WO2005059153A1 - Procede de separation, d'extraction et de purification de poly-$g(b)-hydroxyalcanoates (pha) directement a partir d'un milieu de culture bacterien fermente - Google Patents

Procede de separation, d'extraction et de purification de poly-$g(b)-hydroxyalcanoates (pha) directement a partir d'un milieu de culture bacterien fermente Download PDF

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WO2005059153A1
WO2005059153A1 PCT/CN2003/001092 CN0301092W WO2005059153A1 WO 2005059153 A1 WO2005059153 A1 WO 2005059153A1 CN 0301092 W CN0301092 W CN 0301092W WO 2005059153 A1 WO2005059153 A1 WO 2005059153A1
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fermentation broth
separating
extracting
group
reaction
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PCT/CN2003/001092
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Xuejun Chen
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Tianan Biologic Material Co., Ltd. Ningbo
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Priority to CA2550204A priority Critical patent/CA2550204C/en
Priority to EP03782063.6A priority patent/EP1705250B9/en
Priority to US10/583,587 priority patent/US7582456B2/en
Priority to AU2003292860A priority patent/AU2003292860B2/en
Priority to JP2005512144A priority patent/JP4777778B2/ja
Priority to PCT/CN2003/001092 priority patent/WO2005059153A1/zh
Publication of WO2005059153A1 publication Critical patent/WO2005059153A1/zh

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/62Carboxylic acid esters
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/88Post-polymerisation treatment
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/06Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids

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  • the invention belongs to the technical field of downstream post-processing of biological engineering, and particularly relates to the extraction and separation of bacterial fermentation products, and more particularly, to a method for directly extracting and separating intracellular polyhydroxy fatty acid esters. Background of the invention
  • PHAs Poly- ⁇ -Hydroxyalkanoates
  • n and m are integers of 1-4, usually 1, which is 3-hydroxy fatty acids (3-HAs);
  • R 2 is a substituted or unsubstituted saturated or unsaturated straight or branched C ,. 12 fluorenyl group; X and Y cannot be 0 at the same time, and the size of X and Y determines the content of the component in the copolymer.
  • the weight average molecular weight of PHAs is between 1 and 4 million Da.
  • PHAs are similar to those of polypropylene, but because of its properties that are not available in ordinary petrochemical resins such as biodegradability, biocompatibility, piezoelectricity, and optical activity, PHAs are used in industry, agriculture, medicine, health, and food. , Electronics and other fields have broad application prospects.
  • the cost of PHAs mainly includes the cost of raw materials and the cost of separation and purification.
  • the cost of raw materials depends on the production efficiency of the bacteria and the fermentation process used, while the cost of separation and purification mainly depends on the process used.
  • the current extraction process first uses a high-speed centrifuge to separate cells from the fermentation broth, and then purifies the PHAs in the separated wet bacteria.
  • the purification methods generally used are organic solvent extraction, chemical reagents, and surface activity. Agent + enzymatic method. These methods are either costly or contaminated, making them difficult to implement Industrial production.
  • the invention patent application with publication number CN1328160A discloses a one-step extraction and separation method for directly extracting polyhydroxyalkanoates from a cell fermentation broth containing polyhydroxyphosphonates.
  • this method must use a large amount of sodium hypochlorite, which makes the operating environment harsh and polluted, increases costs due to wastewater treatment, and has a great shear degradation effect on the molecular weight of the extracted product PHAs, which affects the product quality.
  • the object of the present invention is to provide a PHAs extraction method that can effectively reduce the cost of separation and purification, reduce pollution, and is suitable for industrial production. Summary of the invention
  • the invention provides a method for directly separating and purifying intracellular polyhydroxy fatty acid ester from a bacterial fermentation broth.
  • the method of the present invention includes the following steps:
  • the order of adjusting the pH and adding the surfactant can be interchanged.
  • a coagulant in addition to the anionic surfactant, a coagulant may be added.
  • the physical method used for cell wall breaking may be ultrasonic disruption, bead mill grinding, or high-pressure treatment.
  • the alkaline substance used for adjusting the pH may be a solid such as NaOH, Na 2 C0 3 NaHC0 3 or an aqueous solution, or ammonia water.
  • the anionic surfactant used in the present invention may be alkenyl sulfonate (AOS), fatty alcohol sulfate, fatty alcohol polyoxyethylene ether sulfate (AES), fatty alcohol polyoxyethylene ether (AEO), alkylphenol poly
  • AOS alkenyl sulfonate
  • AES fatty alcohol polyoxyethylene ether sulfate
  • AEO fatty alcohol polyoxyethylene ether
  • alkylphenol poly alkenyl sulfonate
  • the amount of oxyethylene ether and the like is 0.5% to 20% (W / V) of the fermentation broth.
  • the coagulant that can be added in the method of the present invention includes sodium polyacrylate, modified starch, polyamine, and the like.
  • the amount of coagulant is 0.5% ⁇ 20% (W / V) of the fermentation broth.
  • reaction temperature 10 ° C ⁇ 70 ° C
  • reaction time 5 ⁇ 60 minutes.
  • the method for separating and extracting the precipitate in the reaction solution can be centrifugation, pressure filtration, vacuum filtration, and the like.
  • the invention has a wide range of applicable treatment objects, and can be applied to the separation and purification of various polyhydroxy fatty acid ester-containing bacteria, their mutants, and genetic engineering bacteria.
  • the applicable bacterial species include: Alcal igenes, Pseudomonas, Azotobacter, Rhodospiri l lum, Methylotrophic bacteria ( Methylotrophs) and Bac il lus.
  • the method of the present invention does not have high requirements on the dry weight of cells in the fermentation broth and the PHAs content in the cells.
  • the invention has simple process, low cost and high extraction yield. And greatly reduce pollution, thereby enabling large-scale industrial production. detailed description

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  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Genetics & Genomics (AREA)
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Description

从细菌发酵液中直接分离提纯胞内聚羟基脂肪酸酯的方法 技术领域
本发明属于生物工程下游后处理技术领域, 具体涉及细菌发酵产 物的提取分离, 更具体涉及胞内聚羟基脂肪酸酯的直接提取分离方 法。 发明背景
聚 _羟基脂肪酸酯 ( Poly- β -Hydroxyalkanoates,简称 PHAs ) 是一类由特定微生物在特殊的生长条件下在胞内积累的生物聚酯,
PHAs 的通式为:
R! O R2 O
I II I II
-f O-CH ― (CH2)n- C- -f- O - CH- (CH2)m- Cty
其中, n,m为 1—4的整数, 通常为 1, 即 3—羟基脂肪酸(3-HAs) ;
R2为取代或未取代的饱和或不饱和的直链或支链 C,.12垸基; X、 Y不能同 时为 0, X、 Y的大小决定了该组份在共聚物中的含量。 一般, PHAs的重 均分子量在 100— 400万 Da之间。
PHAs 的物理性质类似于聚丙烯, 但由于它具有生物降解性、 生 物相容性、 压电性、 光学活性等普通石油化工树脂所不具备的特性, 因此在工业、 农业、 医学、 卫生、 食品、 电子等领域具有广阔的应用 前景。
目前国际上 PHAs的大规模工业化生产仍未实现, 其主要原因是 成本较石油化工树脂高得多。 PHAs成本主要包括原料成本和分离提纯 成本。 原料成本的高低取决于菌种的生产效率及所采用的发酵工艺, 而分离提纯成本则主要取决于采用的工艺。 目前的提取工艺是首先采 用高速离心机将细胞从发酵液中分离出来, 再对分离后的湿菌体中的 PHAs进行提纯,一般采用的提纯方法是有机溶剂萃取法、化学试剂法、 表面活性剂 +酶法等。 这些方法或是成本高、 或是污染严重,难以实现 工业化生产。 公开号 CN1328160A的发明专利申请公开了一种从含有 聚羟基垸酸酯的细胞发酵液中直接提取聚羟基烷酸酯的一步提取分 离方法。 但该方法必须采用大量的次氯酸钠, 使操作环境恶劣、 污染 严重, 由于废水处理而增加成本, 并且对提取产物 PHAs的分子量有 极大的剪切降解作用, 影响产品品质。
因此, 本发明的目的是提供一种可以有效降低分离提纯成本、 减 少污染、 适合于工业化生产的 PHAs提取方法。 发明内容
本发明提供一种从细菌发酵液中直接分离提纯胞内聚羟基脂肪 酸酯的方法。 本发明的方法包括以下步骤:
(1) 用物理方法对发酵液进行细胞破壁预处理;
(2) 将经预处理的发酵液 pH值调节至碱性;
(3) 加入阴离子表面活性剂, 搅拌;
(4) 分离提取反应液中的沉淀;
(5) 洗涤、 干燥,
其中, 调节 pH和加入表面活性剂的顺序可以互换。
步骤 (3) 中除了加入阴离子表面活性剂外还可加入凝聚剂。 本发明方法中, 细胞破壁所用的物理方法可以是超声波破碎、 珠 磨机研磨或高压处理。
预处理后的发酵液 pH调节在 8〜13。 调节 pH所用的碱性物质可 以是 NaOH、 Na2C03 NaHC03等固体或水溶液或氨水等。
本发明所用的阴离子表面活性剂可以是烯基磺酸盐 (AOS) 、 脂 肪醇硫酸盐、 脂肪醇聚氧乙烯醚硫酸盐 (AES) 、 脂肪醇聚氧乙烯醚 (AEO)、烷基酚聚氧乙烯醚等,其用量为发酵液的 0.5%〜20%(W/V)。
本发明方法中可以加的凝聚剂有聚丙烯酸钠、变性淀粉、聚胺等。 凝聚剂的用量为发酵液的 0.5%〜20% (W/V) 。
加入阴离子表面活性剂和凝聚剂后搅拌反应的温度在 10°C〜70 °C, 反应时间为 5〜60分钟。
分离提取反应液中沉淀的方法可以采用离心、压滤、真空抽滤等。 本发明适用处理对象广泛,可应用于各种含聚羟基脂肪酸酯的细 菌及其变异株及基因工程菌等发酵液的分离提纯。所适用的细菌种属 主要包括: 产碱杆菌属(Alcal igenes )、假单胞菌属(Pseudomonas) 、 固氮菌属(Azotobacter)、 红螺菌属(Rhodospiri l lum)、 甲基营养菌 属 (Methylotrophs)和芽抱杆菌属(Bac i l lus)等。
本发明方法对发酵液中细胞干重及其中的 PHAs含量的要求不高。 本发明工艺简单、 成本低、 提取收率高。 并且大大减少污染, 从而能 够实现大规模工业化生产。 具体实施方式
下面用实施例对本发明作进一步描述,但这些实施例并不构成对 权利要求范围的任何限制。本领域技术人员在得到本说明书的揭示后 对本发明所作的任何修改和变动都将落在本说明书所附权利要求的 范围内。 实施例 1
¾ Alcal igenes entrophus 突变株 65- 7的发酵液 1000ml, 细 胞干重为 142克 /升, raBV含量 78. 5%, 用珠磨机(530转 /分, 0. 1誦 钢珠) 预处理 40分钟, 用 30 %氢氧化钠溶液调节 pH值至 12, 加入 13克十二垸基硫酸钠, 调节反应温度至 32 °C, 搅拌反应 5分钟, 用 滤纸抽滤, 所得沉淀用水洗涤至洗液呈中性, 70°C左右烘干至恒重。 所得产品纯度为 98. 2%,重均分子量 5. 2 X 105Da, 提取收率 85. 2%。 抽 滤的废水经厌氧、 好氧处理后, 其 C0D、 B0D分别达到 800mg/l和 30mg/l , 达到国家排放标准。 实施例 2
取 Alcaligenes entrophus菌发酵液 100ml, 细胞干重 147g/l (其中 PHBV含量为 75.2%) , 用超声波 (功率 1500W) 破壁 20分钟, 用 30%Na0H溶液调节 pH值至 8, 加入 0.5g十二垸基硫酸钠和 5g聚 丙烯酸钠, 调节反应温度至 7CTC, 搅拌反应 30分钟。 然后, 用滤纸 抽滤, 将所得沉淀用水洗涤, 洗涤至洗液呈 PH中性后置于 70°C左右 烘箱内干燥至恒重。 所得产品纯度 93.2%, 分子量 4. lX105Da, 提取 收率 80.3%。 实施例 3
取 Alcaligenes entrophus菌发酵液 50ml,细胞干重 102g/l (其 中 PHB含量为 60%) , 用珠磨机 (560转 /分, 0.1讓钢珠) 破壁预处 理 30分钟, 用 ΝΗ3· Η20溶液调节 pH值至 13, 加入 10g十二烷基硫酸 钠和 10g变性淀粉, 调节反应温度至 10Ό, 搅拌反应 10分钟, 用离 心机 (分离因素为 600) 分离, 将所得沉淀用水洗涤, 洗涤至洗液呈 中性后置于 70°C左右烘箱内干燥至恒重。 所得产品纯度 98.2%, 分子 量 4.4X105Da, 提取收率 87%。 实施例 4
取 Alcaligenes entrophus 突变株 65- 7的发酵液 500ml, 细胞 干重为 135克 /升, PHB含量 75.5%, 将其置入特制容器中, 升压至 60Mpa, 10分钟后快速释放压力, 收集液体后重复 2遍。 处理后的发 酵液用 30%氢氧化钠溶液调节 pH值至 10, 加入 9克十二醇聚氧乙烯 醚硫酸钠, 调节反应温度至 38°C, 搅拌反应 8分钟, 用滤纸抽滤, 所 得沉淀用水洗涤至洗液呈中性, 70°C左右烘干至恒重。 所得产品纯度 为 96.7%,重均分子量 4.2X10 a, 提取收率 81.5%。 实施例 5
取 Alcaligenes entrophus菌发酵液 100L,细胞干重 154g/l (其 中 PHBV含量为 80.5%) , 用超声波 (功率 2800W, 连续处理)破壁 40 分钟, 用 30%Na0H溶液调节 pH值至 11, 加入 10 kg十二烷基硫酸钠 和 0.5kg聚丙烯酸钠, 调节反应温度至 50°C, 搅拌反应 60分钟, 用 压滤机压滤, 将所得沉淀用水洗涤, 洗涤至洗液呈 pH中性后置于 70 °C左右烘箱内干燥至恒重。 所得产品纯度 97%, 分子量 5.3X105Da, 提取收率 84%。 实施例 6
取 Pseudomonas菌的发酵液 100ml, 细胞干重为 86克 /升, PHBV 含量 61.5%, 用珠磨机(560转 /分, 0.1匪钢珠) 预处理 50分钟, 用 30%氢氧化钠溶液调节 pH值至 11,加入 3克十二垸基硫酸钠, 调节反 应温度至 24°C, 搅拌反应 10分钟, 用滤纸抽滤, 所得沉淀用水洗涤 至洗液呈中性, 70Ό左右烘干至恒重。所得产品纯度为 94.2%,重均分 子量 3.2X105Da, 提取收率 71.2%。

Claims

权 利 要 求 书
1. 从细菌发酵液中直接分离提纯胞内聚羟基脂肪酸酯的方法, 其特征在于包括以下步骤: ' (1) 用物理方法对发酵液进行细胞破壁预处理;
(2) 将经预处理的发酵液 pH值调节至碱性;
(3) 加入阴离子表面活性剂, 搅拌反应;
(4) 分离提取反应液中的沉淀;
(5) 洗涤、 干燥,
其中, 调节 pH和加入表面活性剂的顺序可以互换。
2. 如权利要求 1所述的方法, 其中步骤 (3) 还可加入凝聚剂。
3. 如权利要求 1所述的方法, 其中所述所述物理方法选自超声 波粉碎、 珠磨机研磨和高压处理。
4. 如权利要求 1所述的方法, 其中预处理后的发酵液 pH调节在 8〜13。
5. 如权利要求 1或 4所述的方法, 其中调节 pH所用的碱性物质 选自 NaOH、 Na2C03、 NaHC03固体 /水溶液和氨水。
6. 如权利要求 1所述的方法, 其中所述阴离子表面活性剂选自 烯基磺酸盐、 脂肪醇硫酸盐、 脂肪醇聚氧乙烯醚硫酸盐、 脂肪醇聚氧 乙烯醚和垸基酚聚氧乙烯醚, 其用量为发酵液的 0.5%〜20% (W/V) 。
7. 如权利要求 1所述的方法, 其中所述凝聚剂选自聚丙烯酸钠、 变性淀粉和聚胺, 其用量为发酵液的 0.5%〜20% (W/V) 。
8. 如权利要求 1所述的方法,其中所述搅拌反应的温度在 10°C〜 70°C, 时间为 5〜60分钟。
9. 如权利要求 1所述的方法, 其中所述分离提取反应液中沉淀 的方法选自离心、 压滤和真空抽滤。
PCT/CN2003/001092 2003-12-19 2003-12-19 Procede de separation, d'extraction et de purification de poly-$g(b)-hydroxyalcanoates (pha) directement a partir d'un milieu de culture bacterien fermente WO2005059153A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA2550204A CA2550204C (en) 2003-12-19 2003-12-19 A method for separating, extracting and purifying poly-.beta.-hydroxyalkanoates (phas) directly from bacterial fermentation broth
EP03782063.6A EP1705250B9 (en) 2003-12-19 2003-12-19 A method for separating, extracting and purifying poly-beta-hydroxyalkanoates (phas) directly from bacterial fermented broth
US10/583,587 US7582456B2 (en) 2003-12-19 2003-12-19 Method for separating, extracting and purifying poly-β-hydroxyalkanoates (PHAs) directly from bacterial fermentation broth
AU2003292860A AU2003292860B2 (en) 2003-12-19 2003-12-19 A method for Separating, Extracting and Purifying Poly- beta -hydroxyalkanoates (PHA's) directly from Bacterial Fermentation Broth
JP2005512144A JP4777778B2 (ja) 2003-12-19 2003-12-19 バクテリア発酵液からポリ−β−ヒドロキシアルカノエート(PHAs)を直接分離、抽出及び精製する方法
PCT/CN2003/001092 WO2005059153A1 (fr) 2003-12-19 2003-12-19 Procede de separation, d'extraction et de purification de poly-$g(b)-hydroxyalcanoates (pha) directement a partir d'un milieu de culture bacterien fermente

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PCT/CN2003/001092 WO2005059153A1 (fr) 2003-12-19 2003-12-19 Procede de separation, d'extraction et de purification de poly-$g(b)-hydroxyalcanoates (pha) directement a partir d'un milieu de culture bacterien fermente

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Cited By (2)

* Cited by examiner, † Cited by third party
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JP2008193940A (ja) * 2007-02-13 2008-08-28 Honda Motor Co Ltd ポリヒドロキシ酪酸精製方法
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CN115786411A (zh) * 2023-01-09 2023-03-14 北京微构工场生物技术有限公司 聚羟基脂肪酸酯的提取方法

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