經濟部中央標準局員工消費合作社印裂 A7 B7 五、發明説明（1) 發_明背暑 發明節瞳 本發明係關於L 一酮己醣之製造，特定言之，本發明 係關於藉L一果糖及L-塔格糖生成酶分別作用於L-阿 洛酮糖及L —山梨糖以製造L —果糖及L 一塔格糖之方法 Ο 先前枝麩的敘沭 L 一果糖及L 一塔格糖是稀有的單糖，其屬於L 一酮 己醣，在自然界的含量極少。這些單糖原則上可以利用有 機化學的方法製得：L -果糖是利用L -葡萄糖及L 一甘 露糖及鹼性試劑（例如：氫氧化鈉、吡啶等）的共存下製 得。而L—塔格糖則是藉L —半乳糖及在鹼性試劑（與L 一果糖例子中使用者相似）的共存下行異構化反應而得。 但是，要得到作爲L 一果糖及L 一塔格糖之原料的L —葡, 萄糖、L 一甘露糖及L -半乳糖並不容易，而且目前並無 適宜的方法大量產製L —果糖及,L —塔格糖。 本人在 Japanese patent Laid-Open No. 3 08, 984/93 中揭亦一種使用克雷伯氏菌屬（genus K 1 e b s i e 1 la)的細 菌自半乳糖醇製得L 一塔格糖的方法。但是，此一方法需 要相當大量的細胞、製備時間、與繁瑣的純化步驟。因此 ，其尙未作實際的應用。 近年來，生化業發展快速，新糖類的硏發在碳水化合 物化學的領域中需求日亟。L —果糖及L —塔格糖的量產 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐）4 — ^---------od------1---^-I-1- (請先閲讀背面之注意事項再填寫本頁) . 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（2 ) 尙未確立。因此，在食品業、醫藥學、化工業等領域中尙 未用這些單糖作爲工業原料。 發明概沭 本人曾經硏究過如何在工業規模及極低成本下利用生 化方法製得L 一酮己醣（例如：L —果糖及L 一塔格糖） 。結果’不經’意地發現（如揭不於japanese patent 1^丨(5-〇?々11^}〇.125,776/94中者）〇—酮己醣3—表異構 酶作用於L —酮己醣時，特別是作用於l —阿洛酮糖及L —山梨糖時，可以自L 一阿洛酮糖製得L —果糖，並可自 L —山梨糖製得L—塔格糖，也由於此一發現，本人方.得 以完成此一發明。 附圖概沭 附圖爲L一塔格糖之紅外線吸收光譜圖。 發明詳沭 本發明所使用之D —酮己醣3 _表異構酶，可以利用 能夠製造此種D —酮己醣3 _表異搆酶之微生物的培養而 得。 此類微生物的實例爲假單胞菌屬微生物，其包括菊苣 假單胞菌31'—24(?£尺]\/1.6?—2 736)與其 突變體（例舉於 Japanese Patent Laid-Open Νο·266,996 /91 )。 本紙張尺度適用中國國家標準（CNS ) Λ4規格（210X297公釐） ~. —>------- *^3.-- (請先閲讀背面之注意事項再填寫本頁) 、言Employees' cooperative of the Central Standards Bureau of the Ministry of Economic Affairs of the People's Republic of China printed A7 B7 V. Description of the invention (1) _ Ming Beishu invention pupil The invention relates to the manufacture of L-ketohexose, in particular, the invention relates to borrowing L- Method for producing fructose and L-tagatose by using fructose and L-tagatose producing enzymes on L-psicose and L-sorbose respectively Lactose is a rare monosaccharide, which belongs to L-ketohexose, and has very little content in nature. These monosaccharides can in principle be prepared by organic chemistry methods: L-fructose is produced by the coexistence of L-glucose and L-mannose and alkaline reagents (such as sodium hydroxide, pyridine, etc.). L-tagatose is obtained through the coexistence of L-galactose and an alkaline reagent (similar to the user in the L-fructose example). However, it is not easy to obtain L-glucose, glucose, L-mannose and L-galactose as raw materials of L-fructose and L-tagatose, and there is currently no suitable method for mass-producing L- Fructose and, L-Tagatose. In Japanese patent Laid-Open No. 3 08, 984/93, I also disclosed a method for preparing L-tagatose from galactitol using bacteria of the genus Klebsiella (genus K 1 e b s i e 1 la). However, this method requires a relatively large number of cells, preparation time, and tedious purification steps. Therefore, its application has not been applied in practice. In recent years, the biochemical industry has developed rapidly, and the emergence of new sugars is in great demand in the field of carbohydrate chemistry. Mass production of L — fructose and L — Tagatose This paper is sized to the Chinese National Standard (CNS) A4 (210 X 297 mm) 4 — ^ --------- od ------ 1 --- ^-I-1- (Please read the notes on the back before filling out this page). Printed by the Consumers' Cooperative of the Central Standards Bureau, Ministry of Economic Affairs, A7 B7 V. Description of Invention (2) 尙 Not established. Therefore, these monosaccharides have not been used as industrial raw materials in the fields of food industry, medicine, chemical industry and the like. Summary of the Invention I have studied how to produce L-ketohexose (such as L-fructose and L-tagatose) by biochemical methods on an industrial scale and at extremely low cost. The result was found 'inadvertently' (such as those disclosed in Japanese patent 1 ^ 丨 (5-〇? 々11 ^). 125,776 / 94). 0-ketohexose 3-epi-isomerase acts on L-ketone For hexose, especially for L-psicose and L-sorbose, L-fructose can be prepared from L-psicose, and L-tagatose can be prepared from L-sorbose. Because of this discovery, I have been able to complete this invention. The outline of the drawings is the infrared absorption spectrum of L-tagatose. Detailed description of the invention D-ketohexose 3 _episolate used in the present invention A constitutive enzyme can be obtained by culturing a microorganism capable of producing such a D-ketohexose 3-episomerase. Examples of such microorganisms are microorganisms of the genus Pseudomonas, including Pseudomonas chicory 31'- 24 (? £ Rule) \ / 1.6? -2 736) and its mutants (for example, Japanese Patent Laid-Open No. 266,996 / 91). This paper size applies the Chinese National Standard (CNS) Λ4 specification (210X297 mm) ~. — ≫ ------- * ^ 3 .-- (Please read the notes on the back before filling this page)
經濟部中央棣準局員工消費合作社印製 A7 B7 五、發明説明（3 ) D -酮己醣3 —表異構酶的製法，通常是將能夠製造 此種D —酮己醣3 —表異構酶的微生物在含有碳源、氮源 、無機鹽、維生素等的營養液培養基中，並且最好在有氧 的條件下，例如，在曝氣攪拌下，培養1 _ 5天，並自生 成的培養物的細胞及/或上澄液回收酵素。一般而言，生 成之培養物可作爲粗製的D —酮己醣3 _表異構酶。如有 需要，培養物在使用前，可以利用習知的方法（例如：過 濾、離心、鹽析、滲析、濃縮及凍乾）.施以部分純化。如 果需要高度純化的產物，則可使用離子交.換器、凝膠過濾 、等電聚焦、電泳、高效能液態層析法（在下文中簡稱％ HP L ）、親和層析法及/或在單克隆抗體（monoc lonal antibody) 上實施吸附及 去吸附 作用將培養释I 施以 吸附及去吸附作用，而任意地將其純化至最髙的含量。 D —酮己酿3 -表異構酶或具有酵素活性之微生物可 以利用習知的方式行固定化作用，以將其重覆地應用L — 阿洛酮糖與L 一山梨糖的轉化反應中，而分別製得L 一果 糖及L 一塔格糖，並且亦可將其應用於連續式反應中。此 種型式的轉化反應通常是在以下的條件實施：基質濃度， 1 — 6 0重量/體稹％，並以5 — 5 0重量/體稹％爲佳 :反應溫度，1 〇 — 7 Ot的溫度範圍內，並以約3 0 — 6 0 °C爲佳；反應之p Η値，5 — 1 0的p Η値範圍內， 並以約7 — 1 0爲佳；乾燥固態基質（d . s . b)上之 酵素量，每克基質至少一單位，並以每克基質1 0 — 5，0 0 0單位爲佳。就經濟的觀點考量，用於批次反應 ----：-0-- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝. 訂 經濟部中央標準局員工消費合作社印製 A7 B7___ 五、發明説明（4 ) 的反應時間可以在5 — 5 0小時的範圍內任意選用。 由是製得的反應混合物含有新生成的L-果糖或L— 塔格糖與完好如初的起始物L _阿洛酮糖與L 一山梨糖， 其利於整體使用作爲增甜劑、水份賦予劑、結晶防止劑與 光澤賦予劑。反應混合物通常會以習知的方式.製成糖漿產 品，即連續地將其施以脫色（使用活性碳）+、鹽析及純化 (利用H —型0H —型離子交換器）與濃縮。生成的濃縮 物利用鹼金屬或鹼土金屬型式之强酸性陽離子交換器可以 極爲容易地以管柱層析術分離成富新生成之L-果糖或L _塔格糖的部分，與富原料之L -阿洛酮糖或L -山梨糖 的部分，繼而將富L -果糖或L —塔格糖的部分施以純.化 與濃縮而製得糖漿產品，如有需要，此·一濃縮物亦可容易 地結晶成結晶形產品。分離出之富L —阿洛酮糖及L —山 梨糖的部份可以再循環使用以作爲下—個轉化反應的起始 物料。 由是製得的L 一果糖及L -塔格糖極適宜作爲增甜劑 用以賦予口服產品（例如：食品、飲料、飼料、寵物食品 、牙粉、口香片、舌下劑與內服藥）適宜的甜味，並可用 以改善其味感。這些單糖極適宜作爲發酵用之碳源，與作 爲化學品與醫藥品之化學試藥、原料及中間物。 以下的試驗用以說明D —酮己醣3 一表異構酶對L 一 酮己醣的作用。 試驗 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）卜' --Printed by A7 B7 of the Consumer Cooperatives of the Central Bureau of Quasi-Economic Bureau of the Ministry of Economic Affairs 5. Description of the invention (3) D-ketohexose 3-epimerase production method, usually will be able to produce such D-ketohexose 3-epiphenone The microorganisms that constitute enzymes are cultured in a nutrient solution medium containing carbon sources, nitrogen sources, inorganic salts, vitamins, etc., and preferably under aerobic conditions, for example, under aeration and agitation, cultured for 1-5 days and self-generated Cells and / or supernatants from cultures recover enzymes. In general, the resulting culture can be used as a crude D-ketohexose 3-epimerase. If necessary, the culture can be partially purified by conventional methods (eg, filtration, centrifugation, salting out, dialysis, concentration, and lyophilization) before use. If highly purified products are required, ion exchange, gel filtration, isoelectric focusing, electrophoresis, high performance liquid chromatography (hereinafter referred to as% HP L), affinity chromatography and / or The monoclonal antibody is subjected to adsorption and desorption, and the culture release I is subjected to adsorption and desorption, and it is optionally purified to the maximum content. D-ketohexanol 3-epi isomerase or microorganisms with enzyme activity can be immobilized in a conventional manner to repeatedly apply it in the conversion reaction of L-psicose and L-sorbose L-fructose and L-tagatose are prepared separately and can also be used in continuous reactions. This type of conversion reaction is usually carried out under the following conditions: substrate concentration, 1-60 weight / body weight%, and preferably 5-50 weight / body weight%: reaction temperature, 10-7 Ot Temperature range, and preferably about 30-60 ° C; p 反应 of the reaction, p Η 値 of 5-10, and preferably about 7-10; dry solid substrate (d. s. b) The amount of enzyme on the substrate is at least one unit per gram of substrate, and preferably 10-5, 000 units per gram of substrate. For economical considerations, for batch reactions ----: -0-- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) Packing. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, printed A7 B7___ 5. The description of the invention (4) The response time can be arbitrarily selected within the range of 5-50 hours. The reaction mixture prepared by this method contains newly formed L-fructose or L-tagatose and the original starting materials L_psicose and L-sorbose, which is beneficial to the overall use as a sweetener, water Giving agent, crystal preventive agent and gloss giving agent. The reaction mixture is usually made into a syrup product in a conventional manner, that is, it is continuously subjected to decolorization (using activated carbon) +, salting out and purification (using H-type 0H-type ion exchanger) and concentration. The resulting concentrate can be easily separated by column chromatography into a newly formed L-fructose or L_tagatose-rich portion and a raw material-rich L- using a strongly acidic cation exchanger of the alkali metal or alkaline earth metal type. The portion of alloxulose or L-sorbose, and then the portion rich in L-fructose or L-tagatose is purified. Concentrated and concentrated to obtain a syrup product. If necessary, this concentrate can also be easily made. Crystallize into a crystalline product. The separated L-psicose-rich and L-sorbose-rich fractions can be recycled as the starting material for the next conversion reaction. The L-fructose and L-tagatose produced from it are very suitable as sweeteners for imparting oral products (such as food, beverages, feed, pet food, tooth powder, chewing tablets, sublinguals and internal medicines). Sweetness and can be used to improve its taste. These monosaccharides are extremely suitable as a carbon source for fermentation and as chemical reagents, raw materials and intermediates for chemicals and pharmaceuticals. The following test is used to illustrate the effect of D-ketohexose 3-epi isomerase on L-ketohexose. Test This paper size applies to China National Standard (CNS) A4 (210X297 mm).
Min- ί^β HI—— ml Bn·— vm HL I ^^1 - - (請先閱讀背面之注意事項再填寫本頁) 訂 ¥ 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（5 ) 在此探究D —酮己醣3 —表異構酶對多·種L 一酮己醣 的相對酵素活性。將利用實例1之方法製得之經純化的D 一酮己醣3 -表異構酶作用於四種不同型態的L 一酮己醣 基質，即L 一阿洛酮糖、L 一果糖、L —山梨糖與L-塔 格糖，以測定D —酮己醣3 —表異構酶對這些基質的酵素 活性。反應條件如下：將1 1單位/克基質的酵素加至濃 度爲0 . 1M的基質溶液（pH7 . 5)中，並在3 0°C 下反應2 4小時。另做—對照組，以L -塔格糖作爲基質 並將相對酵素活性當作1 Q 0。結果示於表1。 表 1 基 質 相對酵素活性 D —塔格糖 10 0 L 一阿洛酮糖 約 2 0 • L 一果糖 約 5 L 一山梨糖 約 2 L 一塔格糖 約 0 · 5 由表1的結果可以很明顯地看出，D —酮己醣3 表 異構酶催化了 L -酮己醣的轉化反應，特別是將L -阿洛 酮糖轉化爲L 一果糖。以上的結果亦顯示該轉化反應是一 _；__；__ ____ ϋΓ張尺度適用中國國家標率"TcNS )八4祕（2獻297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝. 訂 經濟部中夬標準局員工消費合作社印製 A7 B7 五、發明説明（6 ) 種平衡反應，並且平衡位置傾向於L —果糖的生成，因爲 L —阿洛酮糖與L —果糖是以約3 : 7 ( d . s . b .) 的重量比生成，亦即其適於L -果糖的生成。此外，以上 的結果亦顯示L 一山梨糖與L -塔格糖的轉化反應亦是一 種平衡反應。 以下的實例是本發明幾個較佳的實施例。 實例1 將包含0 . 2重量/體積％硫酸銨、〇 . 2 4重量/ 體積％磷酸氫二鉀、0 . 56重量/體積％磷酸二氫鉀、 0 · 0 1重量/體積％七水合硫酸鎂、〇 . 5重量/體稹 %酵母提取物、1重量/體積％D -葡萄糖與去離子水的 螢養培養基置於發酵罐中，12 0°C滅菌2 0分鐘，再以 1重量/體積％菊苣假單胞茼S T - 2 4 ( F E R Μ BP— 2 7 3 6 )之種子培養作無菌接種，繼而在曝氣攪 拌及3 0 °C的條件下培養40小時。自80公升生成之培 養物回收而得的細胞在活性氧化鋁的存在下硏磨粉碎，再 以5〇111]^之1'1'!8—11(：芡緩衝液（口117.5)萃 取目標酵素。 由是製得的粗酵素溶液在氯化錳的存在下以聚乙二醇 6，0 0 0 (在下文中簡稱爲"PEG")及重覆的分段 沉降法施以純化：在0 . 1 Μ氯化錳的存在下及P E G濃 度爲5 — 1 8重量/體積％之粗酵素溶液中生成的沉澱先 經過收集，再溶解於新製備的同一緩衝液中，此一步驟並 ----;-a--- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） J----*-----ί/''r裝--------訂-------..冰 (請先閱讀背面之注意事項再填寫本頁) A7 B7 經濟部中央標準局員工消費合作社印製 五、發明説明（7 ) 重覆兩次。製得的溶液在5 0°C下加熱2 Q分鐘，並以離 心法去除變性的蛋白質。剩餘的蛋白質藉吸 TOYOPAERL® 650M^ (Tosoh Corporation, Tokyo, Japan 之產品）施以純化，吸附的物質並以氯化鉀溶液洗出。由 是製得之經純化的產物利用*Τ0Υ0 ROSHI UK-.1(T (—種 由 Toyo Roshi Kaisha, Ltd·，Tokyo, Japan商業化之薄 膜過濾器）施以超濾以去除礦物質，再利用^Sephadex® G150^ (Pharmacia LKB Biotechο η ο 1 ogy AB, Uppsala, Sweden之產品广施以凝膠過濾以將其濃縮並純化。具酵素 活性的部份利用、AMPHOLINE®〃（Pharmacia LKB Biote-chonology AB, Uppsala, Sweden之產品）施以等電聚焦 而得以濃縮並純化。 由是製得的酵素樣本以下述的方法檢定：在此一檢定 中使用的反應溶液包含1 0 0微升5 0 m Μ T R I S -11(：芡緩衝液（公117.5)、50微升4〇111]^ D — 塔格糖及5 0微升酵素溶液。酵素反應在3 Q°C下實施 6 0分鐘，生成的D —山梨糖並以HPLC定量。一單位 酵素活性的定義爲：每分鐘一微莫耳D -塔格糖表異構化 爲D -山梨糖所須之酵素量。 純化過程如表2所示。 本紙張尺度逋用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 裝· 訂 > 五、發明説明（ 8 表 2 純化步驟 蛋白質 酵素活性 產率 純化作用 (毫克） (xlO3單位） ⑴ (倍數） 粗萃取物 42,115 52,000 100.0 1 PEG(第一次） 8,378 50,580 97.3 4.4 PEG(第二次） 3,763 26,350 50.4 5.7 熱處理 3,207 27,800 53.6 7.0 DEAE-T0Y0PAERL 415 17,220 33.1 33.6 Sephadex® G150 42.3 16,880 32.3 107.0 等電聚焦 3.0 1,078 2 . 1 291.0 (請先閱讀背面之注意事項再填寫本頁} 經濟部中央標準局員工消費合作社印製 由表2的結果可以很明顯地看出，純化過程使酵素的 比活性增加了約2 9 0倍，其產率約爲2 %。5重量/體 積％之1^_阿洛酮糖水溶液（PH7 . 5)與5，000 單位/克L 一阿洛酮糖之經純化D —酮己醣3 —表異構酶 混合，此一混合物並在4 0 °C下行酵素反應3 0小時。待 反應完成後，將生成的反應混合物利用習知的方式以活性 碳脫色，並以、DIAION SK1B(H —型式）#及’ DIAI0NWA30(0H-型式 Γ (Mitsubishi Chemical Industries Ltd.， Tokyo, Japan 之產品 ） 去除擴物質 ，再於眞空 中濃縮而製得透射率約爲6 0 %之含有L 一果糖的糖漿。 ——----— -—~~- 1 1 ...----- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 經濟部中央標準局員工消費合作社印製 Α7 Β7 五、發明説明（9 ) 利用"""DOWEX .5OW X4’（Do.w Chemical Comp.any, Midland ，Michigan, USA製造之强酸性陽子交換器）將此一漿狀 物以管柱層析術分離成富L -果糖與富L -阿洛酮糖的部 份，再以習知的方式施以純化、濃縮，然後經過結晶及分 離而製得結晶性L —果糖，其產率約爲6 0%，d . s . b 〇 此一產物所顯示的物理化學性質與標準L 一果糖（ Tokyo Chemical Industries, Ltd., Tokyo, Japan商業 化之產品）者極爲吻合。該產物可作爲增甜劑、發酵用之 碳源、及化學品與醫藥品之化學試劑、原料與中間物。該 酵素反應是一種可逆反應，並且亦因爲此一原因，當使.用 L —果糖作爲起始物時，可以很容易地製得L 一阿洛酮糖 實例2 .Min- ί ^ β HI—— ml Bn · — vm HL I ^^ 1--(Please read the notes on the back before filling out this page) Order ¥ Printed by the Consumer Standards of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Explanation (5) Here we explore the relative enzyme activity of D-ketohexose 3-epi isomerase to multiple L-ketohexoses. The purified D-ketohexose 3-epi-isomerase prepared by the method of Example 1 was used on four different types of L-ketohexose substrates, namely L-psicose, L-fructose, L-sorbose and L-tagatose to determine the enzyme activity of D-ketohexose 3-epi-isomerase on these substrates. The reaction conditions were as follows: 11 units / gram of substrate enzyme was added to a substrate solution (pH 7.5) at a concentration of 0.1 M, and the reaction was performed at 30 ° C for 24 hours. Do another-the control group, with L-tagatose as the matrix and the relative enzyme activity as 1 Q 0. The results are shown in Table 1. Table 1 Relative enzyme activity of the substrate D-Tagatose 10 0 L-Alloselose about 2 0 • L-Fructose about 5 L-Sorbose about 2 L-Tagatose about 0 · 5 It is clearly seen that the D-ketohexose 3 epimerase catalyzes the conversion reaction of L-ketohexose, especially the conversion of L-psicose to L-fructose. The above results also show that the conversion reaction is a _; __; __ ____ 张 Γ scale is applicable to China's national standard " TcNS) 8 secret (2 297 mm) (Please read the precautions on the back before filling this page ) Order. Printed by the Consumers' Cooperative of the China Standards Bureau of the Ministry of Economic Affairs, printed A7 B7 V. Description of the invention (6) A balanced reaction, and the equilibrium position tends to produce L-fructose, because L-psicose and L-fructose It is produced at a weight ratio of about 3: 7 (d.s.b.), that is, it is suitable for the production of L-fructose. In addition, the above results also show that the conversion reaction between L-sorbose and L-tagatose is also an equilibrium reaction. The following examples are several preferred embodiments of the present invention. Example 1 will contain 0.2 weight / vol% ammonium sulfate, 0.24 weight / vol% dipotassium phosphate, 0.56 weight / vol% potassium dihydrogen phosphate, and 0.1 weight / vol% sulfuric acid heptahydrate. Magnesium, 0.5 weight / body yeast% yeast extract, 1 weight / vol %% D-glucose and deionized water in a culture medium placed in a fermentation tank, sterilized at 120 ° C for 20 minutes, and then at 1 weight / Seeds of volume% Chicory Pseudomonas ST- 2 4 (FER M BP-2 7 3 6) were cultured for aseptic inoculation, and then cultured for 40 hours under aeration and agitation at 30 ° C. Cells recovered from 80 liters of the culture produced were pulverized in the presence of activated alumina, and then the target was extracted with 1′1 ′! 8-11 (: 萃取 buffer solution (mouth 117.5)). Enzyme: The crude enzyme solution was purified in the presence of manganese chloride with polyethylene glycol 6,0 0 0 (hereinafter referred to as " PEG ") and repeated stepwise sedimentation method: The precipitate generated in the presence of 0.1 M manganese chloride and a crude enzyme solution with a PEG concentration of 5 to 18 weight / vol% was first collected and then dissolved in the newly prepared same buffer solution. This step does not- ---;-a --- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) J ---- * ----- ί / '' r installed ------- -Order ------- .. Bing (please read the precautions on the back before filling this page) A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. The invention description (7) is repeated twice. The resulting solution was heated at 50 ° C for 2 Q minutes, and the denatured protein was removed by centrifugation. The remaining protein was purified by suctioning TOYOPAERL® 650M ^ (product of Tosoh Corporation, Tokyo, Japan), The adsorbed material was washed out with a potassium chloride solution. The purified product obtained from the above was filtered using * Τ0Υ0 ROSHI UK-.1 (T (-a kind of membrane filtration commercialized by Toyo Roshi Kaisha, Ltd., Tokyo, Japan The product was subjected to ultrafiltration to remove minerals, and then reused ^ Sephadex® G150 ^ (Pharmacia LKB Biotech ο ο 1 ogy AB, Uppsala, Sweden. Products are widely subjected to gel filtration to concentrate and purify it. Enzyme active Partially used, AMPHOLINE®〃 (product of Pharmacia LKB Biote-chonology AB, Uppsala, Sweden) was concentrated and purified by isoelectric focusing. The enzyme sample thus obtained was tested by the following method: In this test The reaction solution used contained 100 μl of 50 μM TRIS -11 (: 芡 buffer (male 117.5), 50 μl of 4011] ^ D-tagatose and 50 μl of enzyme solution. Enzyme reaction After 60 minutes at 3 Q ° C, the D-sorbose produced was quantified by HPLC. One unit of enzyme activity was defined as: one micromole D-tagatose epimerization to D-sorbose per minute The amount of enzyme required. The purification process is shown in Table 2. This paper is used on a standard scale National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling out this page) Binding and ordering> V. Description of the invention (8 Table 2 Purification steps Protein enzyme activity yield purification effect (mg ) (XlO3 units) ⑴ (fold) Crude extract 42,115 52,000 100.0 1 PEG (first time) 8,378 50,580 97.3 4.4 PEG (second time) 3,763 26,350 50.4 5.7 Heat treatment 3,207 27,800 53.6 7.0 DEAE-T0Y0PAERL 415 17,220 33.1 33.6 Sephadex® G150 42.3 16,880 32.3 107.0 Isoelectric focus 3.0 1,078 2. 1 291.0 (Please read the precautions on the back before filling out this page} Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs It can be clearly seen from the results in Table 2 that the purification The process increased the specific activity of the enzyme by about 290 times, and its yield was about 2%. 5 wt / vol% 1 ^ _oseltose aqueous solution (PH7.5) and 5,000 units / g L-psicose purified D-ketohexose 3-epi isomerase, this one The mixture was reacted for 30 hours at 40 ° C. After the reaction is completed, the resulting reaction mixture is decolorized with activated carbon in a conventional manner, and DIAION SK1B (H-type) # and 'DIAI0NWA30 (0H-type Γ (Mitsubishi Chemical Industries Ltd., Tokyo, Japan) Product) Remove the expanded material and concentrate it in the air to obtain a syrup containing L-fructose with a transmittance of about 60%. --------------~~-1 1 ...---- -This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of Invention (9) Use " " " DOWEX .5OW X4 '( Do.w Chemical Comp.any, Midland, Michigan, USA, strong acidic phallus exchanger) separated this slurry into L-fructose and L-psicose-rich fractions by column chromatography, It is then purified, concentrated in a conventional manner, and then crystallized and separated to obtain crystalline L-fructose. The yield is about 60%. The physical and chemical properties of this product are shown in d.s.b. With standard L-fructose (Tokyo Chemical Industries, Ltd., Tokyo, Japan This product can be used as a sweetener, a carbon source for fermentation, and a chemical reagent, raw material and intermediate for chemicals and pharmaceuticals. The enzyme reaction is a reversible reaction, and because of this The reason is that when using L-fructose as a starting material, L-psicose example 2 can be easily prepared.
1 〇重量/體稹％阿洛酮糖水溶液（pH \ 7 . 0 )與3，0 0 0單位/克L —阿洛酮糖之經部份純 化的酵素溶液（將粗酵素施以實例1之純化步驟，並進行 到第二次P EG分段沉條步驟而得）混合，此一混合物並 在5 0°C下行酵素反應3 0小時。待反應完成後，將生成 的反應混合物利用與實例1類似的方式施以脫色、並去除 礦物質，再於眞空中濃縮而製得含有約7 0%之L 一塔格 糖與L 一果糖（重量比約爲3 : 7 )的透明糖漿，其產率 約爲9 0 %，d . s . b .。該產物適合作爲高品質之增 -：----：-- ΙΟ * —- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) •裝· 經濟部中央標隼局員工消費合作社印製 A7 __^__B7_ 五、發明説明（1G) 甜劑（具高增甜力），並且極適合作爲水份賦予劑、結晶 防止劑、光澤賦予劑及食品與飮料甩增甜劑。 眚例3 . 將經過部份純化的酵素溶液（將粗酵素施以實例1之 純化步驟，並進行到第二次P E G分段沉降步驟而製得者 )固定至 'KITOPEARL BCW250.3〃（Fuji Spinning Co. Ltd., Tokyo, Japan製造）。將該經過部份純化的酵素加 至樹脂，繼而在3 0 °C下攪拌2小時可使其固定。最後， 約8 0%的酵素活性被固定化。5重量/體稹％—阿 洛酮糖水溶液（PH7 . 5 )與5 0 0單位/克L-阿洛 酮糖之經固定化的酵素混合，該混合物並在4 5 °C下行酵 素反應4 0小時。待反應完成後，將生成的反應混合物利 用與實例1類似的方式施以脫色、並去除礦物質，再於眞 空中濃縮而製得含有約7 0 %之L 一塔格糖與L 一果糖（ 重量比約爲3 : 7 )的透明糖漿，其產率約爲9 0 %，d .s . b '··。該產物適宜作爲高品質之增甜劑（具高增甜 力），並且極適合作爲水份賦予劑、結晶防止劑、光澤賦 予劑及食品與飮料用增甜劑。 實例4 將以實例3之方法製得之經固定化的D —酮己醣3 — 表異構酶加至1 0 0毫升含4重量/體積％L —山梨糖之 0 . 05M磷酸鹽緩衝液（pH7 . 0)，並在45°C下 Ί紙張^_適用中國國家標準（CNS ) A4規格（210X297公釐_) 13 = (請先聞讀背面之注意事項再填寫本頁) Γ裝. -訂 i 經濟部中央標準局員工消費合作社印製 細_ A7 ___'___B7 _^_ 五、發明説明（11) 搖動並反應90小時。酵素以過濾法分離之。 ‘上澄液利用習知的方式以活性碳脫色，並以1 DIAION SK1B(H—型式），及 1IAI0N WA30(OH—型式 )# (Mitsubishi Chemical Industries Ltd..Tokyo, Japan之產品）去除礦物質，再於眞空中濃縮而製得透射 率約爲6 0 %之含有完好原料L —山梨糖作爲不純物的糖 漿。利用 ’DOWEX 50WX4( C a —型式），（Dow Chemical· Company, .Midland, Michigan, USA 製造之陽離子交 換器）將此一漿狀物以管柱層析術施以純化，再經濃縮、 結晶及分離而製得結晶性L -塔格糖。L-塔格糖對L 一 山梨糖的產率約爲2 0 %，d . s · b .。 爲了確認此一方法製得的產物，本人曾利用試驗探究 其物理化學性質，並將之與眞實値相較，此外，亦將之與 業已商業化並作爲標準物之D-塔格糖（316^纟（：1^1114&-1 Company, ST.Louis, Mo，USA之產品）所得的數據相較 Ο (1 )熔點 本發明之產物的實測値：1 3 4 — 1 3 5 °C 商業化之D —塔格糖之眞實値：1 3 3 - 1 3 5 °C 參考.文獻：、D i c t i 〇 n a r y 〇 f 0 r g a n i c C 〇 m ρ 〇 u n d s # ，第5版，第3冊，5，0 9 7頁（1 9 8 2年）美國紐 約 C h a p m a n a n d H a Π 出版。 ϋ張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）Μ ' (請先閲讀背面之注意事項再填寫本頁) 訂 i .w· 經濟部中央標準局員工消費合作社印製 S9909S a? B7 ______ 五、發明説明（12 ) (2)比旋光度之量測 產物;的實測値：〔α〕2° = + 6 . 0 7 ( C F 1 〇 % » Η 2 0 ) 商業化之D —塔格糖的量測値：〔α〕2 ° = -5 .78 (C=10%，Η 2〇 ) (3 )紅外線吸收光譜 此一產物藉K B r鹽片法測得的紅外線吸收光譜如附 圖所示。此一光譜與眞實値之間極爲吻合。 (4 )以高效能液態層析術（Η P L C )所實施之分析. 產物的分析以HPLC實施，並使用以下的設備與條 件：、880-PU# (JASCO Co., Tokyo, Japan之產品）；、 GL-C611 COLUMN， （H i tach i Ltd .，Tokyo , Japan之產品 );流洗液，1 0 - 4M氫氧化鈉；溫k，6 0 °C ;液體速 度，1毫升/分鐘；偵測器，％RID-6A" (Shimadzu Corporation, Tokyo, Japan之產品）。分析的結果顯示 產物的流洗時間爲2 2 . 2分鐘，此與D -塔格糖者相近 ，並且此一時間與作爲標準物之d —酮己醣（例如：D — 阿洛酮糖、D —果糖及D —山梨糖）者,不苘。 由以上的結果可以很明顯地看出，此一產物的熔點、 紅外線吸收光譜及利用Η P L C測得的分析値與D -塔格 糖的眞實値或者標準物的數値極爲吻合。測得產物的比旋 光度幾乎與D—塔格糖者一致，但兩者^旋光性（+ , _ ---：-- 1 十----^------ 本紙張尺度適用中國國家標準（CNS ) Μ規格（210Χ297公釐）丄。 (請先閱讀背面之注意事項再填寫本頁) -裝. 訂 399093 at B7 五、發明説明（13 ) )相反。由此得到一個結論，即產物爲L —塔格糖。 依據本發明之產物可以作爲試劑、稀有糖類，並且極 適合作爲食品業、醫藥業及化工業之原料或者中間物。 由以上的敘述可以很明顯地看出，本發明建立了一種 使用生物醫學法的程程，其使以往被認爲難於製得之L 一 果糖及L 一塔格糖的製造變得簡易可行。此一利用D -酮 己醣3 —表異構酶自起始物L—阿洛酮糖製得L_果糖的 方法，對L —果糖的製造有重大的意義，亦即此方法使得 L 一果糖得以以工業的規模製造，因爲作爲起始物的L 一 阿洛酮糖可以藉氧化還原反應自D -塔格糖及D -山梨糖. 製得。自起始物L —山梨糖製得L -塔格糖的方法對於.製 造有極大助益，因爲作爲原料的L-山梨糖可以以工業的 規模及相當低的成本自山梨醇製得。 經濟部中央標準局員工消費合作社印製 (請先聞讀背面之注意事項再填寫本頁) 因此，依據本發明之方法適合作爲工業上製造L 一果 糖及L 一塔格糖的方法，因其可以容易地以工業規模及相 當低的成本供應這些糖類。此外，本發明亦首先將L 一果 糖及L_塔格糖應用於試藥（·作爲稀有糠類）、食品業、 醫藥業及化工業中，而這些用途在以往根本無從想像。 雖然上文僅對現下認爲是本發明之較佳的實施例作敘 述，但是應可理解，在其中可以作各種修飾，在此亦欲將 所有與此有關的修飾涵蓋於所附的申請專利範圍中，只要 此種修飾不背離本發明之眞正精神與範疇。 ----：-- Ifi U - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）10 wt / body 稹% ptoseose aqueous solution (pH \ 7.0) and 3,000 units / g L-a partially purified enzyme solution of ptoseose (the crude enzyme was applied to Example 1 Purification step, and proceed to the second P EG step sinking step) mixing, this mixture and the enzyme reaction at 50 ° C for 30 hours. After the reaction was completed, the resulting reaction mixture was decolorized and minerals were removed in a similar manner to Example 1, and then concentrated in the air to obtain L-tagatose and L-fructose (about 70%). The transparent syrup with a weight ratio of about 3: 7) has a yield of about 90%, d.s.b. This product is suitable as an increase in high quality-: ----:-ΙΟ * --- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page ) • Printing · Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 __ ^ __ B7_ 5. Description of the Invention (1G) Sweetener (with high sweetening power), and is very suitable as a water-imparting agent, crystallization preventive agent, gloss Sweeteners for food additives and foodstuffs. Example 3. A partially purified enzyme solution (made by applying the crude enzyme to the purification step of Example 1 and proceeding to the second PEG staged sedimentation step) was fixed to 'KITOPEARL BCW250.3' (Fuji Spinning Co. Ltd., Tokyo, Japan). The partially purified enzyme was added to the resin and then stirred at 30 ° C for 2 hours to fix it. Finally, about 80% of the enzyme activity was immobilized. 5 weight /% of body weight—a solution of ptoseose in water (PH7.5) mixed with 500 units / gram of L-psicose immobilized enzyme, and the mixture was subjected to enzyme reaction at 4 5 ° C 4 0 hours. After the reaction was completed, the resulting reaction mixture was decolorized and minerals were removed in a similar manner to Example 1, and then concentrated in the air to obtain L-tagatose and L-fructose (about 70%). The transparent syrup with a weight ratio of about 3: 7) has a yield of about 90%, and d.s.b '... The product is suitable as a high-quality sweetener (with high sweetening power), and is very suitable as a water-imparting agent, a crystal preventive agent, a gloss-improving agent, and a sweetener for food and seasonings. Example 4 The immobilized D-ketohexose 3-epi isomerase prepared by the method of Example 3 was added to 100 ml of a 0.05 M phosphate buffer solution containing 4 wt / vol% L-sorbose. (PH7. 0), and paper at 45 ° C ^ _ Applicable to China National Standard (CNS) A4 specifications (210X297 mm_) 13 = (Please read the precautions on the back before filling this page) Γ. -Ordering details printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs _ A7 ___'___ B7 _ ^ _ V. Description of the invention (11) Shake and react for 90 hours. Enzymes are separated by filtration. 'Shangchengye decolorizes with activated carbon in a conventional manner, and removes minerals with 1 DIAION SK1B (H-type) and 1IAI0N WA30 (OH-type) # (product of Mitsubishi Chemical Industries Ltd .. Tokyo, Japan) , And then concentrated in the air to obtain a syrup containing imperfect material L-sorbose as an impure substance with a transmittance of about 60%. This slurry was purified by column chromatography using a 'DOWEX 50WX4 (Ca-type), (Cow exchanger manufactured by Dow Chemical · Company, Midland, Michigan, USA), and then concentrated and crystallized. And separation to obtain crystalline L-tagatose. The yield of L-tagatose to L-sorbose is about 20%, d.s.b. In order to confirm the product made by this method, I have used experiments to explore its physicochemical properties and compared it with 眞 値. In addition, I have compared it with D-tagatose which has been commercialized and used as a standard (316 ^ 纟 (： 1 ^ 1114 & -1 Company, ST.Louis, Mo, USA) data compared with 〇 (1) melting point of the product of the present invention 本: 1 3 4 — 1 3 5 ° C Commercial D of Chemistry—Treatment of Tagatose: 1 3 3-1 3 5 ° C References: Dicti 〇nary 〇f 0 rganic C 〇m ρ 〇unds #, 5th edition, volume 3, 5 Published by Chapmanand H a Π, New York, USA, page 97 (page 1982). The scale of the sheet is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) M '(Please read the notes on the back before filling in This page) Order i.w. Printed by S9909S a? B7 by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs ______ 5. Description of the invention (12) (2) Measurement products of specific optical rotation; measured 値: [α] 2 ° = + 6.7.0 (CF 1 0% »Η 2 0) Commercial D-tagatose measurement 値: [α] 2 ° = -5 .78 (C = 10% (2)) (3) Infrared absorption spectrum The infrared absorption spectrum of this product measured by the KBr salt tablet method is shown in the attached figure. This spectrum is in good agreement with 眞 眞. (4) In a highly efficient liquid Analysis by chromatography (ΗPLC). Product analysis was performed by HPLC using the following equipment and conditions: 880-PU # (product of JASCO Co., Tokyo, Japan); GL-C611 COLUMN, (Products of Hitachi Ltd., Tokyo, Japan); flow washing solution, 10-4M sodium hydroxide; temperature k, 60 ° C; liquid speed, 1 ml / min; detector,% RID- 6A " (product of Shimadzu Corporation, Tokyo, Japan). The analysis results show that the product's flow-washing time is 22.2 minutes, which is similar to that of D-tagatose, and this time is similar to d, which is a standard. Ketohexose (for example: D-psicose, D-fructose, and D-sorbose) are not bad. From the above results, it can be clearly seen that the melting point, infrared absorption spectrum and utilization of this product The analytical value measured by PLC is in good agreement with the actual value of D-tagatose or the number of standards. The specific optical rotation of the measured product is almost the same as that of D-tagatose, but both ^ optical properties (+, _ ---:-1 十 ---- ^ ------ this paper size is applicable Chinese National Standard (CNS) M specifications (210 × 297 mm) 丄 (Please read the precautions on the back before filling out this page)-Binding. Order 399093 at B7 V. Invention Description (13)) Instead. This leads to the conclusion that the product is L-tagatose. The products according to the present invention can be used as reagents, rare sugars, and are very suitable as raw materials or intermediates for the food, pharmaceutical and chemical industries. From the above description, it is clear that the present invention establishes a process using a biomedical method, which makes the manufacture of L-fructose and L-tagatose, which were previously considered difficult to produce, simple and feasible. This method of making L_fructose from D-ketohexose 3-epi isomerase from the starting material L-psicose has great significance for the production of L-fructose, that is, this method makes L-fructose Fructose can be manufactured on an industrial scale because L-psicose as a starting material can be produced from D-tagatose and D-sorbose by redox reactions. The method for preparing L-tagatose from the starting material L-sorbose is extremely helpful for the production because L-sorbose as a raw material can be produced from sorbitol on an industrial scale and at a relatively low cost. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling out this page). Therefore, the method according to the present invention is suitable for industrially manufacturing L-fructose and L-tagatose because of its These sugars can be easily supplied on an industrial scale and at a relatively low cost. In addition, the present invention also first applied L-fructose and L-tagatose to test drugs (as rare bran species), the food industry, the pharmaceutical industry, and the chemical industry, and these uses have never been imagined in the past. Although the above only describes the preferred embodiments of the present invention, it should be understood that various modifications can be made therein, and it is also intended to cover all related modifications in the attached application patent. As long as such modifications do not depart from the true spirit and scope of the present invention. ----:-Ifi U-This paper size applies to China National Standard (CNS) A4 (210X297 mm)