TWI258507B - Process for producing non-proteinogenic L-amino acids by fermentation - Google Patents

Process for producing non-proteinogenic L-amino acids by fermentation Download PDF

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TWI258507B
TWI258507B TW090123402A TW90123402A TWI258507B TW I258507 B TWI258507 B TW I258507B TW 090123402 A TW090123402 A TW 090123402A TW 90123402 A TW90123402 A TW 90123402A TW I258507 B TWI258507 B TW I258507B
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Taiwan
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group
fermentation
strain
amino acid
alanine
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TW090123402A
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Thomas Maier
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Consortium Elektrochem Ind
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Description

1258507 — 1、發明說明（1) " " " '—"" 本發明與藉微生物直接發酵以製造非蛋白原L_胺基酸 之方法及由該方法製得之L-胺基酸有關。 一非蛋白原胺基酸係事實上並非用書作蛋白質生物合成 高分子鏈節之胺基酸，所以可清楚地與2〇種蛋白原胺基酸 加以區分。該等胺基酸最好係取代之L—丙胺（氨）酸衍 生物。 舉例言之’非蛋白原胺基酸係用以製備藥品及農業活 性化合物之新穎化合物。作為活性化合物或作為活性化合 物之一部分，以一種分子擬態模仿自然胺基酸之結構而因 此導致細胞受體交互作用，該等非蛋白原胺基酸可導致自 然反應之抑揚調節作用。此外，如同"掌性（不對稱）庫" 情況下之掌性化合物，用作合成高分子鏈節。 以往製造純淨鏡像異構形式非蛋白原胺基酸之方法， 通常係基於複雜之合成作用，該等方法通常僅可製得經界 定之化合物。僅有少數方法可藉替換起始原料而製得不同 之化合物。 在絕大多數情況下，該等化學合成作用極為複雜，大 多數最初係以掌性高分子鏈節起始，或繼之以消旋混合物 解析。 另一種方式是，採用若干酶催化之方法。因此，利用 轉胺基酶，以L-麩胺酸作為胺基供體，由α—酮酸可製得 不同之非蛋白胺基酸。另一不同之方法係利用乙内醯脲酶 結合胺曱醯酶。但酶催化方法仍屬成本密集，蓋因必須提 供對應之酶類，而且該等酶類可作為催化劑之壽命有限（
1258507 笠、發明說明（2) 雷姆等人，生物技術1 99 6 ;第6卷，第50 5至56 0頁）。 、相反地，藉微生物直接發酵以製造非蛋白原胺基酸之 方法則特別簡單而有利。但該等方法之風險是：所製非蛋 =原胺基酸會干擾天然胺基酸之新陳代謝作用，因而產生 長抑制作用。就此技術領域内，先前曾揭示一種D —胺基 ，直接發酵之方法（世界專利W0 98/ 14602)。該專利申古/ ，曾述及藉重組體微生物以製造D_胺基酸，該等 體月 =内曾導人-D_胺基轉移酶基因及—L_胺基去（脫）胺^ ^_因。再者’齋（西）藤等人（生物藥品公報，1997， 私ω· 53)曾述及藉表現大腸桿菌内之植物性基因以製造植 ，非蛋白原胺基酸L-吡唑基丙胺酸。但以商業生產而+ 為ΐ=(< 1公克/公升），而且以所述L'綠胺(氨)酸； 馬起始原料而言，成本極高。 本發明之目的係提供一種藉直接發酵以製造一系列 蛋白原L-胺基酸之有效方法。 ::習知之方式’使具有去調節半胱胺酸新陳代謝作 於路ΐ 〇之微生物菌株直接發酵可達成此目的，其特徵為 内：：：中’添加適量之親核化合物在該批發酵配合料 内，俾由該微生物菌株製得非蛋白原L_胺基酸。 屌L 止時’最好藉助於習知之方法，將該等非蛋白 原基酸自各批發酵配合料中分離出來。 作用:ί ^ ^』呈發現· *具有去調節半胱胺酸新陳代謝 菌株實施發酵過程中，並非硫化物，而是- ’、-亥化合物極為有效地進入胺基酸新陳代謝作用 ' 1258507 &、發明説明（3) __ 中，而且該等對應反應生成物係分泌至& 是，此處葡萄糖可用作廉價之碳源。培養基内。有利的 在發酵過程中，藉助於本發明所添加 於是形成非蛋白原卜胺基酸。所以，在發合:好 添加一種可進入胺基酸新陳代謝作用之親核化合物。 最好，所加親核化合物包括一個基，該基係 列諸基所組成之族群 卜
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—N—Q =發酵物配合料中所加親核化合物若係選自下述族 砰則更隹· -具有化學通式（1)之硫醇： 取代、最多具有1 5個碳原子 芳基 其中Ri係單價、經取代或未經 之烧基、院氧基、芳烴基或雜 -具有化學通式（2)或（3)之唾
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Ν \ X (3)
1258507 發明說明（4) 及其醋類、醚類或鹽類，
其中X及Y係相同或不同且代表CR4或n，而且R4係-C00H 、-0H、-NH2、-N02-、-SH、-S03-、-F、~C1、-Br、-I、
Ci—Cs -烷基羰基或ri，及ri所代表之意義與化學通式（丨）内 者相同而且 其中R2及R3係相同或不同且係R4或其中化學通式（3)中之Ci 及C2(並非取代基r2及r3)係藉助於μCRSR6 —橋聯接（其中& 係1、2、3或4)而形成一環，其中 R5及R6係相同或不同且係R4及一個或更多個非相鄰基 [_CR5R6-]可由氧、硫、或一亞胺基代換，該亞胺基可未經 取代或業經(：丨-(：5-烷基取代，而且 兩個相鄰[-CR5R6-]基可由一 [_CR5=CR6-]基或一 [一CR5 = N—]基 代換。 -具有化學通式（4)或（5)之異曙.啉酮：
Η I. R2 人 \\ / 0 (5) X-、 〇（4), 及其酯類、醚類或鹽類， 其中X、R1、R2、R3代表之意義與以上所界定者相同且化學 通式（5)中之C1及C2(並非取代基R2及R3)可藉助於化學通式 (3)中所界定之橋聯接而形成一環。 硫醇之實例係選自一個族群之化合物，該族群包括：
1258507 五、發明說明（5) 2=虱硫基乙醇、3_氫硫基丙醇、3氫硫基丙酸、3 上丙績酸、氫硫基乙磺酸、2_氫硫基乙胺、硫經乙酸'基 、硫乙酸、氫硫基琥珀酸、氫硫基丙酮酸、二坊分 糖醇、二硫赤藻糖醇、卜氫琉基丙三醇、硫盼、4_^二蘇 -氫硫基酚、對一鄰甲笨酚、5_硫_2_硝基苯甲酸軋瓜酚 疏基嚷0坐 1，2，4 -三唾 氫 氫硫基噻唑啉、2_氫硫基咪唑、3_氫峻 卜噻吩硫醇、2〜氫硫基吡啶、2-氫硫 :厂：：=酸、2—氫硫基菸噚酸、2一氫硫基一卜曱基味’:定 。一 U笨并㈣、2_氫破基苯并-唾、6-氫硫基'二 1 2-Ϊ:之，實例係選自一個姨群之化合物，該族群包括· 3’脸其叫一甲基吼"圭、4〜甲基吼"坐、3, 5一二甲基吡也· 3酸胺基比;坐、4_胺基㈣、吼唾_[缓酸、㈣_3，匕 曼，’3-三。坐、^,4—三嗓、3_胺基124_三唾、〜竣 1，Λ 3, 4-四唑、吲唑、吲唑 基，唾、笨并三嗤、苯并^坐二酸、°引嗤_5胺、5、胺 、胺基比唾-㈣、8-氣鳥嘴呤、二：二：基本并三 異噚唑啉酮之實例係選自一 ^ 7 包括：異_唑啉-2-酮、4-甲其個族群之化合物，該鴿群 異-嗤啉-2-綱、4, 5_二甲C垒啉+酮、5〜甲基、 二唑啶-3, 5-二酮。 異咚唑啉—2-酮、1，2, 4、鸣 具有去調節半胱胺酸新陳 法之微生物菌株乃既有技術所習1用，可用於本發明方 特點是··可内（源）生0-乙醯基°者。該等微生物菌株 之 絲胺酸[L-半胱胺酸之緊 I麵
1258507 五、發明說明（6) 接生物合成前（驅物）質]’與野生型菌株相較，L—半脱胺 酸有所增加。如所週知的是，於一微生物内，在半胱胺酸 生物合成之最後步驟中’由於〇-乙醯基-絲胺酸-硫氫化酶 之活性，0-乙醯基絲胺酸之乙醯基官能係由一硫醇官 能所替代，於是形成L-半胱胺酸。此一反應類型稱之謂点 -取代作用’蓋因位於該胺基酸之万-碳原子處將一官能基 加以代換。 菌株中之一種： 世界專利W0 97/ 最好本發明方法所用者係下列微生物 具有修飾c y s E對偶基因之菌株，例如·· 15673所述者（併此提出以供參考）或中森等人，ι998 , 應用環境微生物學報，64 : 1 607至1611所述（併此提出 以供參考）或高木（高城）等人’1999，歐洲生物化學學會 聯合會通訊，452 :323至327中所述，或 -含有外流（射）基因之菌株，如歐洲專利EP 08 859 62 A1 [相當於案號SN 09/09 775 9之美國專利申請案中所述（併 此提出以供參考）]’或 -具有修飾CysB活性之菌株，如德國專利申請案DE 1 9949579中所述，或 -利用非特定（異）致突變方法結合半胱胺酸生產過賸或減 低半胱胺酸降解之篩選法所製造之菌株，如世界專利WO 97/ 1 56 73中所述或中森等人，1 9 98，應用環境微生物學 報，64 : 1 60 7至1611中所述。 該等菌株之特點是：在無機硫源（例如··硫酸鹽或硫 代硫酸鹽）之適當供應下，該等菌株將分泌大量L-半胱胺
第10頁 •1258507 豈、發明説明（7) -至培養基内。由於發酵過程中，本發明添加 製ϊϊΐϊΐ/該親核化合物進入該卜取代作用，結果 展仔非蛋白原L-胺基酸。 。禾
(例如用不二有Λ調f半耽胺酸新陳代謝作用之微生物菌株 作用及生長抑制作用。所以，無大量非蛋U 用及〇但ί: LT \之胺菌酸株Λ有去調節半胱胺酸新陳代謝作 量非蛋二胺基酸 仍可碟定有足量L-半胱胺酸形：保證微生物之細胞生長’ 代謝= 好係-種具有去調節半耽胺酸新陳 最好，該等大腸桿菌菌株係世 歐洲專利ΕΡ 0885 962 Α1 也寻利W〇 97/1 5673或 之美國專利申請荦）或辦 田；案號為SN 09/097759 依照該等專利申請案所述之方 9579二斤述$者》 基因及/或外流基因之質4 M 鞛攜有抗回饋cysE對偶 本發明用一微生物菌株以製造 法係以習知之方式於一F_ 卜蛋白原1一胺基酸之方 化合4卜 么酵器内實施，但另外添加一親核 該微生物菌株在發酵器 (不連續)培養基，尤以呈僚：：呈連續培養基或分批 脣刀批培養基更佳。於發酵 1258507 五、發明說明（8) 過程中連續添加-碳源及-親核化合物則最佳。 開始添加親核化合物係在接種之後，尤以初& + i 段之後更佳。發酵作用起始之後尤以初始生長階 至發酵作用終止則最:以至8小時開始添加且持續 卜親核化合物之添加量視其對該微生物 母公升發酵培養基初始體積10至100 係 發酵培養基初始體積5〇至5()()毫莫耳最毫佳莫耳U母公升 發明Γ:Ϊ:之碳源以糖類、糖醇類或有機酸為佳。在本 月之方法中，所用碳源係葡萄糖、乳糖或甘油則 本 以適當之形式添加葡萄糖以確保發二 持W公克/公升為佳 為佳本發明方法所用氮源以氛、按鹽'或蛋白質水解產物 、其他可添加之培養基添加料是：磷、硫、氣、鈉、 声氮、鉀、鈣、鐵等元素之鹽m，及微量(亦即微、錢 又）鉬、硼、鈷、錳、鋅及鎳等元素之鹽類。 滬 此外’培養基内亦可添加有機酸（例如：乙酸曄 樣酸鹽）、胺基酸（例如：#亮胺酸）、及維生素（如檸 、B 6) 〇 · B1 舉例言之，可用之複合營養源是：酵母萃取物 玉米漿、大（黃）豆粉或麥芽萃取物。 賞）、 發酵培養基之酸度值為4至1 〇，但以6至8較佳，+、 6· 5至7· 5最佳。 尤以 第12頁 1258507 主、發明說明（9) " -- 保溫溫度為15至45 °c，尤以30至37 °C更佳。 發酵作用以在有（需）氧生長情況下實施為佳^將氧導 入發酵器係利用壓縮空氣或純氧。 在1至4天之發酵期間内，依照所述方法發酵之微生物 極為有效地將對應之非蛋白原L-胺基酸分泌至培養基内。 酸新程中，添加親核物質時’具有去調節；胱胺 形（型^ 之微生物分泌具有化學通式（6)、呈L—構 之非蛋白原胺基酸： COOH H2N—C一Η CH2 (6) z 選 其中Z係 化學通式（7)至（13)之單價基
(9),
N \ X (10), N.
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第13頁 1258507 酯類、醚類或鹽類， 及尺、矽、P3 所界定者相同Y 、义及¥代表之意義與化學通式（〗）至（5)内 本發明之方法首次可盥 及其衍生物，及ί 2 3 - ΐ =;，四唑基—L—丙胺酸 群化合物。，好：丙胺酸及其衍生物等族 攻好，该等化合物分別為異構物形式之 1，2,3,4-四唑-1 —基吒一丙胺酸（14)及1，2 3 4—四唑—2—美一 L-丙胺酸（15)及其衍生物（包含其酯類、醚類或鹽類）及 1，2, 3-三唑-卜基-L-丙胺酸（16)及！，2, 3—三唑〜2—基-L一丙 胺酸（1 7)及其衍生物（包含其酯類、醚類及鹽類）。 (14)
COOH
I H2N—C—Η
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COOH
I
HjN—C—H (15)
(16)
COOH
I
HjN—C—H ch2 r2、a 丨i
COOH
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HjN--C—H CH2 r2. (17)
1258507 五、發明說明（11) 其中R1、R2、R3及R4代表之意義與化學通式（1)至（5)内所界 疋者相同。 本發明之方法首次可製造S-雜芳基半胱胺酸族群 之化合物。該等化合物總是具有自由胺基及/或羧酸官能 性之胺基酸化合物。s—雜芳基-L-半胱胺酸意謂半胱胺酸 衍生物，其特徵是；S原子之r7基之取代。此處R7係一具有 芳香特性之雜芳基，可能是單環或雙環，而且除碳原子之 外’環内有至少一個雜原子。雜原子之實例是：氮、氧或 硫。雜芳基本身可由一 R4基取代，其中R4代表之意義與化 學通式（2)内所界定者相同。
COOH
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I CH2
I s κ (18) 雜芳基之實例* :唑基、口米唑基、吡唑基、三唑基 '四唑 基、噻吩基、噻唑基、噚唑基、呋喃基、嘧啶 二觀、笨并，嗤基、笨并三唾基、笨并噚唾基= 噻唑基或嘌呤基。 生基本并 所以本發明與該等化合物有關。 特別適合之化合物是： 1，2,3,4-四唑〜1-基一1^丙胺酸(^4 =耵， ^2,3,4—四唑—2-基-L-丙胺酸（γ = Η)，
第15頁 1258507 五、發明說明（12) - 1, 2, 3-苯并三唑-1-基_L-丙胺酸（R2及R3係相同且係 [-CR5 = CR6-]，其中R5及R6係Η及R2及R3係經聯接而形成一 芳烴環）， -1，2, 3 -笨并三唑-2 -基-L-丙胺酸（R2及R3係相同且係 [-CR5=CR6-]，其中R5及R6係Η及R2及R3係經聯接而形成一 芳烴環）， - 5 -羧基-1, 2, 3 -苯并三唑-1-基-L-丙胺酸（R2及R3係不同 且係[-CR5 = CR6-]，其中R5及R6(若係R3)係η及（若係R2)R5 Η及R6係-C00H，而且R2及R3係經聯接而形成一芳烴環）， - 5 -羧基-1，2, 3 -苯并三唑-2 -基-L -丙胺酸（R2及R3係不同 且係[-CR5 = CR6-]，其中R5及R6(若係R3)係Η及（若係R2)R5 係Η及R6係-C00H，而且R2及R3係經聯接而形成一芳烴環） - 1，2,4-三唑-3-基-L-半胱胺酸， -噻唑-2-基-L-半胱胺酸， -咪唑-2-基-L-半胱胺酸， -噻吩-2-基-L-半胱胺酸， 一吡啶-2-基_L-半胱胺酸， 一嘧啶-2-基半胱胺酸， 一本并噻嗤-2-基半胱胺酸， 一本并噻嗤-2-基半胱胺酸。 級與生物質分開之後，最好利用習知之方法（例如： 過渡、離心作用）將該生成物自培養基上清液内分離出來 。分離胺基酸之該等方法亦係精於此項技藝者所習知者。
第16頁 -1258507 五、發明說明（13) Ϊ = 該等分離方法包括：萃取、吸附、離子交換、 色墻層析、沉澱、結晶。 藉助於下列諸實驗例將對本發明進—步之說明。實施 該等實驗例所用之細菌菌株大腸桿菌W3u〇/pACYC184_&
CySEX-GAPDH-ORF3〇6，係依照布達佩斯條約之編號DSM 1 3495儲存在德國微生物及細胞培養基收集公司，D 38 142 勃朗希威格（DSMZ);並於20 01年9月19日寄存於食品工業 發展研究所，編號為CCRC 94037 7。 ' 實驗例1 :製造菌株之初始培養（物）
作為發酵作用之初始培養（物），將2〇毫升之LB培養基 (10公克/公升胰蛋白腺，5公克公升酵母萃取物，1 〇公克/ 公升食鹽）’其中另外含有15毫克/公升四環素，與菌株 W3110/pACYC184-cysEX-GAPDH-ORF3 06 [如歐洲專利EP 0885962 Al(相當於案號為SN 09/097759之美國專利申請 案（併此提出供作參考））實施接種並於溫度30 t：及轉速每 分鐘150轉之情況下在一震盪器内保溫。於七小時之後， 整個培養基轉移成1〇〇毫升SM1培養基（12公克/公升K2 HP 〇4 ;3公克/公升ΚΗ2ΗΡ04 ; 5公克/公升（NH4)2S04 ;0·3公克/公 升MgS04 · 7Η20 ; 〇· 〇15 公克/ 公升CaCl2 · 2Η20 ; 0· 002 公克/ 公升FeS04 · 7Η20 ; 1公克/公升Na3檸檬酸· 2Η20 ; 〇· 1公克/ 公升NaCl ; 1毫升/公升由〇· 15公克/公升Na2MoS04 · 2Η20組 成之微量元素溶液；2· 5公克/公升Na3B03 ; 0· 7公克/公升 CoCl2 · 6H20 ; 0· 25 公克/ 公升CuS04 · 5H20 ; 1· 6 公克/ 公升 MnCl2 · 4H20 ; 0· 3公克/公升ZnS04 · 7H20，其甲並補充以5
第17頁 * 1258507
昱、發明說明（14) 2//Λ葡萄糖；°.5毫克7公升維生素B1及15毫克/公升 溫17 ^時。酿度3〇 ^及轉速每分鐘1 5〇轉之情況下繼續保 藉發酵作用製奶_[2,3_二經基_硫基一丁 暴J—L- +胱胺酸 司（：級用认發广器係最大培養容積2公升、勃朗生物技術公 A艮，德國）出品之B1 os t a t Μ儀器。利用實驗例1 :二述2 :始培養物（m600毫微米t光線下光密度約 f =酵器與900亳升發酵培養基（15公克/公升葡萄 古/ ’ \ 克/公升胰蛋白腺；5公克/公升酵母萃取物；5公
NaCl么.ίΐ(Γ4、)2δ〇4 ; K 5 公克/ 公升KH2P〇4 ; 〇. 5 公克/ 公升 • 9H ή .么克/公升邮〇4 · Μ0 ; 〇. 015公克/公升CaC12 酸·22Η，ΛΓ】公克/公升Μ〇4 ·7Η20 ; 1公克/公升Na3擰檬 吝“2〇及1毫升/公升微量元素溶液（請參考以上所述），5 3^升=生素B1及15毫克/公升四環素，用25%氨水將 25^ΛΛ ) °在發酵過程中，溫度定在32°C並添加 ^水將酸度維持在U而怪常不變。用經消毒、流量 二各積/容積/分鐘之壓縮空氣將該培養物加以處理並以 鐘2二轉之攪拌器速率加以攪拌。於氧飽和度降至5〇% 持4’二二控制器將攪拌速率增至每分鐘1 20 0轉，以保
Di 用一氧分壓探查器測定，並在轉速每分 :〇轉之情況下將其校正至100%飽和）》於八小時後，以 母2時2毫莫耳之速率將1莫耳濃度之二硫蘇糖醇溶液加入 。於發酵器内之含量由初始之15公克/公升降至約
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發酵時間為48小時。於此時間過 心作用將細胞自培養基中分出來。藉(LUN“件=藉離 =費諸門奈克斯公司出品，阿沙芬堡，:) 壓色譜儀將所得培養物上清液加以分級分離 脫液係稀碟酸（0.1毫升濃構酸/公升），流速為〇 5毫升/八 二；ΓΪ;基二經基丁基七半耽胺酸之駐留以 86· 7刀知。產率為2. 5公克/公升。 實驗例3 :藉發酵作用製造卜苯基_L-半胱胺酸 、依照實驗例1及2所述將細菌加以培養。於八小時之後 ，以2毫莫耳/小時之速率添加丨莫耳濃度之硫酚鈉懸浮液 矛J用實驗例2内所述之高壓液體色譜法，以μ分鐘之駐 留時間洗脫S -苯基一 L 一半胱胺酸。產率為21公克/公升。 實驗例4 ·藉發酵作用製造1，2 — 卩比唾基 — l-丙胺酸依照實 驗例1及2所述將細菌加以培養。於八小時之後，以4毫莫 耳/小時之速率添加1莫耳濃度之1,2-吡唑溶液。 、 利用實驗例2内所述之高壓液體色譜法，以8· 4分鐘之 駐留時間洗脫1，2-S-吡唑基-L-丙胺酸。產率為6· 1公克/ 公升。 實驗例5 ··藉發酵作用製造1，2, 4 -三唑基-L_丙胺酸
m 第19頁 _ 1^· 1258507 ~五、發明說明（16) 依照實驗例1及2所述將細菌加以培養。於八小時之 後’以4毫莫耳/小時之速率添加1莫耳濃度之丨，2，4 -三唑 溶液。 利用實驗例2内所述之高壓液體色譜法，以5. 8分鐘之 駐留時間洗脫1，2, 4 -三唑基-1-L -丙胺酸。產率為4· 6公克 /公升。 實驗例6 :藉發酵作用製造丨，2, 3, 4一四唑基_L—丙胺酸 依照貫驗例1及2所述將細菌加以培養。於八小時之後 ’以4毫莫耳/小時之速率添加1莫耳濃度之1，2, 3, 4 -四唑 溶液。 在發酵過程中形成丨，2, 3, 4_四唑基气—丙胺酸及 1，2, 3, 4-四唑—2-基-L-丙胺酸兩種異構物。利用實驗例2 内所述之局壓液體色譜法，分別以5 · 4分鐘及5. 7分鐘之駐 留時間洗脫該兩種異構物。該兩種異構物合計之產率為 3 · 9公克/公升。 實驗例7 :藉發酵作用製造5-羥基-1, 2, 3 -苯并三唑基-L-丙胺酸 、依」照實驗例1及2所述將細菌加以培養。於八小時之後 ’以4毫莫耳/小時之速率添加1莫耳濃度之1，2, 3-苯并三 唑一5—羧酸在0· 5莫耳濃度NaOH中之懸浮液。 、在發酵過程中形成5-羧基-1,2, 3-苯并三唑-i_基-L — 丙、5 -羧基—1，2, 3-苯并三唑—2-基丙胺酸及5-羧 基’ 2, 3笨并二唾基—l—丙胺酸所有異構物，但5-羧 基一，2, 3-苯并三唑—2—基—L—丙胺酸為主要生成物。利用 第20頁 1258507 五、發明說明（17) "" 實驗例2内所述之高壓液體色譜法，以67· 5分鐘之駐留時 間洗脫該主要生成物。產率為5 · 2公克/公升。 實驗例8 :藉發酵作用製造1，2, 4-噚二唑啶-2, 5_二酮基-L-丙胺酸（=使君子酸） 依照κ驗例1及2所述將細菌加以培養。於八小時之後 ’以2毫莫耳/小時之速率添加2莫耳濃度之丨，2, 4_噚二唑 啶-2, 5 -二酮在二甲亞楓中之溶液。 利用實驗例2内所述之高壓液體色譜法，以5. 6分鐘之 駐留時間洗脫使君子酸。產率為2 · 2公克/公升。 實驗例9 :自發酵液分離Li吡唑基―丨-丙胺酸 首先藉離心作用，以5〇〇〇 g之重力加速度將細胞自 0 · 6公升發酵液中分離出來。於上清液内添加丨〇公克活性 碳加以脫色，在室溫下攪拌該混合物2小時，隨後加以過 濾。用2莫耳濃度NaOH將所得溶液之酸度調整至6. 〇，並塗 敷於一陽離子交換柱Amber jet 1 200 /H +(羅姆及哈斯公司 ，曉尼、法國；250毫升凝膠床體），並以j莫耳濃度“以 洗脫黏結物質。將洗脫之諸部分加以組合（5〇〇毫升），用2 莫耳濃度NaOH將酸度調整6·0並將其漢縮至iQ〇毫升。在 4 C狐度下將該試樣儲存1 6小時。藉過濾分出所得之晶體 ’用50毫升乙醇洗滌後並加以烘乾。 實驗例10 ··藉發酵作用製造S-噻唑-2-基-L-半胱胺酸 依照實驗例1及2所述將細菌加以培養。於八小時之後 ，以2毫莫耳/小時之速率添加1莫耳濃度2—氫硫基噻唑溶 液。利用實驗例2内所述之高壓液體色譜法，以33· 7分鐘
1258507 五、發明說明（18) 之駐留時間洗脫S-噻唑-2-基-L-半胱胺酸。產率為6· 1公 克/公升。 實驗例11 :藉發酵作用製造S-1，2,4_三吐_3 -基-L-半胱胺 酸 依照實驗例1及2所述將細菌加以培養。於八小時之後 ，以2毫莫耳/小時之速率添加1莫耳濃度3-氫硫基三唑溶 液。利用實驗例2内所述之高壓液體色譜法，以8 · 2分鐘之 駐留時間洗脫S-噻唑-2-基-L-半胱胺酸。產率為5· 5公克/ 公升。
第22頁 -1258507
第23頁

Claims (1)

1258507
1. 一種使具有修飾cys-E-對偶基因之 酵以製造非蛋白原L-胺基酸之方法因菌株直接發 程中，將適量之親核化合物添加在發醅、1為，於發酵過 生物菌株製造非蛋白原L-胺基酸。X酵&料内，俾由微 2· 如申請專利範圍第1項之方法，发 時，係用習知之方法將該非蛋白原L /、在發酵作用終止 料中分離出來。 〜胺基酸自發酵物配合 3· 如申請專利範圍第1或2項之方法， 物之一個基係選自一個由下列諸美斛，其中所加親核化合 土斤組成之族群 Η -Ν—ο- Η Η—S- I —Ν—Ν: 4.'如申請專利範圍第丨或2項之方丰。 物係選自由下列諸物所組成之族群古，其中所加親核化合 一具有化學通式（1)之硫醇： Η—S—-Ri (1) 其中R1係單價、經取代或未經取代息 之烷基、烷氧基、芳烴基或雜芳最多具有15個碳原子 一具有化學通式（2)或（3)之唑 土
第24頁 1258507
R2\ •c/N\n \\ // 丫一 X (2), r3. H I C1^\ (3) 及其酯類、_類或鹽類， 其中X及Y係相同或不同且代表CR4或N，而且r4係—η、 、-0Η、-ΝΗ2、-Ν02-、-SH、-S03-、-F、-Cl、—Br、—！、 q-C5-烷基羰基或Ri，及Ri所代表之意義與化學通 者相同而且 ％ U )内 其中R2及R3係相同或不同且係R4或其中化學通式（3)中之p 及C2(並非取代基R2及r3)係藉助於卜CR5R6_]a橋聯接（其中& 係1、2、3或4)而形成一環，其中驴及反6係相同或不同且係 R4及一個或更多個非相鄰基[ — Cr5r6 — ]可由氧、硫、或一亞 胺基代換，該亞胺基可未經取代或業經匕—Cs-烷基取代， 而且 兩個相鄰[-CR5R6-]基可由一[-CR5 = CR6 —]基或一[-邙54一]基 代換， -具有化學通式（4)或（5)之異噚啉酮
⑷，
(5) 及其酯類、醚類或鹽類
第25頁 1258507 六、申請專利範圍 其中X、R1、R2、R3代表之意義與以上所界定者相同且化學 通式（5)中之C1及C2(並非取代基R2及R3)可藉助於化學通式 (3)中所界定之橋聯接而形成一環。 5 ·如申請專利範圍第1或2項之方法，其中本發明方法所 用之微生物菌株係選自由下列諸菌株所組成之族群 :具有經修飾cysE對偶基因之菌株；含有外流基因之菌株 ;具有經修飾Cy,sB活性之菌株；利用非特定致突變方法結 合半胱胺酸生產過賸或減低半胱胺酸降解之篩選法所製造 之菌株。 ’其中所用微生物菌 ’其中该微生物菌株 分批培養基或银養分 6 · 如申请專利範圍第1或2項之方法 株係一大腸桿菌菌株。 7· 如申請專利範圍第1或6項之方法 在發酵器内之生長係呈連續培養基、 批培養基狀。 其中於發酵過程中 8·如申請專利範圍第1或2項之方法 連續添加一碳源及一親核化合物。 9係=:!專利範圍第1或2項之方法’其中該親核化合物 用終1 起始6至8小時之後開始添加且持續至發酵作 添加i I:專利乾圍第1或2項之方法，其中親核化合物之 ^、里、、母公升發酵培養基初始體積1 〇至1 0 0 0毫莫耳。 類、或2項之方法，其中所用碳源係糖 糖％類或有機酸類。 囉 12.如申請專利範圍第"項之方法，其中所加碳源係呈適 1258507 申請專利範圍 =形式以確保發酵器内之葡萄糖含量係維持在〇· 1至50公 克/公升。 1 3.如申請專利範圍第1或2項之方法，其中所用氮源係 、銨鹽或蛋白質水解產物。 14·如申請專利範圍第1或2項之方法，其中發酵培養美 酸度為4至1〇及保溫溫度為15至45t;。 氨 之 15·如申請專利範圍第1或2項之方法，其中該方法係 (需）氧生長之情況下實施。 係在有 16·如申請專利範圍第1或2項之方法，其中藉過濾或離心 作用將生物質自該發酵配合料内移開之後，該非蛋白原匕一 胺基酸係藉助於萃取、吸附、離子交換色譜層析、沉澱或 結晶自培養基上清液内分離出來。 17·如申請專利範圍第1或2項之方法，其中該非蛋白原L 一 胺基酸係一具有化學通式（6 )且呈L構形之胺基酸：” COOH
CH2 1 2 (6) 其中Z係一選自化學通式（7 )至（1 3 )之單價基 —S一R1 (7)
N (9),
X \
(10),
第27頁 1258507 六、申請專利範圍 .N X R2』 •c/N、o \\ / X 一 C、 (11) 0 (12), ‘、V/N \〇 (13) 及其酯類、醚類或鹽類， 及R1、R2、R3、R4、X及Y代表之意義與化學通式（1)至（5)内 所界定者相同。 18. —種選自一個族群之化合物，該族群包括1，2, 3, 4-四 °坐一 1一基一L—丙胺酸（14)及1，2，3，4一四ϋ坐一2-基一L —丙胺酸 (15)及其衍生物（包含其酯類、醚類或鹽類）及1，2, 3-三 σ坐-1-基-L-丙胺酸（16)及1，2，3 -三口坐-2-基-L-丙胺酸（17) 及其衍生物（包含其酯類、醚類及鹽類）， COOH ! Η2Ν—C—Η
R心 C—Ν (15)
第28頁 1258507 六、申請專利範圍 COOH I H2N—C—Η Η2Ν- R2、 r3/ ch2 -N \ //N COOH 1 C—H I ch2 I -N \ N N IIC、〆 (16) (17) 其中R1、R2、R3及R4代表之意義與申請專利範圍第4項内所 界定者相同。 19. 化學通式之S-雜芳基-L -半光胺酸衍生物 C00H H2N—C—Η S R7 (18) 其中R7具有下述意義：吡咯基、吡唑基、噻唑基、噚唑基 、呋喃基、吡啶基、吡嗪基、苯駢咪唑基、苯駢三唑基或 嘌呤基。
第29頁
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