and as a practical matter, many plastic waste treatment methods cannot be handled by incineration, landfill, and recycling alone, and many are left in the natural environment. Therefore,
Biodegradable plastics that are taken into the natural material cycle after disposal and whose decomposition products do not become harmful substances are drawing attention, and their development is underway. As such a plastic, in particular, polyester produced by microorganisms in the cells is expected to be incorporated into the natural carbon cycle process to stabilize the ecosystem. Also,
In the medical field as well, it can be used as an implant material or a drug carrier that does not require recovery.
【０００３】しかし、このポリエステルをプラスチック
として使用するためには、微生物の菌体内から分離して
取り出す必要がある。バイオポリエステル含有微生物か
らバイオポリエステルを得る方法として、クロロホルム
をはじめとする有機溶媒による抽出法、次亜塩素酸ソー
ダ（Ｗｉｌｌｉａｍｓｏｎ，Ｄ．Ｈ．，ａｎｄ Ｗｉｌ
ｋｉｎｓｏｎ，Ｊ．Ｆ．（１９５８），Ｊ．Ｇｅｎ．Ｍ
ｉｃｒｏｂｉｏｌ．１９，１９８−２０３．）またはリ
ゾチームを用いて菌体を溶解し、残存したポリマーを顆
粒として回収する方法が知られている。その他、リゾチ
ーム以外の特定の酵素による菌体の溶解によってポリマ
ーを回収する方法（特開昭６０−１４５０９７）、１０
０℃超の高圧の水蒸気等の圧力の開放により菌体を破壊
し、菌体破片屑とポリマーとに分離する方法（特開昭５
７−１７４０９４）等もある。However, in order to use this polyester as a plastic, it is necessary to separate it from the microbial cells and take it out. As a method for obtaining a biopolyester from a biopolyester-containing microorganism, an extraction method using an organic solvent such as chloroform, sodium hypochlorite (Williamson, DH, and Wil)
kinson, J .; F. (1958), J. Gen. M
icrobiol. 19, 198-203. ) Or lysozyme is used to dissolve bacterial cells and the remaining polymer is recovered as granules. In addition, a method of recovering a polymer by dissolving bacterial cells with a specific enzyme other than lysozyme (JP-A-60-145097), 10
A method in which cells are destroyed by releasing the pressure of steam or the like having a high pressure of more than 0 ° C. and separated into cell debris and a polymer (Japanese Patent Laid-Open No. Sho 5).
7-174094) and the like.
【０００４】[0004]
【発明が解決しようとする課題】しかし、クロロホルム
等による溶媒抽出法は、当該抽出溶媒だけでなく、再沈
澱のための貧溶媒も大量に必要とする。したがって、溶
媒を各々再利用しようとすれば、２種の溶媒を分離する
ことが必要である。さらに、一般に溶媒抽出に先立って
菌体全体を完全に乾燥することが必要なため、多大の熱
エネルギーを要することにもなるので、バイオポリエス
テルを工業的に生産するためには、多くのプロセス用設
備やエネルギーが必要となり、事実上不利である。次亜
塩素酸ソーダで処理した場合は、溶媒抽出法の欠点を回
避することはできるが、一方、ポリエステルの分子量低
下が起こり（Ｊ．Ａ．Ｒａｍｓａｙ，Ｅ．Ｂｅｒｇｅ
ｒ，Ｂ．Ａ．Ｒａｍｓａｙ ａｎｄ Ｃ．Ｃｈａｖａｒ
ｉｅ（１９９０），Ｊ．Ｂｉｏｔｅｃｈｎｏｌｏｇｙ
Ｔｅｃｈｎｉｑｕｅｓ ４，４，２２１−２２６）、ポ
リマーの品質に問題が生じる。リゾチームのような酵素
は、少量の実験的利用には効果的であるが、大量に確保
するのが困難なため、バイオポリエステルの量産には適
切でない。特開昭６０−１４５０９７の酵素法では、酵
素処理前後の操作が多段階になり、量産のためには、な
お改善の余地が大きい。特開昭５７−１７４０９４の圧
力の解放による方法は、得られたポリエステルの純度や
収量が未記載のため、効果が不明である。本発明は、有
機溶媒を用いないで、水性媒体中で１００℃未満で剪断
力をかけることにより、バイオポリエステルを含む微生
物からバイオポリエステルを分離する方法を提供するこ
とを目的とする。However, the solvent extraction method using chloroform or the like requires a large amount of not only the extraction solvent but also a poor solvent for reprecipitation. Therefore, if each solvent is to be reused, it is necessary to separate the two solvents. Furthermore, since it is generally necessary to completely dry the whole microbial cell prior to solvent extraction, a large amount of heat energy is required. Equipment and energy are required, which is a practical disadvantage. When treated with sodium hypochlorite, the drawbacks of the solvent extraction method can be avoided, but on the other hand, a decrease in the molecular weight of the polyester occurs (JA Ramsay, E. Berge.
r, B. A. Ramsay and C.I. Chavar
ie (1990), J. Am. Biotechnology
(Techniques 4, 4, 221-226), which causes problems with polymer quality. Enzymes such as lysozyme are effective for experimental use in small quantities, but are not suitable for mass production of biopolyesters because they are difficult to secure in large quantities. In the enzyme method disclosed in JP-A-60-145097, the operations before and after the enzyme treatment have multiple stages, and there is still a lot of room for improvement for mass production. The effect of the method of releasing pressure of JP-A-57-174094 is unclear because the purity and yield of the obtained polyester are not described. An object of the present invention is to provide a method for separating a biopolyester from a microorganism containing the biopolyester by applying a shearing force in an aqueous medium at a temperature of less than 100 ° C. without using an organic solvent.
【０００５】[0005]
【課題を解決するための手段】本発明は、バイオポリエ
ステル含有微生物の水性懸濁液に１mmol／kg菌体〜１mo
l ／菌体、好ましくは２．５mmol／kg菌体〜２００mmo
l／kg菌体、特に好ましくは５０mmol〜２００mmol／kg
菌体の量のアルカリを添加し、該懸濁液を耐圧性容器に
導入し、もしくは予め該懸濁液を４０〜１００℃の範囲
に加熱して耐圧性容器に導入し、４０〜１００℃の範囲
内で加熱、保温し、該懸濁液を噴出させることによって
微生物に流体剪断力を作用させ、顆粒状のバイオポリエ
ステルを分離することを特徴とするバイオポリエステル
含有微生物からのバイオポリエステルの分離方法に関す
る。なお、特開昭５７−１７４０９４の方法では、同じ
く高圧を用いているが、これが、高圧を急激に低圧化し
た際の圧力ショックで菌体を破壊するのに対し、本発明
は、高圧液体の微小口噴射時の剪断力によって、菌体破
壊とバイオポリエステルの分離を促進する方法である。The present invention provides an aqueous suspension of a biopolyester-containing microorganism in an amount of 1 mmol / kg microbial cell to 1 mo.
l / cell, preferably 2.5 mmol / kg cell-200 mmo
l / kg bacterial cells, particularly preferably 50 mmol-200 mmol / kg
Alkali of the amount of microbial cells is added and the suspension is introduced into a pressure resistant container, or the suspension is previously heated to a range of 40 to 100 ° C. and introduced into the pressure resistant container, Separation of biopolyester from biopolyester-containing microorganisms, characterized in that a bioshear is exerted on the microorganisms by heating and incubating within the range of the above, and the suspension is ejected to separate granular biopolyesters. Regarding the method. Incidentally, in the method of JP-A-57-174094, the high pressure is also used, but this destroys the microbial cells by the pressure shock when the high pressure is suddenly reduced, whereas the present invention uses the high pressure liquid. This is a method of promoting cell destruction and separation of biopolyester by the shearing force at the time of jetting with a fine mouth.
【０００６】本発明に用いる微生物は、細胞内にバイオ
ポリエステルを蓄積しているバクテリア（細菌）であ
る。例えば、アルカリゲネス属（Ａｌｃａｌｉｇｃｎｅ
ｓ）の菌、Ａ．ｌｉｐｏｌｙｔｉｃａ ＡＫ２０１（特
開平５−６４５９２）、Ａ．ｅｕｔｒｏｐｈｕｓ、Ａ．
ｌａｔｕｓ等、シュウドモナス属（Ｐｓｅｕｄｏｍｏｎ
ａｓ）、バシルス属（Ｂａｃｉｌｌｕｓ）、アゾトバク
ター属（Ａｚｏｔｏｂａｃｔｅｒ）、ノカルディア属
（Ｎｏｃａｒｄｉａ）等の菌株が示されるが、その種類
に限定されるものではない。ここで、バイオポリエステ
ルとは、ポリ−Ｄ−３−ヒドロキシブチレート〔以下、
Ｐ（３ＨＢ）と略称する〕をはじめとするポリヒドロキ
シアルカノエート〔以下、Ｐ（ＨＡ）と略称する〕と称
される微生物産生ポリエステルを指す。Ｐ（３ＨＢ）以
外の代表的な例として、３ＨＢとＤ−３−ヒドロキシバ
レレート（３ＨＶ）との共重合体〔Ｐ．Ａ．Ｈｏｌｍｅ
ｓ ｅｔ ａｌ（ＩＣＩ），Ｅｕｒ．Ｐａｔ．Ａｐｐ
ｌ，００５２４５９（１９８１）〕、３ＨＢと４−ヒド
ロキシブチレート（４ＨＢ）との共重合体〔Ｙ．Ｄｏｉ
ｅｔ ａｌ．，Ｍａｃｒｏｍｏｌｅｃｕｌｅｓ，２
１，２７２２（１９８８）〕が挙げられる。細胞内に蓄
積しているバイオポリエステルは、微小な顆粒として存
在することが知られている。The microorganism used in the present invention is a bacterium which accumulates biopolyester in the cell. For example, the genus Alcaligcne
s), A. lipolytica AK201 (Japanese Patent Laid-Open No. 5-64592), A.L. eutrophus, A .;
Latus, etc., Pseudomonas (Pseudomon)
As), Bacillus genus, Azotobacterium genus, Nocardia genus, and the like strains are shown, but the strains are not limited thereto. Here, bio-polyester means poly-D-3-hydroxybutyrate [hereinafter,
P (3HB)] and other polyhydroxyalkanoates [hereinafter abbreviated as P (HA)]. As a typical example other than P (3HB), a copolymer of 3HB and D-3-hydroxyvalerate (3HV) [P. A. Holme
s et al (ICI), Eur. Pat. App
1, 0052459 (1981)], a copolymer of 3HB and 4-hydroxybutyrate (4HB) [Y. Doi
et al. , Macromolecules, 2
1, 722 (1988)]. Biopolyester accumulated in cells is known to exist as fine granules.
【０００７】処理される細胞内のバイオポリエステル含
有率（以下、ポリマー含有率という）は、高いほうが好
ましい。一般に、乾燥菌体としてポリマー含有率が２０
重量％以上がよい。アルカリ添加量、処理時間、分離操
作の効率、分離ポリマーの純度等を考慮すると、５０重
量％以上のポリマー含有率が特に好ましい。水性懸濁液
とは、培養終了後の培養懸濁液そのもの、または培養液
から遠心等で分離した菌体を水に懸濁させたものを指
す。菌体の懸濁濃度は、乾燥菌体換算で１５０ｇ／ｌ以
下、好ましくは１００ｇ／ｌ以下である。使用するアル
カリとしては、ＮａＯＨを始めとしてＬｉＯＨ、ＫＯＨ
等を含めたアルカリ金属の水酸化物、あるいはＮＨ4 Ｏ
Ｈが用いられる。アルカリの使用量は１mmol／kg菌体〜
１mol ／kg菌体、好ましくは２．５mmol／kg菌体〜２
００mmol／kg菌体、特に好ましくは５０mmol／kg菌体〜
２００mmol／kg菌体で、これを微生物の水性懸濁液に添
加する。It is preferable that the biopolyester content in the cells to be treated (hereinafter referred to as polymer content) is high. Generally, the polymer content is 20 as dry cells.
Weight% or more is preferable. Considering the amount of alkali added, the treatment time, the efficiency of the separation operation, the purity of the separated polymer and the like, a polymer content of 50% by weight or more is particularly preferable. The aqueous suspension refers to the culture suspension itself after completion of the culture, or a suspension of cells separated from the culture solution by centrifugation or the like in water. The suspension concentration of the microbial cells is 150 g / l or less, preferably 100 g / l or less in terms of dry cells. Alkali used is NaOH, LiOH, KOH
Alkali metal hydroxides, etc., or NH 4 O
H is used. The amount of alkali used is 1 mmol / kg
1 mol / kg microbial cell, preferably 2.5 mmol / kg microbial cell-2
00 mmol / kg bacterial cells, particularly preferably 50 mmol / kg bacterial cells
At 200 mmol / kg cells, this is added to an aqueous suspension of microorganisms.
【０００８】本発明の方法では、アルカリを添加後は、
水性懸濁液は、微小開口部を有する耐圧性容器に導入さ
れ、高圧をかけられる。このようにして開口部から押し
出される菌体には、大きな剪断力が働くため、菌体は破
壊され、バイオポリエステルの分離が促進されると推定
される。このような耐圧性容器と加圧機構からなる装置
は、循環装置付高圧ホモジナイザーによって代表され
る。したがって、本発明のバイオポリエステル分離法
は、高圧ホモジナイザーの利用によって実施可能とな
る。高圧ホモジナイザーの温度設定は４０〜１００℃、
好ましくは６０〜１００℃にする。該懸濁液の加熱は、
高圧ホモジナイザーの導入前に、設定温度に加熱してお
くことも望ましい。高圧ホモジナイザー内に導入した該
懸濁液にかける圧力は、装置によるが、５００〜１５０
０ｋｇｆ／ｃｍ2 で作用させるのが好ましい。In the method of the present invention, after the alkali is added,
The aqueous suspension is introduced into a pressure resistant container having a minute opening and is subjected to high pressure. It is presumed that the bacterial cells pushed out through the opening in this way are subjected to a large shearing force, so that the bacterial cells are destroyed and the separation of biopolyester is promoted. A device composed of such a pressure-resistant container and a pressurizing mechanism is represented by a high-pressure homogenizer with a circulation device. Therefore, the biopolyester separation method of the present invention can be carried out by using a high-pressure homogenizer. The temperature setting of the high pressure homogenizer is 40 to 100 ° C,
The temperature is preferably 60 to 100 ° C. The heating of the suspension is
It is also desirable to heat to a set temperature before introducing the high pressure homogenizer. The pressure applied to the suspension introduced into the high-pressure homogenizer depends on the device, but is 500 to 150.
It is preferable to operate at 0 kgf / cm 2 .
【０００９】循環装置付高圧ホモジナイザーとしては、
マントンゴーリン（独国ＡＰＶ・ゴーリン社製）、ミニ
ラボ（デンマーク ＡＰＶラニー社製）、ブランリュー
ベ連続式細胞破砕機（独国Ｂｒａｎ＋Ｌｕｅｂｂｅ社
製）、マイクロフルイダイザー（米国Ｍｊｃｒｏｆｌｕ
ｉｄｉｃｓ社製）等を用いることができる。これらの装
置は、一般的に液体を加圧することによって、乳化・分
散・細胞破砕等に用いられることがよく知られている。
本発明では、高圧ホモジナイザー内での加熱が必須なの
で、類似の高圧ホモジナイザーの一種であるが非加熱型
であるフレンチプレスは、本発明に不適当である。フレ
ンチプレスを用いて微生物中のバイオポリエステルを分
離することは知られているが（Ｈｅｌｍｕｔ Ｂｒａｎ
ｄｌ ｅｔａｌ．，Ａｄｖａｎｃｅｓ ｉｎ Ｂｉｏｃ
ｈｅｍｉｃａｌ Ｅｎｇｉｎｅｅｒｉｎｇ，Ｂｉｏｔｅ
ｃｈｎｏｌｏｇｙ（１９９０），４１，７７−９
３．）、本発明の技術的特徴であるアルカリ添加や、加
熱による分離の協同効果を実現した例は知られていな
い。As a high pressure homogenizer with a circulation device,
Manton Gorlin (German APV Gorlin), Minilab (Denmark APV Rani), Bran-Ruebe cell crusher (Bran + Luebbe, Germany), Microfluidizer (Mjcroflu USA)
idics company) or the like can be used. It is well known that these devices are generally used for emulsification / dispersion / cell crushing by pressurizing a liquid.
Since heating in the high-pressure homogenizer is essential in the present invention, a similar type of high-pressure homogenizer, but a non-heating type French press, is not suitable for the present invention. It is known to use a French press to separate biopolyesters in microorganisms (Helmut Bran
dl et al. , Advances in Bioc
chemical engineering, biote
chnology (1990), 41, 77-9.
3. ), There is no known example of realizing the cooperative effect of the addition of alkali or the heating, which is the technical feature of the present invention.
【００１０】以上の処理操作により、短時間で効率よく
菌体壁を破壊し、バイオポリエステルを顆粒状で菌体か
ら分離できる。菌体壁が破壊されると、核酸のような水
溶性の高分子物質が細胞外に溶出するために、該懸濁液
の粘度は一旦上昇するが、剪断力によって核酸分子の切
断も起こるためか、該懸濁液の粘度が再び低下し、その
後の遠心操作、ろ過操作等でのバイオポリエステルの分
離が容易に行える。処理前の該懸濁液の菌体濃度は、乾
燥菌体換算で１５０ｇ菌体／ｌまで処理可能であるた
め、通常培養後の菌体濃度を薄める必要がない。本発明
により、短時間で効率良く菌体壁が破壊され、バイオポ
リエステルを顆粒状で分離できる。By the above treatment operation, the bacterial cell wall can be efficiently destroyed in a short time, and the biopolyester can be separated from the bacterial cells in a granular form. When the cell wall is destroyed, a water-soluble polymer substance such as nucleic acid is eluted out of the cell, so that the viscosity of the suspension temporarily increases, but the shearing force also causes cleavage of the nucleic acid molecule. Alternatively, the viscosity of the suspension is reduced again, and the biopolyester can be easily separated by the subsequent centrifugation operation, filtration operation and the like. Since the cell concentration of the suspension before treatment can be up to 150 g of cells / l in terms of dry cells, it is not usually necessary to dilute the cell concentration after culturing. INDUSTRIAL APPLICABILITY According to the present invention, the bacterial cell wall can be efficiently destroyed in a short time, and biopolyester can be separated in the form of granules.
【００１１】[0011]
【実施例】本実施例で用いた微生物は、アルカリゲネス
属に属する微生物アルカリゲネス・リポリティカ（Ａｌ
ｃａｌｉｇｅｎｅｓ ｌｉｐｏｌｙｔｉｃａ）ＡＫ２０
１（特開平５−６４５９２）で、培養後、Ｐ（３ＨＢ）
を約５０ｗｔ％含有している菌を遠心（８０００ｒｐ
ｍ，１０ｍｉｎ，遠心分離機はＫＵＢＯＴＡ製６８１０
使用）によって培養液から分離後、ペースト状菌体に水
を加えて４０ｇ菌体／ｌの水性懸濁液とした。この水性
懸濁液を用いて、以下に示す実施例１、２および比較例
１〜４を行った。EXAMPLE The microorganism used in this example is a microorganism belonging to the genus Alcaligenes Alcaligenes liporitica (Al
caligenes lipolytica) AK20
1 (JP-A-5-64592), after culturing, P (3HB)
Centrifuge the bacteria containing about 50wt% (8000rp
m, 10 min, centrifugal separator made by KUBOTA 6810
After being separated from the culture solution according to (use), water was added to the paste-like microbial cell to obtain an aqueous suspension of 40 g microbial cell / l. Using this aqueous suspension, Examples 1 and 2 and Comparative Examples 1 to 4 shown below were performed.
【００１２】実施例１、２および比較例１〜４の操作で
得たＰ（３ＨＢ）は、純度を調べるためにガスクロマト
グラフィー、分子量分布の決定にゲルパーミエーション
クロマトグラフィー（ＧＰＣ）を用いて分析を行った。
なお、ガスクロマトグラフィーには、実施例１、２およ
び比較例１〜４で得られた沈澱物を乾燥（１０５℃，２
４ｈｒ）した後、メタノール／硫酸（８５／１５ ｗｔ
％／ｗｔ％）によりメタノリシスして菌体内ポリエステ
ルをモノマーのメチルエステルとしたものを分析して、
ポリマー含有率を求めた。これは、〔Ｈ．Ｂｒａｎｄｌ
ｅｔ ａｌ，ｌｎｔ．Ｊ．Ｂｉｏｌ．Ｍａｃｒｏｍｏ
ｌ．，１１，４９−５５（１９８９）〕に示される方法
に従った。ＧＰＣには、試料（約１００ｍｇ）中のポリ
エステルを熱クロロホルム１５０ｍｌで抽出後、溶液を
濃縮してヘキサンを加えて再沈し、沈澱を濾過、真空乾
燥（２ｈｒ）して１０ｍｇ／１０ｍｌのクロロホルム溶
液にして測定した。P (3HB) obtained by the operations of Examples 1 and 2 and Comparative Examples 1 to 4 was subjected to gas chromatography to check the purity and gel permeation chromatography (GPC) to determine the molecular weight distribution. Analysis was carried out.
For gas chromatography, the precipitates obtained in Examples 1 and 2 and Comparative Examples 1 to 4 were dried (105 ° C, 2 ° C).
After 4 hours, methanol / sulfuric acid (85/15 wt
% / Wt%) was used for the methanolysis to analyze intracellular polyesters as monomeric methyl esters,
The polymer content was determined. This is [H. Brandl
et al, lnt. J. Biol. Macromo
l. , 11, 49-55 (1989)]. For GPC, the polyester in the sample (about 100 mg) was extracted with 150 ml of hot chloroform, the solution was concentrated and hexane was added to reprecipitate, and the precipitate was filtered and vacuum dried (2 hr) to give a 10 mg / 10 ml chloroform solution. Was measured.
【００１３】（実施例１）４．０ｍＭとなるように０．
１ＭのＮａＯＨ水溶液を加え、Ｐ（３ＨＢ）含有菌体の
該懸濁液５００ｍｌを作成した。予め該懸濁液を９０℃
で約５分間加熱後、ＡＰＶ・ゴーリン社製マントンゴー
リンに投入する。この装置内で、該懸濁液をゲージ圧約
１０００ｋｇｆ／ｃｍ2 に加圧し（このとき、装置内の
温度は予備加熱された懸濁液の温度に制御する）、瞬時
（約１０-6〜１０-5秒）に約０．０２ｍｍの隙間を通過
させて空気中に放出し、流体剪断力をかけた。この操作
を、懸濁液を自動的に循環させることにより５回繰り返
した。処理後の懸濁液を遠心分離（２７００ｒｐｍ，１
０ｍｉｎ）して沈澱物を得た。 （実施例２）該懸濁液を７０℃、約５分間予備加熱する
こと、およびマントンゴーリン内の加熱温度を７０℃と
する以外は、実施例１と同様に操作した。(Embodiment 1) The concentration was adjusted to 4.0 mM.
A 1 M NaOH aqueous solution was added to prepare 500 ml of the suspension of P (3HB) -containing cells. The suspension was previously heated to 90 ° C
After heating for about 5 minutes at above, it is put into Manton Gorin manufactured by APV Gorin. In this device, the suspension was pressurized to a gauge pressure of about 1000 kgf / cm 2 (at this time, the temperature inside the device was controlled to the temperature of the preheated suspension), and immediately (about 10 −6 to 10 −6 ). -5 seconds), it was passed through a gap of about 0.02 mm, discharged into the air, and subjected to fluid shear force. This operation was repeated 5 times by automatically circulating the suspension. The treated suspension is centrifuged (2700 rpm, 1
0 min) to obtain a precipitate. (Example 2) The same operation as in Example 1 was carried out except that the suspension was preheated at 70 ° C for about 5 minutes, and the heating temperature in the manton-goulin was 70 ° C.
【００１４】（比較例１）本例では、該懸濁液にＮａＯ
Ｈ水溶液を加えないこと以外は、実施例１と同様に操作
した。 （比較例２）本例では、該懸濁液にＮａＯＨ水溶液を加
えないこと以外は、実施例２と同様に操作した。 （比較例３）本例では、該懸濁液を加熱しないこと以外
は、実施例１と同様に操作した。 実施例１、２および比較例１〜４の分離条件を表１に示
す。Comparative Example 1 In this example, NaO was added to the suspension.
The same operation as in Example 1 was performed except that the H aqueous solution was not added. (Comparative Example 2) In this example, the same operation as in Example 2 was performed except that no aqueous NaOH solution was added to the suspension. Comparative Example 3 In this example, the same operation as in Example 1 was performed except that the suspension was not heated. Table 1 shows the separation conditions of Examples 1 and 2 and Comparative Examples 1 to 4.
【００１５】[0015]
【表１】 実施例、比較例のガスクロマトグラフィー、ＧＰＣで得
られた結果を表２に示した。[Table 1] The results obtained by gas chromatography and GPC in Examples and Comparative Examples are shown in Table 2.
【００１６】[0016]
【表２】 [Table 2]
【００１７】[0017]
【発明の効果】本発明により、従来の各方法の欠点を克
服した新しい分離方法を開発した。すなわち、有機溶媒
を用いないで、水性媒体中に少量のアルカリを添加し、
１００℃未満の加熱下で高圧ホモジナイザーを作動して
菌体に剪断力をかけることにより、バイオポリエステル
を含む微生物からバイオポリエステルを分離できた。According to the present invention, a new separation method has been developed which overcomes the drawbacks of the conventional methods. That is, without using an organic solvent, add a small amount of alkali in an aqueous medium,
By operating the high-pressure homogenizer under heating at less than 100 ° C and applying a shearing force to the cells, the biopolyester could be separated from the microorganism containing the biopolyester.
Claims (2)
濁液に１mmol／ｋｇ菌体〜１mol ／kg菌体の量のアル
カリを添加した後、該懸濁液を耐圧性容器に導入し、４
０〜１００℃の範囲内で加熱、保温し、該懸濁液に高圧
をかけ、該容器の微小開口部から該懸濁液を噴出させる
ことによって微生物に液体剪断力を作用させ、顆粒状の
バイオポリエステルを分離することを特徴とするバイオ
ポリエステル含有微生物からのバイオポリエステルの分
離方法。1. An alkaline suspension in an amount of 1 mmol / kg to 1 mol / kg is added to an aqueous suspension of biopolyester-containing microorganisms, and the suspension is introduced into a pressure-resistant container.
The suspension liquid is heated and kept warm within the range of 0 to 100 ° C., a high pressure is applied to the suspension liquid, and the suspension liquid is ejected from the minute opening portion of the container to exert a liquid shearing force on the microorganisms to form a granular form. A method for separating biopolyester from a microorganism containing biopolyester, which comprises separating biopolyester.
液を４０〜１００℃の範囲に加熱する請求項１に記載の
方法。2. The method according to claim 1, wherein the aqueous suspension is heated in the range of 40 to 100 ° C. before being introduced into the pressure resistant container.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5196671A JPH0731489A (en) | 1993-07-15 | 1993-07-15 | Separation of bio-polyester from bio-polyester-containing microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5196671A JPH0731489A (en) | 1993-07-15 | 1993-07-15 | Separation of bio-polyester from bio-polyester-containing microorganism |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0731489A true JPH0731489A (en) | 1995-02-03 |
Family
ID=16361661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5196671A Pending JPH0731489A (en) | 1993-07-15 | 1993-07-15 | Separation of bio-polyester from bio-polyester-containing microorganism |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0731489A (en) |
Cited By (10)
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|---|---|---|---|---|
| US5821299A (en) * | 1996-02-16 | 1998-10-13 | The Proctor & Gamble Company | Solvent extraction of polyhydroxy-alkanoates from biomass facilitated by the use of marginal nonsolvent |
| US5942597A (en) * | 1995-08-21 | 1999-08-24 | The Procter & Gamble Company | Solvent extraction of polyhydroxyalkanoates from biomass |
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1993
- 1993-07-15 JP JP5196671A patent/JPH0731489A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5942597A (en) * | 1995-08-21 | 1999-08-24 | The Procter & Gamble Company | Solvent extraction of polyhydroxyalkanoates from biomass |
| US5821299A (en) * | 1996-02-16 | 1998-10-13 | The Proctor & Gamble Company | Solvent extraction of polyhydroxy-alkanoates from biomass facilitated by the use of marginal nonsolvent |
| WO2003091444A1 (en) * | 2002-04-26 | 2003-11-06 | Kaneka Corporation | Method of separating poly-3-hydroxyalkanoic acid |
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| WO2004033700A1 (en) * | 2002-09-30 | 2004-04-22 | Kaneka Corporation | Method of coagulating poly-3-hydroxyalkanoic acid |
| US7435566B2 (en) | 2002-09-30 | 2008-10-14 | Kaneka Corporation | Method of purifying 3-hyroxyalkanoic acid copolymer |
| JPWO2004065608A1 (en) * | 2003-01-20 | 2006-05-18 | 株式会社カネカ | Method for recovering high-purity polyhydroxyalkanoate from microbial cells |
| EP1609868A1 (en) * | 2003-01-20 | 2005-12-28 | Kaneka Corporation | Method of collecting highly pure polyhydroxyalkanoate from microbial cells |
| US7393668B2 (en) | 2003-01-20 | 2008-07-01 | Kaneka Corporation | Method of collecting highly pure polyhydroxyalkanoate from microbial cells |
| WO2004065608A1 (en) * | 2003-01-20 | 2004-08-05 | Kaneka Corporation | Method of collecting highly pure polyhydroxyalkanoate from microbial cells |
| EP1609868A4 (en) * | 2003-01-20 | 2011-05-25 | Kaneka Corp | Method of collecting highly pure polyhydroxyalkanoate from microbial cells |
| US7435567B2 (en) | 2004-03-04 | 2008-10-14 | Kaneka Corporation | Method for degradation of nucleic acids and use thereof |
| EP2265659A2 (en) | 2008-03-28 | 2010-12-29 | The Coca-Cola Company | Bio-based polyethylene terephthalate polymer and method of making the same |
| EP2403894A2 (en) | 2009-03-03 | 2012-01-11 | The Coca-Cola Company | Bio-based polyethylene terephthalate packaging and method of making thereof |
| WO2022215335A1 (en) | 2021-04-09 | 2022-10-13 | 株式会社カネカ | Method for producing recycled water |
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