TWI408234B - 對映異構選擇性酶催化還原羥基酮化合物類之方法 - Google Patents
對映異構選擇性酶催化還原羥基酮化合物類之方法 Download PDFInfo
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- TWI408234B TWI408234B TW095147521A TW95147521A TWI408234B TW I408234 B TWI408234 B TW I408234B TW 095147521 A TW095147521 A TW 095147521A TW 95147521 A TW95147521 A TW 95147521A TW I408234 B TWI408234 B TW I408234B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/002—Nitriles (-CN)
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Description
本發明關於一種用於對映異構選擇性酶催化還原通式I羥基酮
(其中R1
=C1
-C6
烷基且R2
=-Cl、-CN、-OH、-H或C1
-C6
烷基)為通式II對掌性二醇(chiral diol)
(其中R1
及R2
具有式I中之相同意義)之方法;其中,該羥基酮係在輔因子(cofactor)的存在下經氧化還原酶(oxidoreductase)予以還原。
通式II對掌性二醇皆為醫藥品製造中之重要中間產物,特別是在HMG-CoA還原酶抑制劑之製造中。此等對掌性二醇為,例如,(3R,5S)-6-氯-3,5-二羥基己酸第三丁基酯,(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯或(5S,3R)-3,5,6-三羥基-3,5-己酸第三丁基酯。
此類型二醇特別係經由對映異構選擇性還原相應的式I羥基酮,例如(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯,(5S)-6-氯-5-羥基-3-合氧基己酸第三丁基酯或(5S)-5,6-二羥基-3-合氧基己酸第三丁基酯而生成。在如此作法中,該化學催化還原具有缺點,一方面,可能由於嚴格的反應條件導致副產物,且於另一方面,會分別產生令人不滿意的對映異構超量及非對映異構超量,且只能用很大的努力才在技術上可行。
為此理由,已有相當的時間,力圖開發生物催化方法以用於上面提及的羥基酮之對映異構選擇性還原。生物催化方法通常在溫和條件下運作,此為彼等可預期促成總為相當不穩定的5-羥基-3-酮基己酸酯衍生物之還原,而不形成其他副產物之原因。不過,至目前為止,都尚未能發現任何適合的生物催化劑以用來使上面提及的5-羥基-3-合氧基己酸酯衍生物之酶催化還原以有效方式且用經分離的酶實行。
例如,美國專利說明書6,001,615及國際專利申請WO 01/85975 A1分別述及(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯及(5S)-5,6-二羥基-3-合氧基己酸第三丁基酯之還原,其中係使用白僵菌屬(Beauvaria
)、畢赤酵母屬(Pichia
)、假絲酵母屬(Candida
)、克魯維酵母屬(Kluyveromyces
)及有孢圓酵母屬(Torulaspora
)等屬別之各種酵母。不過,由此,該等轉化僅在野生株的完整細胞下發生且因此僅可在遠低於5%的非常低濃度之下進行。至目前為止,尚未能鑑定出促成該轉化的酵素及DNA序列。
此外,結構相似的化合物之微生物轉化經載於EP 0 569 998 B1中。在其中,業已可能從乙酸鈣不動桿菌(Acinetobacter calcoaceticus
)ATCC 33305純化出NADH-依賴性酵素,其係以分離狀態與輔酶(coenzyme)再生用之葡萄糖脫氫酶(glucose dehydrogenase)一起使用。在所述方法中,基質係以1%之濃度使用及達到僅為10的NADH“總轉換值”。尚未提出工業可應用的方法。
在美國專利說明書6,645,746 B1中,揭示一種來自木蘭假絲酵母(Candida magnoliae
)之胺基酸序列,其可藉助NADPH用於還原(5S)-6-氯-5-羥基-3-合氧基己酸第三丁基酯至(3R,5S)-6-氰基-3,5-二羥基己酸第三丁基酯。在該文件之專利說明書中,該酵素較佳地在與來自巨大芽胞桿菌(Bacillus megaterium
)之葡萄糖脫氫酶的共表現之狀態中使用,其中係藉助葡萄糖脫氫酶且用葡萄糖作為輔受質而發生該輔因子NADPH的再生。
本發明之目的為提供一種方法,其可促成以高產率及高對映異構純度而沒有任何副產物之下經濟地製造通式II對映異構地純二醇。
根據本發明,此目的係經由最初提及類型之方法達到,其中氧化還原酶係在作為輔因子的NADH或NADPH之存在中還原且其特徵在於a)該羥基酮係以≧50克/升之濃度提供於反應中,b)已經生成的被氧化輔因子NAD或NADP係經由通式RX
RY
CHOH二級醇的氧化而持續再生,其中RX
和RY
各別表示氫、支鏈或非支鏈C1
-C8
-烷基且Ctotal
≧3,且,c)羥基酮之還原及二級醇之氧化皆以相同的氧化還原酶催化。
該方法的一較佳具體實例之特徵在於該氧化還原酶a)包含胺基酸序列,其中至少50%之胺基酸係與胺基酸序列SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14之胺基酸相同,b)係由核酸序列SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:15所編碼,或c)係由在嚴格條件下(stringent condition)與SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:15雜交之核酸序列所編碼。
根據本發明,上面提及的目的也可由最初提及類型之方法達到,其中該氧化還原酶a)包含胺基酸序列,其中至少50%之胺基酸係與胺基酸序列SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14之胺基酸相同,b)係由核酸序列SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:15所編碼,或c)係由在嚴格條件下(stringent condition)與SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:15雜交之核酸序列所編碼。
業經發現包含胺基酸序列SEQ ID NO:1、SEQ ID NO:8及SEQ ID NO:11的多肽(polypeptide)顯示出氧化還原酶活性且可用於還原(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯成為具有>99%之非對映異構超量的(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯。其他通式I羥基酮可利用胺基酸序列SEQ ID NO:1、SEQ ID NO:8及SEQ ID NO:11以相同的方式還原。
此外,前面提及的氧化還原酶具有能經由還原二級醇再生在還原期間形成的氧化輔因子之優點。如此,該氧化還原酶一項特別的經濟優點亦在於,相異於先前技藝方法(US 6,645,746 B及EP 0 569 998 B)者,不需其他酵素用於輔因子之再生。
編碼包含SEQ ID NO:1的多肽之DNA序列SEQ ID NO:2,可得自,例如,生物Rubrobacter xylanophilus
DSM 9941之基因組,此外,業經發現DNA序列SEQ ID NO:3,可用於在大腸桿菌屬(Escherichia)中表現SEQ ID NO:1多肽。
編碼包含SEQ ID NO:8的多肽之DNA序列SEQ ID NO:9可得自,例如,生物Geobacillus thermodenitrificans
DSM 465之基因組。此外,業經發現DNA序列SEQ ID NO:10可用於在大腸桿菌屬中表現SEQ ID NO:8多肽。
編碼包含SEQ ID NO:11的多肽之DNA序列SEQ ID NO:12可得自,例如,生物綠絲菌(Chloroflexus aurantiacus
)DSM 635之基因組。
編碼包含SEQ ID NO:14的多肽之DNA序列SEQ ID NO:15可得自,例如,生物木蘭假絲酵母CBS 6396。
如此,本發明亦關於一種還原通式I羥基酮成為通式II二醇之方法,其中使用包括胺基酸序列SEQ ID NO:1,SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14的多肽,或使用展現出胺基酸序列SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14至少50%相同之胺基酸序列的多肽,即,可從至少一種胺基酸序列SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14經由取代、嵌入、刪除或添加而衍生的一種多肽,或使用由核酸序列SEQ ID NO:2、SEQ ID NO:9、SEQ ID NO:12、或SEQ ID NO:15所編碼或由在嚴格條件下與SEQ ID NO:2、SEQ ID NO:9、SEQ ID NO:12、或SEQ ID NO:15雜交的核酸序列所編碼之多肽。
經由在嚴格條件下雜交例如SEQ ID NO:2的核酸序列,其意為一種聚核苷酸(polynucleotide),其可透過群落雜交法,溶菌斑雜交法、南方雜交法或可比較之方法,使用SEQ ID NO:2作為DNA探針(probe)予以鑑定。
為此目地,係將固定化到濾器上的聚核苷酸在0.7-1 M NaCl溶液中於60℃下雜交到例如SEQ ID NO:2。雜交係按照,例如在Molecular Cloning,A Laboratory Manual,第二版(Cold Spring Harbor Laboratory Press,1989)或類似的公開中所述進行。隨後,用0.1至2-倍SSC溶液在65℃洗滌該濾器,其中1-倍SSC溶液要瞭解為150 mM NaCl及15mM檸檬酸鈉所組成之混合物。
於本發明方法中,包括序列SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14的多肽及可衍生自該等多肽之多肽,可分別,以完全純化狀態,部份純化狀態或以含有多肽SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14的細胞之形式使用。藉此,所用細胞可用天然、經滲透或經溶裂的狀態使用。較佳地,包括序列SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14的多肽及可衍生自該等之衍生物,分別的,在適合的宿主生物諸如,例如,大腸桿菌(Escherichia coli),中過度表現且使用重組型多肽還原通式I羥基酮。
每公斤要反應(向上地打開)的式I化合物係分別使用5,000至10 Mio U之氧化還原酶SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14或其衍生物。藉此,該酶單位1 U係相當於每分鐘要反應1微莫耳(μmol)式I羥基酮所需的酵素之量。
根據本發明方法之酶催化還原係在溫和條件下進行使得可以大幅避免常為不穩定之羥基酮之降解及因而非所欲副產物之形成。根據本發明之方法具有一高滞留時間及通常>99%所產生的對掌性二醇之一非對映異構純度。
本發明一較佳具體實例的特徵在於方法中使用的輔因子係用一輔受質連續地還原。較佳地,使用NAD(P)H作為輔因子,在還原反應中形成的NAD(P)再利用輔受質予以還原成NAD(P)H。
二級醇諸如2-丙醇、2-丁醇、2-戊醇、3-戊醇、4-甲基2-戊醇、2-庚醇、2-辛醇或環己醇由此較佳地用為輔受質。根據一特別較佳具體實例,係使用2-丙醇於輔酶再生。用於再生的輔受質之量,以體積為基準,可從5至95體積%。
輔酶再生可,例如,透過包含SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14的多肽同樣地完成。
於根據本發明之方法中,通式I羥基酮的用量,以總體積為基準,較佳者為從5至50重量%(50克/升至50克/升),較佳者為從8至40重量%,特別者從10至25重量%。
一特別較佳具體實例之特徵在於使用(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯、(5S)-6-氯-5-羥基-3-合氧基己酸第三丁基酯或(5S)-5,6-二羥基-3-合氧基己酸第三丁基酯作為通式(I)羥基酮。
較佳者,根據本發明之方法係在一種水有機二相系統中進行。
於其中進行酶催化反應的反應混合物水部分較佳地含有緩衝液,例如,磷酸鉀、tris/HCl或三乙醇胺緩衝液,具有自5至10之pH值,較佳的自6至9之pH。此外,該緩衝液可含有用於穩定化或活化該酵素之離子,諸如,例如,鋅離子或鎂離子。
在實施根據本發明之方法的期間,溫度適合範圍為自約10℃至70℃,較佳者自20℃至45℃。
於根據本發明方法之另一較佳具體實例中,該酶催化轉化係在有機溶劑存在中進行,該有機溶劑係不與水混溶或僅可與水混溶到一有限程度。該溶劑係,例如,對稱或不對稱的二(C1
-C6
)烷基醚,線形鏈或支鏈型烷或環烷或水不溶性二級醇,其同時,代表輔受質。較佳有機溶劑為二乙醚、第三丁基甲基醚、二異丙基醚、二丁基醚、乙酸丁酯、庚烷、已烷、2-辛醇、2-庚醇、4-甲基-2-戊醇及環己醇。於最後提及的二級醇之情況中,該溶劑也同時作為輔受質用於輔因子再生。
若分別使用水不溶性溶劑及輔受質,則組反應批料係由水相及有機相所組成。根據其溶解度,該羥基酮會分散於有機相與水相之間。通常,有機相具有,以總反應體積計,自5至95%,較佳者自10至90%之比例。該二液體相較佳地經機械地混合使得,於彼等之間,產生大表面積。在此具體實例中,也在酶催化還原中形成的NAD,可以例如,用輔受質再還原成NADH,如上所述者。
該輔因子,特別者NADH或NADPH,分別在水相中之濃度通常為自0.001 mM至10 mM,特別者自0.01 mM至1 mM。
於根據本發明之方法中,亦可使用氧化還原酶/脫氫酶之安定劑。適合的安定劑為,例如,甘油、山梨糖醇、1,4-DL-二硫蘇糖純(1,4-DL-dithiothreitol)(DTT)或二甲亞碸(DMSO)。
根據本發明之方法係在,例如,一玻離或金屬製成之密閉容器內進行。為此目的,係將各成分個別地轉移到反應容器中且在,例如,氮氣或空氣之氣體環境中攪拌。
根據本發明之另一可能的具體實例,該氧化過的輔受質(例如,丙酮)可連續地移除及/或可將輔受質(例如,2-丙醇)以連續方式重新添加以使反應平衡朝向反應產物(通式II二醇)偏移。
在另一具體實例中,根據SEQ ID NO:1、SEQ ID NO:8、SEQ ID NO:11或SEQ ID NO:14及/或輔受質的氧化還原酶之添加亦可在程序過程中逐漸地完成。
在還原完成後,處理反應混合物。為此目的,將該水相隨意地自有機相分離出且過濾含有產物之有機相。隨意地,也可萃取該水相且類似有機相地進一步處理。隨即,將溶劑自有機相蒸發而獲得粗產物形式的通式II二醇。該粗產物可接著進一步純化或直接用於所得產物之合成。在下文中,要藉由實施例進一步闡明本發明。
自Rubrobacter xylanophilus
DSM 9941的氧化還原酶之選殖A)Rubrobacter xylanophilus
DSM 9941之培養在下述培養基內於50℃(pH7.2)及在細菌搖動培養器中以140 rpm培養Rubrobacter xylanophilus
DSM 9941之細胞:0.1%酵母萃取物、0.1%胰蛋白腖、0.004% CaSO4
x 2H2
o、0.02% MgCl2
x 6H2
O、0.01%氮基三醋酸、100毫升磷酸鹽緩衝液[5.44克/升KH2
PO4
、43克/升Na2
HPO4
x 12H2
O]、500微升/升0.01 M檸檬酸Fe、500微升/升微量元素[500微升/升H2
SO4
、2.28克/升MnSO4
x H2
O、500毫克/升ZnSO4
x 7H2
O、500毫克H3
BO3
、25毫克/升CuSO4
x 5H2
O、25毫克/升Na2
MoO4
x 2H2
O、45毫克/升CoCl2
x 6H2
O]。培養之第6天,經由離心自培養基中分離出細胞且貯存在-80℃。
B)編碼選擇性氧化還原酶的基因之擴增根據Manniatis &Sambrook在“Molecular Cloning”中所述方法萃取出基因組DNA。以所得核酸用於聚合酶鏈型反應(PCR)中作為模板,於該PCR中用到引子係衍生自NCBI資料庫中第46106817號下公開之基因序列。在如此做之中,引子係經提供在5’-端位置中分別具有核酸內切酶Nde I及Hind III或Sph I之限制部位(SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:7),供後續選殖到表現載體內所用。
擴增係在PCR緩衝液[10 mM tris-HCl(pH8.0);50 mM KCl;10 mM MgSO4
;1 mM dNTP混合物;每20 pMol之引子及2.5 U之Platinum Pfx DNA聚合酶(Invitrogen)]中與500 ng之基因組DNA及下列溫度循環進行:循環1:94℃、2分鐘循環2x30:94℃、15秒54℃、30秒68℃、60秒循環3:68℃、7分鐘4℃、∞
具有約750 bp之大小的所得PCR產物在通過1%瓊脂糖凝膠純化之後分別藉助核酸內切酶Nde I及Hind III或Sph I予以再串接,並分別連接到pET21a載體(Novagen)或pQE70載體(Qiagen)之骨幹中,其骨幹業經用相同的核酸內切酶處理過。在將2微升的連接批料轉形到大腸桿菌(E.coli)Top 10F‵細胞(Invitrogen)內之後,經由分別使用核酸內切酶Nde I及Hind III或Sph I進行限制分析以檢驗抗-安比西林(ampicillin-resistant)菌落之質體DNA中具有750bp大小的插入物之存在。對從對該片段為陽性的菌落所得質體製備物施以序列分析且隨後分別轉形到大腸桿菌(大腸桿菌)BL21 Star(Invitrogen)和大腸桿菌(E.coli)RB791(genetic stock,Yale)之內。
多肽SEQ ID NO:1在大腸桿菌細胞內之有效表現為了多肽SEQ ID NO:1在大腸桿菌細胞內之有效表現,使用要選殖到表現載體中的編碼DNA SEQ ID NO:3作為在PCR反應中之模板。在第一組160鹼對之區域中,此DNA序列與先前已知DNA序列(SEQ ID NO:2)有51鹼對不同。此改質係保守性者且不會導致胺基酸序列之改變。
擴增係在PCR緩衝液[10 mM tris-HCl(pH8.0);50 mM KCl;10 mM MgSO4
;1 mM dNTP混合物;每20 pMol之引子(SEQ ID NO:6,SEQ ID NO:5)及2.5 U之Platinum Pfx DNA聚合酶(Invitrogen)]中用50 ng作為模板之DNA SEQ ID NO:3及下列溫度循環進行:循環1:94℃、2分鐘循環2x30:94℃、40秒56℃、30秒68℃、60秒循環3:68℃、7分鐘4℃、∞
具有約750 bp之大小的所得PCR產物在通過1%瓊脂糖凝膠純化之後分別藉助核酸內切酶Nhe I及Hind III予以連接到pET21a載體(Novagen)之骨幹中,其骨幹業經用相同的核酸內切酶處理過。在將2微升的連接批料轉形到大腸桿菌(E.coli)Top 10F‵細胞(Invitrogen)內之後,經由使用核酸內切酶Nhe I及Hind III進行限制分析以檢驗抗-安比西林菌落之質體DNA中具有750 bp大小的插入物之存在。對從對該片段為陽性的菌落所得質體製備物施以序列分析且隨後分別轉形到大腸桿菌(大腸桿菌)BL21 Star(Invitrogen)之內。
自Rubrobacter xylanophilus
DSM 9941製備氧化還原酶將業經表現構成物轉形過的大腸桿菌菌株BL21 Star(Invitrogen)及RB791(E.coli
genetic stock,Yale,USA)分別用有安比西林(50微克/毫升)的培養基中(1%胰蛋白腖、0.5%酵母萃取物、1% NaCl)培養到達到在550奈米測量為0.5的光學密度為止。經由添加濃度0.1 mM之異丙基硫代半乳糖苷(IPTG)誘發該重組蛋白質之表現。在25℃及220 rpm誘導16小時後,收穫細胞且冷凍在-20℃。
為了回收酵素,將30克細胞懸浮在150毫升之三乙醇胺緩衝液中(100 mM、pH=7、2mM MgCl2
、10%甘油)及利用高壓勻化器予以溶解化。隨後,用150毫升甘油混合酵素溶液且貯存在-20℃。
使用如此獲得之酵素溶液合成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯。
在稀釋狀態中,所得酵素溶液亦用於對應的酶催化之測量。由此,依照下述構成活性檢驗:870微升之100 mM TEA緩衝液、pH 7.0、160微克NAD(P)H、10微升稀釋細胞溶裂物。反應係經由添加100微升之100 mM受質溶液(5R)-6-氰基-5-羥基-3-酮基己酸第三丁基酯到反應混合物中而起始。
利用氧化還原酶SEQ ID NO:1轉化(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯成為(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯對於(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯變成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯之轉化,係將900微升緩衝液(100 mM TEA、pH=7、1 mMMgCl2
)、100微升2-丙醇、10微升之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯粗產物(對映異構純度>99%)、0.1毫克DNA及100微升酶懸浮液(參考實施例3)的混合物在Eppendorf反應容器中室溫下溫置24小時,同時不停地混合。24小時後,96%所用的(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯業經還原成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯。其非對映異構超量達>99%。
轉化率和非對映異構超量之測定係以對掌性氣體層析法實施。為此目的,係使用一來自Shimadzu包括對掌性分離管柱CP-Chirasil-Dex CB之氣體層析儀(Varian Chrompack,Darmstadt,Germany)、一火焰離子化偵測器及使用氦作為載體。
6-氰-3,5-二羥基己酸第三丁基酯之分離出現在0.72巴(bar)及50℃ 10分鐘,5℃/分鐘→200℃ 10分鐘。
滯留時間為:(R-BCH)4.4分鐘;(R,R-BCH)47.1分鐘及(R,S-BCH)48.2分鐘。
利用氧化還原酶SEQ ID NO:1自(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯合成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯對於(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯變成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯的進一步轉化,係將550微升緩衝液(100 mM TEA、pH=7、1 mM MgCl2
)、150微升2-丙醇、200微升之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯粗產物(對映異構純度>99%)、0.1毫克NAD及200微升酶懸浮液(參考實施例3)的混合物在Eppendorf反應容器中溫置。反應過程中,經由導入氮氣,重復地蒸發所形成的丙酮/2-丙醇混合物,且添加新的2-丙醇及50微升之酶,在24小時間隔的2至3次重復後,>90%所用之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯業經還原成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯。其非對映異構超量達>99%。
利用氧化還原酶SEQ ID NO:1自(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯合成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯對於(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯變成(3R,5R)-6-氰-3,5-二羥基己酸第三丁基酯的進一步轉化,係將6.7毫升緩衝液(100 mM TEA、pH=9)、1.7毫升2-丙醇、2毫升之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯粗產物(對映異構純度>99%)、1.0毫克NAD及150毫克冷凍細胞大腸桿菌BL21 Star(含有氧化還原酶SEQ ID NO:1)(參考實施例3)之混合物在反應容器中於45℃下溫置。24小時後,>90%所用之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯業經還原成為(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯。其非對映異構超量總計為>99%。
利用氧化還原酶SEQ ID NO:8自(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯合成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯對於(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯變成(3R,5R)-6-氰-3,5-二羥基己酸第三丁基酯的進一步轉化,係將5.0毫升緩衝液(100 mM TEA、pH=7.5)、2.0毫升2-丙醇、4.0毫升之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯粗產物(對映異構純度>99%)、1.0毫克NAD及250毫克冷凍細胞大腸桿菌BL21 Star(含有氧化還原酶SEQ ID NO:8)(對應於實施例2及3)之混合物在反應容器中於40℃溫置。在24小時後,>90%所用之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯業經還原成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯。其非對映異構超量總計為>99%。
利用氧化還原酶SEQ ID NO:11自(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯合成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯對於(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯變成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯的進一步轉化,係將350微升之緩衝液(100 mM磷酸鉀、pH=7)、150微升2-丙醇、50微升之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯粗產物(對映異構純度>99%)、0.025毫克NAD及15微升酶懸浮液SEQ ID NO:11(參考實施例3)的混合物在Eppendorf反應容器中室溫下溫置48小時,同時被不停地混合。在24小時後,>80%所用之(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯業經還原成(3R,5R)-6-氰基-3,5-二羥基己酸第三丁基酯。其非對映異構超量總計為>99%。
SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4
GGGAATTCCATATGATGCTCGAGGGGAAGGTCG SEQ ID NO:5
CCCAAGCTTATTACTTGAACTCCGCGTAGCCGTC SEQ ID NO:6
CCTAGCTAGCATGCTGGAAGGTAAAGTGGC SEQ ID NO:7
CACATGCATGCGAATGCTCGAGGGGAAGGTC SEQ ID NO:8 Geobacillus thermodenitrificans
DSM 465蛋白質序列碳基還原酶 SEQ ID NO:9 Geobacillus thermodenitrificans
DSM 465核酸序列碳基還原酶 SEQ ID NO:10 Geobacillus thermodenitrificans
DSM 465核酸序列合成基因 SEQ ID NO:11
綠絲菌(Chloroflexus auratiacus)DSM635蛋白質序列碳基還原酶 SEQ ID NO:12
綠絲菌DSM635核酸序列碳基還原酶 SEQ ID NO:13
綠絲菌DSM635核酸序列合成基因 SEQ ID NO:14
木蘭假絲酵母(Candida magnoliae)CBS 6396蛋白質序列碳基還原酶 SEQ ID NO:15
木蘭假絲酵母CBS 6396核酸序列合成基因
<110> IEP GmbH <120> Verfahren zur enantioselektiven enzymatischen Reduktion von Hydrixyketoverbindungen <130> I 9845 <150> AT A 2027/2005 <151> 2005-12-19 <160> 15 <170> PatentIn version 3.3 <210> 1 <211> 249 <212> PRT <213> Rubrobacter xylanophilus DSM 9941 <400> 1 <210> 2 <211> 750 <212> DNA <213> Rubrobacter xylanophilus DSM 9941 <400> 2<210> 3 <211> 753 <212> DNA <213> 合成構成物<400> 3 <210> 4 <211> 33 <212> DNA <213> 合成構成物<400> 4<210> 5 <211> 34 <212> DNA <213> 合成構成物<400> 5<210> 6 <211> 30 <212> DNA <213> 合成構成物<400> 6<210> 7 <211> 31 <212> DNA <213> 合成構成物<400> 7<210> 8 <211> 250 <212> PRT <213> Geobacillus thermodenitrificans DSM 465 <400> 8 <210> 9 <211> 753 <212> DNA <213> geobacillus thermodenitrificans DSM 465 <400> 9 <210> 10 <211> 753 <212> DNA <213> 合成構成物<400> 10<210> 11 <211> 252 <212> PRT <213> 綠絲菌(Chloroflexus aurantiacus)DSM 635 <400> 11 <210> 12 <211> 759 <212> DNA <213> 綠絲菌(Chloroflexus aurantiacus)DSM 635 <400> 12 <210> 13 <211> 756 <212> DNA <213> 合成構成物<400> 13<210> 14 <211> 243 <212> PRT <213> 木蘭假絲酵母(Candida magnoliae)CBS 6396 <400> 14 <210> 15 <211> 732 <212> DNA <213> 木蘭假絲酵母(Candida magnoliae)CBS 6396 <400> 15
Claims (11)
- 一種用於對映異構選擇性酶催化還原通式I羥基酮
- 根據申請專利範圍第1項之方法,其中該輔因子係由輔受質持續還原。
- 根據申請專利範圍第2項之方法,其中使用2-丙醇、2-丁醇、2-戊醇、4-甲基-2-戊醇、2-庚醇或2-辛醇作為輔受質。
- 根據申請專利範圍第1項之方法,其中該羥基酮的用量以總反應體積為基準計為5至50重量%。
- 根據申請專利範圍第4項之方法,其中該羥基酮的用量為8至40重量%。
- 根據申請專利範圍第5項之方法,其中該羥基酮的用量為10至25重量%。
- 根據申請專利範圍第1至6項中任一項之方法,其中使用(5R)-6-氰基-5-羥基-3-合氧基己酸第三丁基酯、(5S)-6-氯-5-羥基-3-合氧基己酸第三丁基酯或(5S)-5,6-二羥基-3-合氧基己酸第三丁基酯作為通式(I)羥基酮。
- 根據申請專利範圍第1至6項中任一項之方法,其中TTN(總週轉數=所還原的羥基酮莫耳數/所用輔因子莫耳數)係≧103 。
- 根據申請專利範圍第1至6項中任一項之方法,其中該方法係在水有機二相系統中進行。
- 根據申請專利範圍第1至6項中任一項之方法,其中使用有機溶劑。
- 根據申請專利範圍第10項之方法,其中使用二乙醚、第三丁基甲基醚、二異丙基醚、二丁基醚、乙酸乙酯、乙酸丁酯、庚烷、已烷或環己烷作為有機溶劑。
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CN109913514A (zh) * | 2019-03-12 | 2019-06-21 | 江苏理工学院 | 一种固定化酶流化床连续催化合成ats-7的方法 |
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EP1152054A1 (en) * | 1999-12-03 | 2001-11-07 | Kaneka Corporation | Novel carbonyl reductase, gene thereof and method of using the same |
WO2003078615A1 (en) * | 2002-03-18 | 2003-09-25 | Ciba Specialty Chemicals Holding Inc. | Alcohol dehydrogenases with high solvent and temperature stability |
WO2004111083A2 (de) * | 2003-06-18 | 2004-12-23 | Iep Gmbh | Oxidoreduktase aus pichia capsulata |
WO2005108593A1 (de) * | 2004-05-10 | 2005-11-17 | Iep Gmbh | Verfahren zur herstellung von 2-butanol |
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PT1963516E (pt) | 2011-05-25 |
US8980592B2 (en) | 2015-03-17 |
AT503017A4 (de) | 2007-07-15 |
EP1963516A1 (de) | 2008-09-03 |
CA2633583A1 (en) | 2007-07-05 |
EP1963516B1 (de) | 2011-02-23 |
AT503017B1 (de) | 2007-07-15 |
WO2007073875A1 (de) | 2007-07-05 |
DK1963516T3 (da) | 2011-06-06 |
KR101344605B1 (ko) | 2013-12-26 |
TW200801196A (en) | 2008-01-01 |
AU2006331069A1 (en) | 2007-07-05 |
KR20080087000A (ko) | 2008-09-29 |
JP5328365B2 (ja) | 2013-10-30 |
PL1963516T3 (pl) | 2011-07-29 |
DE502006008961D1 (de) | 2011-04-07 |
US9228214B2 (en) | 2016-01-05 |
ZA200805336B (en) | 2009-12-30 |
US20150152451A1 (en) | 2015-06-04 |
JP2009519722A (ja) | 2009-05-21 |
CN101432435A (zh) | 2009-05-13 |
ATE499447T1 (de) | 2011-03-15 |
AU2006331069B2 (en) | 2012-04-05 |
SI1963516T1 (sl) | 2011-06-30 |
US20090221044A1 (en) | 2009-09-03 |
CA2633583C (en) | 2016-06-21 |
ES2363374T3 (es) | 2011-08-02 |
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