200914620 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種使用微生物從甘油製造蘋果酸之方 法。 【先前技術】 生質柴油燃料（BDF)為一種碳中性的輕油替代燃料，且 目前作爲一種助於解決環境問題（例如能源枯竭、全球暖 化及空氣污染）的燃料已逐漸受到關注。 生質柴油燃料係從植物油、動物脂肪、廢油及類似物中 製造。如今，含有甘油的廢液以副產物被製造。對於該廢 油沒有特定的用途，如今只能丟棄。 關於利用生質柴油廢液之方法，日本未審查專利公開號 2006-1807 82揭示一種藉由腸桿菌屬細菌從廢液的甘油製 造氫氣及乙醇之方法。 關於有機酸之製造，日本未審查專利公開號2003-235592揭示了一種藉由需氧細菌（尤其棒桿細菌）從作爲碳 源的葡萄糖製造蘋果酸之方法。PCT國際公開案WO 02/00846 A1揭示了一種從葡萄糖中製造多種有機酸的菌株 (曼海姆菌屬（Mannheimia Sp.) 55E)。 曰本未審查專利公開號平10-75772揭示了 一種藉由馬來 酸懸浮睾丸酮叢毛單胞菌（Comamonas testosteroni) FERM P-15714菌株的細菌細胞而製造蘋果酸之方法。日本專利 公開號昭44-14786揭示了一種在含富馬酸的培養基中培養 需氧細菌而製造蘋果酸之方法。Biotechnology 131378.doc 200914620200914620 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a method for producing malic acid from glycerin using a microorganism. [Prior Art] Biodiesel fuel (BDF) is a carbon-neutral light oil alternative fuel and is currently receiving attention as a fuel to help solve environmental problems such as energy depletion, global warming and air pollution. Biodiesel fuels are manufactured from vegetable oils, animal fats, waste oils, and the like. Today, waste liquids containing glycerol are produced as by-products. There is no specific use for this waste oil and it can only be discarded today. Regarding the method of using the biomass diesel waste liquid, Japanese Unexamined Patent Publication No. Hei No. 2006-1807 82 discloses a method of producing hydrogen gas and ethanol from glycerin of waste liquid by Enterobacter bacteria. Regarding the production of an organic acid, Japanese Unexamined Patent Publication No. 2003-235592 discloses a method of producing malic acid from glucose as a carbon source by an aerobic bacterium (especially a rod-shaped bacterium). PCT International Publication WO 02/00846 A1 discloses a strain (Mannheimia Sp. 55E) which produces a variety of organic acids from glucose. A method for producing malic acid by suspension of bacterial cells of the Comamonas testosteroni FERM P-15714 strain by maleic acid is disclosed in Japanese Unexamined Patent Publication No. Hei No. Hei 10-75772. Japanese Patent Publication No. Sho 44-14786 discloses a method for producing a malic acid by culturing an aerobic bacterium in a medium containing fumaric acid. Biotechnology 131378.doc 200914620
LetterS(1993)15(3):272-276揭示一種藉由 D，L_蘋果酸懸浮 細函細胞而製造D-蘋果酸之方法。但是，至今仍不了解是 否以下細菌能從甘油製造蘋果酸：選自由木糖氧化無色桿 菌（Achromobacter xylosoxydans)、天牛微桿菌 (Miicrobacterium saperdae) 、moderatus 鏈黴菌 (Streptomyces m〇deratus)及莓實假單胞菌（Pseud〇m〇nas fragi)組成之菌種群中之菌種的細菌；或睾丸酮叢毛單胞 菌 ATCC 2791 1 菌株。 〇 【發明内容】 欲有效利用資源，希望從生質柴油廢液中製造有用的物 質。 本發明之目的係提供一種從甘油中製造蘋果酸之方法， 係在包含甘油的培養基中培養選自由木糖氧化無色桿菌、 天牛微桿菌、m〇deratus鏈黴菌及莓實假單胞菌組成之菌種 群之菌種的細菌；或睾丸酮叢毛單胞菌ATCC 2791 1菌株 V (以下，該等細菌統稱為”用於本發明之製造蘋果酸的細菌”， 〃 或簡稱為”製造蘋果酸的細菌"），特別係在需氧條件下培養 肖細菌’或係將該細菌細胞、該細菌之經加工細胞或其經 固定產物與甘油接觸。 即’本發明係提供以下内容·· 種從甘油製造蘋果酸之方法，該方法包含： 在含甘油的培養基中培養具有從甘油製造蘋果酸之能力 且屬於選自由木糖氧化無色桿菌、天牛微桿菌、则如咖 鏈黴菌及每實假單胞菌組成之菌種群之菌種的細菌；或 131378.doc 200914620 將該細菌細胞、該細菌之經加工細胞或其經固定產物與 甘油接觸； 2. 如上述1之方法，其中屬於選自由木糖氧化無色桿 菌、天牛微桿菌、m〇deratUS鏈黴菌及莓實假單胞菌組成 之菌種群中之菌種的該細菌為木糖氧化無色桿菌nbrc 15126菌株、天牛微桿菌JCM 1352菌株、m〇deratus鏈黴菌 NBRC 13432菌株或每實假單胞菌jcm 205 52菌株； 3. —種從甘油製造蘋果酸之方法，該方法包含： 在含甘油的培養物培養基中培養睾丸酮叢毛單胞菌 ATCC 2791 1 菌株，或 將該細菌細胞，該細菌之經加工細胞或其經固定產物與 甘油接觸； 4·如上述1至3中任一項之方法，其中該細菌在需氧條件 下培養； 5·如上述1至4中任一項之方法，進一步包含從培養所得 之培養物中回收蘋果酸；及 6.如上述1至5中任一項之方法，其中甘油係來自於生質 柴油廢液。 、 【實施方式】 ‘ 用於本發明之製造蘋果酸的細菌之實例為上述1至3中所 述的菌屬、菌種及菌株。在該等製造蘋果酸的細菌中，較 佳細菌為能以更高收率從甘油製造蘋果酸者。該等細菌之 實例包含天牛微桿菌JCM 1352菌株、木糖氧化無色桿菌 NBRC 15126菌株、睾丸酮叢毛單胞菌ATCC 279ιι菌株、 131378.doc 200914620 moderatus鏈黴菌NBRC 13432菌株及莓實假單胞菌JCM 20552菌株，但不限於此。木糖氧化無色桿菌NBRC 15126 菌株、睾丸_叢毛單胞菌ATCC 2791 1菌株、moderatus鍵 黴菌NBRC 13432菌株及莓實假單胞菌JCM 20552菌株較 佳，且睾丸酮叢毛單胞菌ATCC 27911菌株、m〇deratus鏈 黴菌NBRC 13432菌株及莓實假單胞菌JCM 20552菌株更 * 佳。 文中，以”更高收率”製造蘋果酸表示在培養物培養基中 以約〇. 1 g/L或更高之收率從甘油中累積蘋果酸。一般，收 率越高越好。例如，在培養物培養基中以約0.2 g/L或更 高，較佳為約0.5 g/L或更高之收率累積蘋果酸。 木糖氧化無色桿菌NBRC 1 5 126菌株及moderatus鍵徽菌 NBRC 13432 菌株購自 Chiba 292-0818 日本，Kisarazu-shi， 2-5-8 Kazusakamatari，國家技術與評價學院的合併管理機 構，生物技術部門，生物資源中心。睾丸酮叢毛單胞菌 ATCC 2791 1 菌株購自 VA 20108 USA ，Manassas ，P.O.-LetterS (1993) 15(3): 272-276 discloses a method for producing D-malic acid by suspending fine cells of D,L-malic acid. However, it is still unknown whether the following bacteria can produce malic acid from glycerol: from Achromobacter xylosoxydans, Miicrobacterium saperdae, Streptomyces m〇deratus and raspberry Bacteria of the species in the population of bacteria consisting of Pseud〇m〇nas fragi; or strains of C. testosteroni ATCC 2791 1 . 〇 [Summary of the Invention] In order to use resources efficiently, it is desirable to produce useful substances from biodiesel waste liquid. The object of the present invention is to provide a method for producing malic acid from glycerol, which comprises culturing a medium selected from the group consisting of Achromobacter xylosoxidans, Microbacterium oxysporum, Streptomyces mstderatus and Pseudomonas granulosus in a medium containing glycerol. a bacterium of the species of the bacterium; or a strain of C. testosteroni ATCC 2791 1 (hereinafter, these genus are collectively referred to as "the bacterium for producing malic acid of the present invention", 或 or simply "to make an apple" The acid bacteria "), in particular, the culture of the bacterium under aerobic conditions or the bacterial cells, the processed cells of the bacterium or the immobilized product thereof are contacted with glycerin. That is, the present invention provides the following contents·· A method for producing malic acid from glycerol, the method comprising: cultivating a glycerol-containing medium having the ability to produce malic acid from glycerol and belonging to a bacterium selected from the group consisting of Achromobacter xylosoxidans, Microbacterium bacillus, such as Streptomyces fabri, and each a bacterium of the species of the bacterium of the genus Pseudomonas; or 131378.doc 200914620 contacting the bacterial cell, the processed cell of the bacterium, or the immobilized product thereof with glycerin 2. The method according to the above 1, wherein the bacterium belonging to the genus selected from the group consisting of Achromobacter xylosoxidans, Microbacterium oxysporum, M〇deratUS Streptomyces, and Pseudomonas syringae is xylose Achromobacter oxidans nbrc 15126 strain, Microbacterium bacillus JCM 1352 strain, M〇deratus Streptomyces NBRC 13432 strain or each Pseudomonas sp. jcm 205 52 strain; 3. A method for producing malic acid from glycerol, the method comprising : culturing the C. testosteroni ATCC 2791 1 strain in a glycerol-containing culture medium, or contacting the bacterial cell, the processed cell of the bacterium or the immobilized product thereof with glycerin; 4· as in the above 1 to 3 Or a method of any one of the above 1 to 4, further comprising recovering malic acid from the culture obtained by the culture; and 6. A method according to any one of the preceding claims, wherein the glycerin is derived from a biomass diesel waste liquid. [Embodiment] The examples of the bacteria for producing malic acid used in the present invention are the genus and bacteria described in the above 1 to 3. And strains Among the bacteria producing malic acid, preferred bacteria are those capable of producing malic acid from glycerol in a higher yield. Examples of such bacteria include Mycobacterium annua JCM 1352 strain, Achromobacter xylosoxidans NBRC 15126 strain, testosterone tube hair Monocytogenes ATCC 279 ι strain, 131378.doc 200914620 moderatus Streptomyces NBRC 13432 strain and Pseudomonas syringae JCM 20552 strain, but not limited thereto. Xylose oxidizing Achromobacter NBRC 15126 strain, Testicular Phytophthora 2791 1 strain, Moderatus sclerotium NBRC 13432 strain and Pseudomonas syringae JCM 20552 strain are preferred, and C. testosteroni ATCC 27911 strain, M〇deratus Streptomyces NBRC 13432 strain and Pseudomonas syringae JCM The 20552 strain is better*. Herein, the production of malic acid in "higher yield" means that malic acid is accumulated from glycerol in a culture medium at a yield of about 1 g/L or more. In general, the higher the rate, the better. For example, malic acid is accumulated in the culture medium in a yield of about 0.2 g/L or higher, preferably about 0.5 g/L or higher. Achromobacter xylosoxidans NBRC 1 5 126 strain and Moderatus bacillus NBRC 13432 strain purchased from Chiba 292-0818 Japan, Kisarazu-shi, 2-5-8 Kazusakamatari, the merger of the National Institute of Technology and Evaluation, Biotechnology Department , the Biological Resource Center. The testosterone testosterone ATCC 2791 1 strain was purchased from VA 20108 USA, Manassas, P.O.-
G BOX 1549，美國菌種寄存中心。天牛微桿菌JCM 1352菌 株及莓實假單胞菌JCM 20552菌株購自日本，8&以111&351-0198，Wako，2-1 Hirosawa，RIKEN生物資源中心，曰本 - 微生物寄存中心/微生物部門。 用於本發明之製造蘋果酸的細菌不僅可為野生型菌株， 亦為任意給定的天然產生或人工突變株，包含經X-射線輻 射、紫外線輻射或化學誘變劑（例如N -甲基-Ν'硝基-N-亞 硝基胍）處理得到者，或經基因工程技術（例如細胞融合及 131378.doc 200914620 口因重！）知·到的重組體。重組體的宿主可屬於任意菌， 只要其為可轉化的微生物。但是，宿主較佳屬於與產生標 土因之親株相同的ϋ。宜選擇具有從甘油轉化成韻果酸 之增強轉化能力的製造蘋果酸之細菌。 、含甘油的培養物培養基可為藉由加人純甘油或含甘油的 & σ物所得到之培養基。在含甘油的混合物中，除甘油以 外之組分或其含量較佳對用於本發明之製造蘋果酸的細菌 不具有有害作用。含甘油的混合物來源不特別限制，但是 為有效利用資源較佳使用生質柴油廢液。 -種製造生質柴油燃料之方法為藉由驗催化劑醇解甘油 三醋製造脂肪酸甲醋（FAME)。在該方法中，含甘油的廢 液係以副產物（稱爲生質柴油廢液）被製造。該廢液一般受 催化劑 '未轉化的脂肪酸（其隨所使用的油而不同）及類似 物污染。例如，下述實例中使用的生質柴油廢液的組成爲 甘油：5”/。'曱醇：11%、氫氧化鉀：8%、水：作。，其他 例如甘油醋· 26%。亦可使用含有不定組成的生質柴油廢 液，例如在S. Papanikolaou等人，生物資源技術 (2002)82:43-49中描述之甘油：65%、鉀/納鹽：4至5%、 甲醇.1/〇、水.28%，及在M. Gonzalez-Pajuelo等人，j IndMierobiol Biotechnol(2004)31:442-446 中描述之甘油. 65%、鈉鹽：5%或更少。 當生質柴油廢液添加於本發明方法之培養基中時，可產 生與加入純甘油情況下相同或更高收率及轉化率之蘋果 酸0 131378.doc 200914620 务月方法的培養基可為任意種類， 養細菌所必需的一般组分，日要/、包3培 "以㈣特定料基。在本發 養基中 具有含碳源、兔源及無機鹽的簡單組成的培 養基中仍可得到蘋果酸。 用於本發明方法的培養基包含甘油作爲碳源。培養基中 含甘油濃度可在對細菌生長及顏 圍内適當選擇，是，無副作用的範 至遍/τ 疋n約0.1至500 g/L，較佳為約！ gL。當生質柴油廢液作爲甘油源使用時，根據廢液 :㈣度而定’可稀釋廢液或加甘油於其中直至培養基 中甘油含量落入上述範圍内。 。養基可含有除甘油以外的物質作爲碳源，但苴含 :制在不影響由甘油製造成蘋果酸之程度。用於ς的 碳源實例為葡萄糖、果糖、殿粉、乳糖、阿拉伯糖、： :旦，精、糖蜜、及麥芽提取物，但不限於此。其他碳源 ::較佳為甘油的㈣重量㈣更少…圭為㈣輯 少。培養基最佳含有甘油作爲單一碳源。 硝=源實例包含無機氮化合物例如氨、硫酸鞍、氯化錢及 銨、尿素及類似物。亦可於培養物培養基中加入有機 酵源=如，縠朊粉、棉籽粉、大豆粉、玉米漿、乾酵母、 纟、提取物、蛋白脒、肉提取物及酪蛋白胺基酸。 曰2合使用碳源及氮源較有利。因包含微量生長因子及大 =機營養物的低純度來源亦適用’所以無需以純態使用 口系等〇 據两要，可使用無機鹽，例如磷酸二氫鉀、磷酸氫二 131378.doc 200914620 鉀、氯化鈉、硫酸鎂、硫酸錳、碳酸鈣、氯化鈣、碘化 鈉、碘化鉀、及氯化鈷。根據需要，亦可加入消泡劑，例 如液體石蠟、高級醇、植物油、礦物油及矽酮，特別在培 養基顯著發泡之情況下。 其他組分例如多種維生素可根據需要加入培養基中。 氮源、無機鹽及其他組分爲熟悉該技術者所知。 在本發明中，可在厭氧條件下培養製造蘋果酸的細菌， 但是在需氧條件下進行較佳。需氧條件指在分子氧存在下 培養。可進行通氣、料及振盪提供減。可使用培養微 生物的任意常用裝置。在需氧條件下進行培養之情況，本 發明方法可不使用產生厭氧條件所必需的任意裝置以簡易 方式培養細菌並製造蘋果酸。 乂厭氧條件下培養細菌可藉由引人二氧化碳或惰性氣體 (氮氣、氬氣等）或無需通氣進行。 頻果酸的大量製造較佳在液體淹沒培養條件下進行。杂 細菌在大槽中繁殖時，較佳在生長期將該等細菌接種至二 造槽中’以避免在製造蘋果酸方法期間延遲繁殖。即，該 等細菌較佳先接種至相對少量的培養基中，且在 : :以製造菌種，然後以無菌方式將該等菌種轉移至大槽 坧養溶液的攪拌及通氣 螺旋槳或類似於螺旋槳的機械性攪拌可藉由 或振盪、㈣置、發酵槽的旋轉 …，滅菌空氣通入培養溶 進仃。在操作中，通氣亦可產生攪掉效果。 131378.doc 200914620 在液體淹沒培養基中培養的情況下，培養方法例如分抵 培養、床分批培養及連續培養，可適t選擇及使用。 培養條件為任意的，^要其適㈣翻於本發明之製造 顏果酸的細菌。例如，培養溫度為約4至40〇C ’較佳為約 20至3rc。培養基pH為約5至9，較佳為約6至8。當培養基 PH隨蘋果酸的製造下降時，可根據需要，加入驗⑽如氨 水溶液、碳_、氫氧化鈉及氫氧化卸）至培養系統中， 以調整其pH落入以上範圍内。 Ο 培養基組成及其他培養條件可由熟悉該技術者適當調 整。亦可考慮調整條件以進__步提高蘋果酸收率。 用於本發明之方法的細菌可採用該細菌細胞、經加工細 菌細胞或其經固定產铷。七+ 文中，、，座加工細菌細胞為經破裂 的細菌細胞或從經培養的物質（包含細菌細胞及培養上層 液）中提取的酶。經力& A ^ 、 、、、田邊細胞的實例包含經有機酸（例G BOX 1549, American Type Culture Center. Microbacterium bacillus JCM 1352 strain and Pseudomonas syringae JCM 20552 strain purchased from Japan, 8 & 111 & 351-0198, Wako, 2-1 Hirosawa, RIKEN Biological Resource Center, 曰本 - microbial deposit center / microorganism department. The malic acid producing bacteria used in the present invention may be not only a wild type strain but also any given naturally occurring or artificial mutant strain, including X-ray radiation, ultraviolet radiation or a chemical mutagen (for example, N-methyl group). - Ν 'Nitro-N-nitrosoguanidine) treatment of the recipient, or recombinants known by genetic engineering techniques (such as cell fusion and 131378.doc 200914620 mouth weight!). The host of the recombinant may belong to any bacteria as long as it is a transformable microorganism. However, the host preferably belongs to the same sputum as the parent strain producing the standard. It is preferred to select a malic acid-producing bacterium having an enhanced conversion ability from glycerol to vicinogenic acid. The glycerol-containing culture medium may be a medium obtained by adding pure glycerin or glycerin-containing & σ. In the glycerin-containing mixture, components other than glycerin or a content thereof are preferably not harmful to the bacteria for producing malic acid of the present invention. The source of the glycerin-containing mixture is not particularly limited, but it is preferable to use the raw diesel waste liquid for efficient use of resources. A method of producing a biodiesel fuel is to produce fatty acid methyl vinegar (FAME) by calcination of glycerol triacetate by a catalyst. In this method, the glycerin-containing waste liquid is produced as a by-product (referred to as a biomass diesel waste liquid). This effluent is generally contaminated by the catalyst 'unconverted fatty acids, which vary with the oil used, and the like. For example, the composition of the biodiesel waste liquid used in the following examples is glycerin: 5" /. 'sterol: 11%, potassium hydroxide: 8%, water: for others. Others such as glycerin vinegar · 26%. A biomass diesel waste liquid having an indefinite composition can be used, for example, glycerin described in S. Papanikolaou et al., Bioresource Technology (2002) 82: 43-49: 65%, potassium/salt salt: 4 to 5%, methanol .1/〇, water. 28%, and glycerin described in M. Gonzalez-Pajuelo et al., j Ind Mierobiol Biotechnol (2004) 31: 442-446. 65%, sodium salt: 5% or less. When the diesel fuel waste liquid is added to the medium of the method of the present invention, the same or higher yield and conversion rate of malic acid can be produced in the case of adding pure glycerin. The medium of the method can be any kind, and the medium can be raised. The general components necessary for bacteria, daily, and 3 cultivating " to (4) specific material base. Malic acid can still be obtained in a medium having a simple composition of carbon source, rabbit source and inorganic salt in the present hair base. The medium used in the method of the present invention contains glycerin as a carbon source. The medium contains glycerol in the concentration of bacteria. Appropriate selection in the length and the skin circumference is: the range of no side effects / τ 疋 n is about 0.1 to 500 g / L, preferably about! gL. When the raw diesel waste liquid is used as a glycerin source, according to the waste liquid : (4) Degrees may be diluted with waste liquid or glycerin until the glycerin content in the medium falls within the above range. The nutrient may contain substances other than glycerol as a carbon source, but the : contains: does not affect the glycerin The degree of manufacture of malic acid. Examples of carbon sources for strontium are glucose, fructose, house powder, lactose, arabinose,: denier, molasses, and malt extract, but are not limited thereto. Other carbon sources: Preferably, the weight of the glycerin is (4), the weight is less, and the weight is less. The medium is preferably glycerol as a single carbon source. The nitrate source example includes inorganic nitrogen compounds such as ammonia, sulfuric acid saddle, chlorinated money and ammonium, urea and Analogs. Organic yeast sources can also be added to the culture medium = for example, glutinous rice flour, cottonseed meal, soy flour, corn syrup, dry yeast, alfalfa, extract, peptone, meat extract and casein amino acid. It is advantageous to use carbon sources and nitrogen sources together. Low-purity sources containing micro-growth factors and large-sized nutrients are also suitable for 'so there is no need to use oral systems in pure form. Inorganic salts such as potassium dihydrogen phosphate, hydrogen phosphate II 131378.doc 200914620 potassium, Sodium chloride, magnesium sulfate, manganese sulfate, calcium carbonate, calcium chloride, sodium iodide, potassium iodide, and cobalt chloride. Defoaming agents such as liquid paraffin, higher alcohols, vegetable oils, mineral oils, etc. may also be added as needed. Anthrone, especially in the case of significant foaming of the medium. Other components such as multivitamins may be added to the medium as needed. Nitrogen sources, inorganic salts and other components are known to those skilled in the art. In the present invention, malic acid-producing bacteria can be cultured under anaerobic conditions, but it is preferably carried out under aerobic conditions. Aerobic conditions refer to culture in the presence of molecular oxygen. Ventilation, material and oscillation can be provided to reduce. Any conventional device for culturing microorganisms can be used. In the case of cultivation under aerobic conditions, the method of the present invention can culture bacteria and produce malic acid in a simple manner without using any means necessary for producing anaerobic conditions. The culture of bacteria under anaerobic conditions can be carried out by introducing carbon dioxide or an inert gas (nitrogen, argon, etc.) or without aeration. The mass production of frequency fruit acid is preferably carried out under liquid submerged culture conditions. When the bacteria are propagated in a large tank, it is preferred to inoculate the bacteria into the second tank during the growing period to avoid delaying the propagation during the method of producing malic acid. That is, the bacteria are preferably first inoculated into a relatively small amount of medium, and are: : to produce strains, and then aseptically transfer the strains to a stirred and ventilated propeller of a large tank solution or similar to a propeller The mechanical agitation can be carried out by oscillating, oscillating, (four) setting, rotation of the fermentation tank, and sterilizing air into the culture solution. In operation, ventilation can also produce a scuffing effect. 131378.doc 200914620 In the case of culturing in a liquid submerged medium, the culture method can be selected and used, for example, by subculture, bed batch culture, and continuous culture. The culture conditions are arbitrary, and it is necessary to (4) turn over the bacteria for producing anaphoric acid of the present invention. For example, the culture temperature is about 4 to 40 ° C ', preferably about 20 to 3 rc. The pH of the medium is from about 5 to 9, preferably from about 6 to 8. When the pH of the medium decreases with the production of malic acid, the test (10) such as aqueous ammonia solution, carbon_, sodium hydroxide and hydroxide can be added to the culture system as needed to adjust the pH to fall within the above range.培养基 The composition of the medium and other culture conditions can be appropriately adjusted by those skilled in the art. It is also conceivable to adjust the conditions to increase the yield of malic acid. The bacteria used in the method of the present invention may be produced by the bacterial cells, the processed bacterial cells or the fixed calves. In the seventh + text, the processing bacterial cells are ruptured bacterial cells or enzymes extracted from cultured substances (including bacterial cells and culture supernatant). Examples of meridian & A ^ , , , , and Tanabe cells include organic acids (eg
如丙剩及乙醇^冷康乾燥或驗處理經培養的細菌細胞得 到者、經物理或酶化學破裂細菌細胞得到者、或從中分離 或提取的粗製酶。具體言之，經培養的細®經離心處理， 收集的細胞經物理磨碎方法（例如超聲波、Dyno_millA 理)或使用表面活性劑或分解酶(例如溶菌酶） 六子Ιέ方法破裂。所得溶液經離心或膜過滤以去除不 冷物2所侍的無細胞提取物經分離/純化方法 離子交換層析法、陰離子交 過遽層析法及金屬養層析法、凝膠 頌耸口層析法，以分餾及純化酶。 入 用；層析法的载體實例包含不溶性聚合物載體例如引 131378.doc •12- 200914620 叛甲基（CM)、二乙胺基乙基（deae)、苯基或丁基的纖維 素糊精及填脂糖。亦可使用市售的填有載體的管柱。細 菌細胞的破裂及酶的提取可藉由熟悉該技術者所知之方法 及上述方法進行。 將細菌細胞、經加工的細菌細胞或其經固定產物與甘油 接觸之實例如下。 使用細菌細胞或經加工細菌細胞從甘油製造蘋果酸之方For example, the residual bacteria and ethanol are used to dry or test the cultured bacterial cells, the physical or enzymatically ruptured bacterial cells, or the crude enzyme isolated or extracted therefrom. Specifically, the cultured fine® is centrifuged, and the collected cells are disrupted by a physical grinding method (for example, ultrasonic wave, Dyno_millA) or by a surfactant or a decomposing enzyme (for example, lysozyme). The obtained solution is subjected to centrifugation or membrane filtration to remove the cell-free extract of the non-cold material 2 by separation/purification method, ion exchange chromatography, anion exchange chromatography and metal growth chromatography, gel enthalpy Chromatography to fractionate and purify the enzyme. An example of a carrier for chromatography includes an insoluble polymer carrier such as a cellulose paste of 131378.doc •12-200914620 demethyl (CM), diethylamine (deae), phenyl or butyl. Fine and fat-filled sugar. Commercially available columns filled with a carrier can also be used. The disruption of the bacterial cells and the extraction of the enzyme can be carried out by a method known to those skilled in the art and the above methods. Examples of contacting bacterial cells, processed bacterial cells or immobilized products thereof with glycerin are as follows. The use of bacterial cells or processed bacterial cells to produce malic acid from glycerol
法實例為在含甘油的受質溶液中懸浮細«細胞並反應之方 法。細菌細胞經培養製造蘋果酸的細菌，隨後離心而製 備。受質溶液中甘油濃度較佳為約〇 〇1至5〇重量％。反應 溫度一般為約4至，且較佳為約20至3rc。反應溶液 之pH -般為約5至9’且較佳為約6至8。當培養基爾頻 果酸的製造下降時，可根據需要，加入驗(例如氨水溶 液、碳酸弼、氫氧化鈉及氫氧化卸）至培養系統中，以調 整其pH落入以上範圍内。 使用經固定細菌細胞或經蚊的加工細菌細胞從甘油製 造蘋果酸之方法係例如將經㈣之細菌細胞或經固定的加 工細菌細胞填入管柱中’使含甘油的受質溶液通過該管柱 之方法。細菌細胞或經加工細菌細胞係藉由培養製造蘋果 酸的細菌，然後離心而得到。固定細菌細胞的方法例如使 用凝膠的廣泛固定法、及擔持離子交換材料的固定法。可 使用的凝膠實例包含鹿角菜膠、瓊脂、甘露聚糖、Μ及 聚丙稀醯胺凝膠。凝膠的適宜粒度以直徑計為約聰， 但其大小隨㈣種類而異。離子交換材料的實例包含以纖 131378.doc -13- 200914620 維素爲主的材料、以苯乙烯二乙烯基苯爲主的材料及以笨 齡甲路爲主的離子交換材料。受質溶液中甘油濃度較佳為 約0.01至50重量％。亦可加人纽化合物（例如驗基乙醇、半 胱胺酸及穀胱甘肽）、還原劑（例如亞硫酸鹽）、及酶活化劑 (例如鎖離子及㈣子）。該溶液通過的速度隨管柱大小及 經固定物質#而變化。作爲處理溶液的速度指帛，空間速 度（ml/ml樹脂.hr)適宜為〇 〇5至1〇。 蘋果酸的分離及純化按照傳統已知方法進行。例如，蘋 果酸可藉由以下方法分離並純化，例如溶劑提取、離子交 換層析、藉由不溶性處理的分級沉澱、藉由結晶的分級結 晶、藉由逆滲透膜的膜分離、及濃縮結晶方法。具體言 之，例如在培養結束後進行培養液的過濾或分離，以得到 上層液。從該上層液中，蘋果酸經濃縮結晶，或在視需要 酸處理該上層液後使用活性碳而純化，但是蘋果酸的分離 及純化不限於此。可根據日本未審查專利公開號2〇〇1_ 1 97897中描述的方法分離及純化蘋果酸。 本發明經以下實例進一步闡述。應理解本發明不限於該 等實例’可在本發明範圍内作多種修改。 實例 實例1 木糖氧化無色桿菌NBRC 15126菌株塗佈於瓊脂培養基A 上，該培養基用於平板培養（組成：3 g磷酸二氫鉀、6 §磷 酸氫二鈉、0.5g氯化鈉、1 g氯化銨、492 mg七水合硫酸 鎂、147 mg二水合氣化鈣、100 mg酵母提取物、1〇 §甘 131378.doc -14· 200914620 油、20 g瓊脂及1 L蒸餾水（最終pH為7 4))，並使其在3〇<)(： 下靜置4天。在上述平板上生長的菌株藉由鉑環接種至3 mL培養基B中，該培養基B用於試管培養（組成··與瓊脂培 養基A相同的組成’但不含氣化鈣、酵母提取物及璦脂）， 且在30°C下以200 rpm振盪培養（預培養小時。3〇叫上 述生長的菌株培養液轉移至3 mL培養基C中，該培養基C 用於試管培養（組成：與用於試管培養的上述培養基B相同 的組成，但代替10 g甘油含有19 6 g之製造生質柴油燃料 的副產物的甘油顧分（甘油：5 1 %、甲醇：11 %、氫氧化 鉀：8%、水：4%、及其他包含甘油酯：26%))，並在30°C 下以200 rpm振盪培養（主培養）。開始反應4天後，消耗〇4 g甘油且每1升累積〇·1 g蘋果酸。 實例2 木糖氧化無色桿菌NBRC 15126菌株塗佈於瓊脂培養基a 上’ s亥培養基用於平板培養（組成：3 g雄酸二氫鉀、6 g麟 酸氫二納、0.5 g氣化鈉、1 g氯化銨、492 mg七水合硫酸 鎂、147 mg二水氯化鈣、100 mg酵母提取物、1〇 g甘油、 2〇 g瓊脂及1 L蒸餾水（最終pH為7.4))，並使其在3(TC下靜 置4天。在上述平板上生長的菌株藉由鉑環接種至3 培 養基D中’該培養基D用於試管培養（組成：與瓊脂培養基 A相同的組成，但不含瓊脂）’並在3〇。〇下以2〇〇 rpm振盪 培養（預培養）6天。30 μί上述生長的菌株培養液轉移至3 mL培養基£中，該培養基Ε用於試管培養（組成··與用於試 ^培養的上述培養基D相同的組成’但代替1 〇 g甘油含有 131378.doc -15- 200914620 19.6 g之製造生質柴油燃料的副產物的甘油餾分（甘油： 51 %、甲醇：11 %、氫氧化鉀：8%、水：4%、及其他包含 甘油酯：26。/。））’並在3(TC下以200 rpm振盪培養（主培 養）。開始反應6天後，消耗1.3 g甘油且每1升累積〇 4 g頻 果酸。 實例3 睾丸酮叢毛單胞菌ATCC 2791 1菌株以如實例1的相同方 式反應。結果，消耗2.6 g甘油且每1升累積〇.5 g蘋果酸。 實例4 天牛微桿菌JCM 1352菌株塗佈於瓊脂培養基a上，該培 養基用於平板培養（組成：3 g磷酸二氫鉀、6 §磷酸氫二 鈉、〇·5 g氣化鈉、1 g氣化銨、492 mg七水合硫酸鎂、147 mg二水合氣化鈣、1〇〇 mg酵母提取物、1〇 g甘油、2〇吕瓊 脂及1 L蒸餾水（最終？11為7.4))，並使其在3〇t：下靜置4 天。在上述平板上生長的菌株藉由鉑環接種至3 培養基 D中，該培養基D用於試管培養（組成：與瓊脂培養基a相 同的組成’但不含瓊脂），且在30°C下以200 rpm振盪培養 (預培養）2天。30吣上述生長的菌株培養液轉移至3 mL培 養基D中’該培養基D用於試管培養，並在3(TC下以200 P振盈培養（主培養）。開始反應4天後，消耗0.4 g甘油且 每1升累積0.1 g蘋果酸。 實例5 舉丸_叢毛單胞菌ATCC 2791 1菌株以如實例3的相同方 式反應但預培養期間從2天改爲6天，且主培養期間從4 131378.doc •16- 200914620 天改爲6天。結果 酸0 消耗2.3 g甘油且每1升 累積0.2 g蘋果 實例6 莓實假單㈣觀则巧株^實例_目同方式反 應。結果’消耗4.4g甘油且每！升累積〇3蜱 實例7An example of a method is a method of suspending fine «cells in a glycerol-containing substrate solution and reacting. Bacterial cells are cultured to produce malic acid bacteria, which are then centrifuged to prepare. The concentration of glycerin in the substrate is preferably from about 1 to about 5 % by weight. The reaction temperature is usually from about 4 to about 1, and preferably from about 20 to 3 rc. The pH of the reaction solution is generally from about 5 to 9' and preferably from about 6 to 8. When the production of the medium is decreased, the test (e.g., aqueous ammonia solution, cesium carbonate, sodium hydroxide, and hydroxide) may be added to the culture system as needed to adjust the pH to fall within the above range. A method for producing malic acid from glycerol using a fixed bacterial cell or a processed bacterial cell of a mosquito, for example, by filling a bacterial cell (4) or a fixed processed bacterial cell into a column, and passing the glycerin-containing substrate through the tube The method of the column. Bacterial cells or processed bacterial cell lines are obtained by culturing bacteria producing malic acid and then centrifuging. The method of immobilizing bacterial cells is, for example, a method of immobilizing a gel and a method of immobilizing an ion exchange material. Examples of gels that can be used include carrageenan, agar, mannan, guanidine, and polyamidamide gel. The suitable particle size of the gel is about the diameter of the rod, but its size varies with the type of (4). Examples of the ion exchange material include a material mainly composed of fiber 131378.doc -13-200914620, a material mainly composed of styrene divinylbenzene, and an ion exchange material mainly composed of an aging road. The concentration of glycerin in the substrate is preferably from about 0.01 to 50% by weight. It is also possible to add human compounds (such as arginine, cysteine and glutathione), reducing agents (such as sulfites), and enzyme activators (such as locked ions and (iv)). The rate at which the solution passes varies with the size of the column and by the immobilized material #. As the speed index of the treatment solution, the space velocity (ml/ml resin.hr) is suitably 〇 5 to 1 Torr. The separation and purification of malic acid is carried out in accordance with a conventionally known method. For example, malic acid can be isolated and purified by, for example, solvent extraction, ion exchange chromatography, fractional precipitation by insoluble treatment, fractional crystallization by crystallization, membrane separation by reverse osmosis membrane, and concentrated crystallization method. . Specifically, for example, filtration or separation of the culture solution is carried out after completion of the culture to obtain an supernatant liquid. From the supernatant liquid, malic acid is concentrated and crystallized, or purified by using activated carbon after acid treatment of the supernatant liquid as necessary, but separation and purification of malic acid are not limited thereto. The malic acid can be isolated and purified according to the method described in Japanese Unexamined Patent Publication No. Hei No. Hei. The invention is further illustrated by the following examples. It is to be understood that the invention is not limited to the examples, and various modifications may be made within the scope of the invention. EXAMPLES Example 1 Achromobacter xylosoxidans NBRC 15126 strain was spread on agar medium A for plate culture (composition: 3 g potassium dihydrogen phosphate, 6 § disodium hydrogen phosphate, 0.5 g sodium chloride, 1 g Ammonium chloride, 492 mg magnesium sulfate heptahydrate, 147 mg calcium carbonate dihydrate, 100 mg yeast extract, 1 〇 甘 131378.doc -14· 200914620 oil, 20 g agar and 1 L distilled water (final pH 7 4)), and allowed to stand at 3 〇 <) (: 4 days. The strain grown on the above plate was inoculated into 3 mL of medium B by a platinum ring, and the medium B was used for test tube culture (composition· · The same composition as agar medium A 'but not containing calcium carbonate, yeast extract and rouge", and shaking culture at 200 rpm at 30 ° C (pre-culture hour. 3 〇 called the above-mentioned growth of the strain culture solution transfer To 3 mL of medium C, the medium C was used for tube culture (composition: the same composition as the above-mentioned medium B for tube culture, but instead of 10 g of glycerol containing 19 6 g of glycerin which is a by-product of the manufacture of biodiesel fuel Points (glycerol: 5 1 %, methanol: 11%, potassium hydroxide: 8%, water: 4%, and others containing glycerides: 26%)), and shaking culture at 200 rpm at 30 ° C (main culture). After 4 days from the start of the reaction, 〇 4 g of glycerol was consumed and accumulated per liter·1 g Malic acid. Example 2 Xylose-oxidized Achromobacter NBRC 15126 strain was coated on agar medium a 'shai medium for plate culture (composition: 3 g potassium dihydrogen dihydrogenate, 6 g hydrogen dihydrogen hydride, 0.5 g gas Sodium, 1 g ammonium chloride, 492 mg magnesium sulfate heptahydrate, 147 mg calcium chloride dihydrate, 100 mg yeast extract, 1 g g glycerol, 2 g g agar and 1 L distilled water (final pH 7.4) And allowed to stand at 3 (TC for 4 days. The strain grown on the above plate was inoculated into 3 medium D by a platinum ring'. This medium D was used for tube culture (composition: the same composition as agar medium A, But without agar)' and at 3〇. shake culture (pre-culture) at 2 rpm for 6 days. 30 μί of the above-mentioned strain culture was transferred to 3 mL of medium, which was used for tube culture. (Composition··The same composition as the above-mentioned medium D for the test culture' but instead of 1 〇g glycerol contains 131378.doc -15 - 200914620 19.6 g of glycerin fraction of by-products of biodiesel fuel production (glycerol: 51%, methanol: 11%, potassium hydroxide: 8%, water: 4%, and others containing glycerides: 26%) 'and cultured at 3 rpm with shaking at 200 rpm (main culture). After 6 days from the start of the reaction, 1.3 g of glycerol was consumed and 4 g of cretroic acid was accumulated per liter. Example 3 The C. testosteroni ATCC 2791 1 strain was reacted in the same manner as in Example 1. As a result, 2.6 g of glycerin was consumed and .5 g of malic acid was accumulated per 1 liter. Example 4 Microbacterium Bacillus subtilis JCM 1352 strain was spread on agar medium a for plate culture (composition: 3 g potassium dihydrogen phosphate, 6 § disodium hydrogen phosphate, 〇·5 g sodium gas, 1 g Ammonium vapor, 492 mg of magnesium sulfate heptahydrate, 147 mg of calcium carbonate dihydrate, 1 mg of yeast extract, 1 g of glycerol, 2 g of agar and 1 L of distilled water (final? 11 is 7.4)), and Let it sit for 4 days at 3〇t:. The strain grown on the above plate was inoculated into 3 medium D by a platinum ring, which was used for test tube culture (composition: the same composition as agar medium a but no agar), and 200 at 30 ° C The culture was incubated by shaking at rpm for 2 days. 30 吣 The above-mentioned grown strain culture medium was transferred to 3 mL of medium D. The medium D was used for tube culture, and cultured at 200 P in 3 (TC) (main culture). After 4 days from the start of the reaction, 0.4 g was consumed. Glycerol and 0.1 g of malic acid per 1 liter. Example 5 Pills - C. mobilis ATCC 2791 1 strain was reacted in the same manner as in Example 3 but changed from 2 days to 6 days during pre-culture, and during the main culture period 4 131378.doc •16- 200914620 days changed to 6 days. Results acid 0 consumes 2.3 g of glycerol and accumulates 0.2 g per 1 liter of apple. Example 6 Raspberry real fake single (four) view is a good case ^ instance _ the same way to react. Results ' Consumes 4.4 g of glycerol and accumulates per liter of 蜱3蜱 Example 7
莓實假單胞㈣Μ則巧株以如實例2的相同方式反 應。結果’消耗4.5 g甘油且每!升累積〇8§蘋果酸。 實例8 莓實假單胞菌JCM 20552菌株以如實例4的相同方式反 應。結果，消耗8.8 g甘油且每1升累積〇 4 g蘋果酸。 實例9 m〇deratus鏈黴菌NBRC ! 3432菌株以如實例2的相同方式 反應。結果，消耗6.3 g甘油且每1升累積〇.5 §蘋果酸。 工業應用 根據本發明，蘋果酸藉由微生物以簡易方式從甘油（生 質柴油廢液）製造。蘋果酸作爲食品添加劑、除臭劑、助 洗劑、pH調節劑、沐浴粉及類似物使用。因此，本發明用 於從廢物料中製造有用的物質。 131378.docRaspberry real cells (4) were then reacted in the same manner as in Example 2. As a result, 4.5 g of glycerin was consumed and 8 § malic acid was accumulated per liter. Example 8 The Pseudomonas syringae JCM 20552 strain was reacted in the same manner as in Example 4. As a result, 8.8 g of glycerin was consumed and 4 g of malic acid was accumulated per 1 liter. Example 9 M. cerevisiae NBRC! 3432 strain was reacted in the same manner as in Example 2. As a result, 6.3 g of glycerin was consumed and 〇.5 § malic acid was accumulated per 1 liter. Industrial Applicability According to the present invention, malic acid is produced from glycerol (biodiesel waste liquid) in a simple manner by microorganisms. Malic acid is used as a food additive, deodorant, builder, pH adjuster, bath powder and the like. Accordingly, the present invention is useful for making useful materials from waste materials. 131378.doc