TW202412627A - Process for preparing (1r,2s)-2,6-dimethyl-1-indanamine - Google Patents
Process for preparing (1r,2s)-2,6-dimethyl-1-indanamine Download PDFInfo
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- TW202412627A TW202412627A TW112129495A TW112129495A TW202412627A TW 202412627 A TW202412627 A TW 202412627A TW 112129495 A TW112129495 A TW 112129495A TW 112129495 A TW112129495 A TW 112129495A TW 202412627 A TW202412627 A TW 202412627A
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Abstract
Description
本發明係關於一種用於製備實質性對映體純(1R,2S)-2,6-二甲基-1-胺基茚烷之方法,(1R,2S)-2,6-二甲基-1-胺基茚烷係用於合成除草活性成分茚嗪氟草胺(indaziflam)之有價值之建構組元。具體言之,本發明係關於一種用於藉由外消旋2,6-二甲基-1-胺基茚烷之酶催化之立體選擇性醯化製備實質性對映體純(1R,2S)-2,6-二甲基-1-胺基茚烷之方法。The present invention relates to a method for preparing substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindanes, which is a valuable building block for the synthesis of the herbicidal active ingredient indaziflam. Specifically, the present invention relates to a method for preparing substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindanes by enzyme-catalyzed stereoselective acylation of racemic 2,6-dimethyl-1-aminoindanes.
根據先前技術,迄今為止已知用於製備實質性對映體純(1R,2S)-2,6-二甲基-1-胺基茚烷之唯一方法係彼等由於使用昂貴之反應組分及觸媒而僅適用於實驗室規模,但不適用於工業用途之方法。例如,WO 2004/69814 A1揭示一種特徵在於下列五個反應步驟之方法: 1. 藉由相應肟之鈀催化還原製備2,6-二甲基-1-胺基茚烷之四種立體異構體之混合物。 2. 將該等四種立體異構體管柱層析術分離成順式及反式異構體。 3. 反式異構體混合物與2-甲氧基乙酸甲酯在酶Novozym 435®之存在下反應以形成相應之乙醯化(1R,2S)-2,6-二甲基-1-胺基茚烷。 4. 分離該乙醯化(1R,2S)-2,6-二甲基-1-胺基茚烷。 5. 酸性水解該乙醯化(1R,2S)-2,6-二甲基-1-胺基茚烷以形成游離(1R,2S)-2,6-二甲基-1-胺基茚烷。 According to the prior art, the only methods known so far for preparing substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindanane are those which are only suitable for laboratory scale but not for industrial use due to the use of expensive reaction components and catalysts. For example, WO 2004/69814 A1 discloses a method characterized by the following five reaction steps: 1. Preparing a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindanane by palladium-catalyzed reduction of the corresponding oxime. 2. Separating the four stereoisomers into cis- and trans-isomers by column chromatography. 3. The trans-isomer mixture is reacted with methyl 2-methoxyacetate in the presence of the enzyme Novozym 435® to form the corresponding acetylated (1R,2S)-2,6-dimethyl-1-aminoindan. 4. The acetylated (1R,2S)-2,6-dimethyl-1-aminoindan is separated. 5. The acetylated (1R,2S)-2,6-dimethyl-1-aminoindan is hydrolyzed with acid to form free (1R,2S)-2,6-dimethyl-1-aminoindan.
Tetrahedron 2007, 63 (29), 6755-6763描述一種用於製備(1R,2S)-2,6-二甲基-1-胺基茚烷之方法,其特徵在於下列兩個反應步驟: 1. 藉助於對掌性釕觸媒非鏡像異構體選擇性(96:4 d.r.)及對映體選擇性(98:2 e.r.)還原外消旋1,6-二甲基茚-1-酮以形成產率為80%之相應(1S,2S)-2,6-二甲基茚-1-醇。 2. 因此獲得之(1S,2S)-2,6-二甲基茚-1-醇與二苯基磷醯基疊氮化物反應及隨後用氫化鋰鋁還原產生產率為76%之(1R,2S)-2,6-二甲基-1-胺基茚烷。 Tetrahedron 2007, 63 (29), 6755-6763 describes a process for the preparation of (1R,2S)-2,6-dimethyl-1-aminoindanane, which is characterized by the following two reaction steps: 1. Non-imagewise isomer-selective (96:4 d.r.) and enantioselective (98:2 e.r.) reduction of racemic 1,6-dimethylindan-1-one with the aid of a chiral ruthenium catalyst to form the corresponding (1S,2S)-2,6-dimethylindan-1-ol in 80% yield. 2. The (1S,2S)-2,6-dimethylindene-1-ol thus obtained was reacted with diphenylphosphatidyl azide and subsequently reduced with lithium aluminum hydroxide to produce (1R,2S)-2,6-dimethyl-1-aminoindane in a yield of 76%.
認為此方法之缺點不僅在於使用昂貴之試劑,且亦在於總計八天之長反應時間。It is believed that the disadvantage of this method is not only the use of expensive reagents, but also the long reaction time of eight days in total.
本發明之目的係提供一種用於製備實質性對映體純(1R,2S)-2,6-二甲基-1-胺基茚烷之方法,其克服自先前技術已知的缺點。The object of the present invention is to provide a process for preparing substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindanane which overcomes the disadvantages known from the prior art.
吾人發現一種用於製備實質性對映體純(1R,2S)-2,6-二甲基-1-胺基茚烷之方法,其特徵在於使2,6-二甲基-1-胺基茚烷之四種立體異構體之混合物與醯化劑或羧化劑在酶之存在下反應。We have found a method for preparing substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindanane, which comprises reacting a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindanane with an acylation agent or a carboxylation agent in the presence of an enzyme.
本發明提供一種用於製備實質性對映體純(1R,2S)-2,6-二甲基-1-胺基茚烷之方法,其特徵在於:The present invention provides a method for preparing substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindanane, which is characterized by:
1. 於第一步驟中,使2,6-二甲基-1-胺基茚烷(I)之四種立體異構體之混合物與醯化劑或羧化劑R-C(=O)R 1在具有脂肪酶活性之蛋白質之存在下反應以形成相應之醯胺或胺甲酸酯(II)及2,6-二甲基-1-胺基茚烷之未反應立體異構體之混合物(III): , 其中該蛋白質係由選自由以下組成之群之胺基酸序列編碼: I.與在SEQ ID No. 1顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之蛋白質, II.與在SEQ ID No. 1顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之蛋白質,除與在SEQ ID No. 1顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列具有選自由以下組成之群之修飾的事實外: i. 於位置186處之胺基酸不同於L; ii. 於位置280處之胺基酸不同於L; iii. 於位置312係處之胺基酸不同於P; iv. 於位置3處之胺基酸不同於M; v. 於位置29處之胺基酸不同於N; vi. 於位置17處之胺基酸不同於L; vii. 於位置4處之胺基酸不同於S; viii. 於位置18處之胺基酸不同於V; ix. 於位置202處之胺基酸不同於A; x. 於位置301處之胺基酸不同於D; xi. 於位置309處之胺基酸不同於P; xii. 於位置31處之胺基酸不同於Q; xiii. 於位置111處之胺基酸不同於Q; xiv. 於位置85處之胺基酸不同於W; xv. 於位置8處之胺基酸不同於K; xvi. 於位置79處之胺基酸不同於E; xvii. 於位置40處之胺基酸不同於K; 1. In the first step, a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan (I) is reacted with an acylation agent or carboxylation agent RC(=O) R1 in the presence of a protein having lipase activity to form the corresponding amide or carbamate (II) and a mixture of unreacted stereoisomers of 2,6-dimethyl-1-aminoindan (III): , wherein the protein is encoded by an amino acid sequence selected from the group consisting of: I. a protein having at least 80%, preferably 85%, better 90%, even better 95%, even better 96%, even better 97%, particularly better 98%, and best 99% identity to the amino acid sequence shown in SEQ ID No. 1, II. a protein having at least 80%, preferably 85%, better 90%, even better 95%, even better 96%, even better 97%, particularly better 98%, and best 99% identity to the amino acid sequence shown in SEQ ID No. 1, except for the fact that the amino acid sequence having at least 80%, preferably 85%, better 90%, even better 95%, even better 96%, even better 97%, particularly better 98%, and best 99% identity to the amino acid sequence shown in SEQ ID No. 1 has a modification selected from the group consisting of: i. The amino acid at position 186 is different from L; ii. The amino acid at position 280 is different from L; iii. The amino acid at position 312 is different from P; iv. The amino acid at position 3 is different from M; v. The amino acid at position 29 is different from N; vi. The amino acid at position 17 is different from L; vii. The amino acid at position 4 is different from S; viii. The amino acid at position 18 is different from V; ix. The amino acid at position 202 is different from A; x. The amino acid at position 301 is different from D; xi. The amino acid at position 309 is different from P; xii. The amino acid at position 31 is different from Q; xiii. The amino acid at position 111 is different from Q; xiv. The amino acid at position 85 is different from W; xv. The amino acid at position 8 is different from K; xvi. The amino acid at position 79 is different from E; xvii. The amino acid at position 40 is different from K;
2. 於第二步驟中,藉由結晶自殘餘2,6-二甲基-1-胺基茚烷(III)及次要組分分離該醯胺或胺甲酸酯(II),2. In a second step, the amide or carbamate (II) is separated from the residual 2,6-dimethyl-1-aminoindanane (III) and the secondary components by crystallization,
3. 於第三步驟中,使用酸或鹼將該醯胺或胺甲酸酯(II)轉化為實質性對映體純(1R,2S)-2,6-二甲基-1-胺基茚烷(IV), 其中R意謂選自由以下組成之群之基團:CH 2OCH 3、CH 2OCH 2CH 3、CH 3、OCH 3、OCH 2CH 3、OCH(CH 3) 2、OCH 2CH 2CH 2CH 3、OCH 2CHCH 2及OCH 2(C 6H 5), 及 其中R 1意謂選自由以下組成之群之基團:OCH 3、OCH 2CH 3、OCH(CH 3) 2、OCH 2CH 2CH 2CH 3、OCH 2CHCH 2及OCH 2(C 6H 5)。 3. In the third step, the amide or carbamate (II) is converted into substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindanane (IV) using an acid or a base. wherein R means a group selected from the group consisting of CH 2 OCH 3 , CH 2 OCH 2 CH 3 , CH 3 , OCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 CH 2 CH 2 CH 3 , OCH 2 CHCH 2 , and OCH 2 (C 6 H 5 ), and wherein R 1 means a group selected from the group consisting of OCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 CH 2 CH 2 CH 3 , OCH 2 CHCH 2 , and OCH 2 (C 6 H 5 ).
需作為用於根據本發明之方法之起始材料之2,6-二甲基-1-胺基茚烷(I)之四種立體異構體之混合物係已知的且可(例如)如WO 2004/69814中描述製備。The mixture of the four stereoisomers of 2,6-dimethyl-1-aminoindanane (I) required as starting material for the process according to the invention is known and can be prepared, for example, as described in WO 2004/69814.
較佳地,R意謂選自由以下組成之群之基團:OCH 3及OCH 2CH 3, 及 R 1意謂選自由以下組成之群之基團:OCH 3及OCH 2CH 3。 Preferably, R means a group selected from the group consisting of OCH 3 and OCH 2 CH 3 , and R 1 means a group selected from the group consisting of OCH 3 and OCH 2 CH 3 .
在根據本發明之方法中,使用具有解脂酶或脂肪酶活性之蛋白質。In the method according to the invention, a protein having lipolytic or lipase activity is used.
SEQ ID No. 1係解脂野生型蛋白之胺基酸序列。野生型脂肪酶係來源於來自環境樣本之非培養細菌,其可來源於GenPept (PDB),登錄號為QRD81023 (版本ORD81023.1)。在SEQ ID No. 1中顯示之胺基酸序列與上文資料庫條目中顯示之序列之間存在疑問之情況下,SEQ ID No. 1具有優先權。SEQ ID No. 1 is the amino acid sequence of the wild-type protein of lipolysis. The wild-type lipase is derived from an uncultivated bacterium from an environmental sample and is available from GenPept (PDB) under accession number QRD81023 (version ORD81023.1). In the event of a doubt between the amino acid sequence shown in SEQ ID No. 1 and the sequence shown in the database entry above, SEQ ID No. 1 has priority.
本發明進一步描述具有解脂酶或脂肪酶活性之蛋白質,其中此等蛋白質之胺基酸序列係具有解脂酶或脂肪酶活性之已知蛋白質之變體。特定言之,本文描述之具有脂肪酶活性之蛋白質之胺基酸序列係由SEQ ID No. 1中顯示之胺基酸描述之胺基酸序列之變體,其中,在SEQ ID No. 1顯示之胺基酸序列中,至少於位置186處之胺基酸、於位置280處之胺基酸、於位置312處之胺基酸、於位置3處之胺基酸、於位置29處之胺基酸、於位置17處之胺基酸、於位置4處之胺基酸、於位置18處之胺基酸、於位置202處之胺基酸、於位置301處之胺基酸、於位置309處之胺基酸、於位置31處之胺基酸、於位置111處之胺基酸、於位置85處之胺基酸、於位置8處之胺基酸、於位置79處之胺基酸或於位置40處之胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處存在之胺基酸。The present invention further describes proteins having lipolytic or lipase activity, wherein the amino acid sequences of these proteins are variants of known proteins having lipolytic or lipase activity. Specifically, the amino acid sequence of the protein having lipase activity described herein is a variant of the amino acid sequence described by the amino acids shown in SEQ ID No. 1, wherein, in the amino acid sequence shown in SEQ ID No. 1, at least the amino acid at position 186, the amino acid at position 280, the amino acid at position 312, the amino acid at position 3, the amino acid at position 29, the amino acid at position 17, the amino acid at position 4, the amino acid at position 18, the amino acid at position 202, the amino acid at position 301, the amino acid at position 309, the amino acid at position 31, the amino acid at position 111, the amino acid at position 85, the amino acid at position 8, the amino acid at position 79 or the amino acid at position 40 is different from the amino acid at position 186, the amino acid at position 280, the amino acid at position 312, the amino acid at position 313, the amino acid at position 314, the amino acid at position 315, the amino acid at position 316, the amino acid at position 317, the amino acid at position 318, the amino acid at position 319, the amino acid at position 319, the amino acid at position 310, the amino acid at position 311, the amino acid at position 312, the amino acid at position 313, the amino acid at position 314, the amino acid at position 315, the amino acid at position 316, the amino acid at position 317, the amino acid at position 318, the amino acid at position 319, the amino acid at position 319, the amino acid at position 318, the amino acid at position 319, the amino acid at position 319, the amino acid at position 310 1 is the amino acid present at the corresponding amino acid position in the sequence shown.
如本文使用,術語「變體」意謂一種與根據先前技術已知的實體不同之實體。關於核酸分子及蛋白質,應瞭解變體意謂偏離相應已知序列,但編碼具有相同功能或催化相同反應之蛋白質(例如編碼具有脂肪酶活性之蛋白質之功能)之核酸序列或胺基酸序列。核酸分子序列及胺基酸序列自已知核酸序列及蛋白質序列之「偏離」意謂相較於相應已知核酸序列或胺基酸序列,該等序列包含核苷酸或胺基酸之取代(置換)及/或缺失及/或插入。As used herein, the term "variant" means an entity that is different from an entity known according to the prior art. With respect to nucleic acid molecules and proteins, it is understood that a variant means a nucleic acid sequence or an amino acid sequence that deviates from the corresponding known sequence but encodes a protein having the same function or catalyzing the same reaction (e.g., a function of encoding a protein having lipase activity). "Deviation" of nucleic acid molecule sequences and amino acid sequences from known nucleic acid sequences and protein sequences means that, compared to the corresponding known nucleic acid sequences or amino acid sequences, these sequences contain substitutions (replacements) and/or deletions and/or insertions of nucleotides or amino acids.
通常使用具有脂肪酶活性之蛋白質,其中該等蛋白質係由與在SEQ ID No. 1顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列編碼。Typically, proteins having lipase activity are used, wherein the proteins are encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity to the amino acid sequence shown in SEQ ID No. 1.
同樣使用具有脂肪酶活性之蛋白質,其中該等蛋白質係由與在SEQ ID No. 1顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列編碼,除與在SEQ ID No. 1顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列具有選自由以下組成之群之修飾的事實外: i. 於位置186處之胺基酸不同於L,較佳於位置186處之胺基酸係F、W、Y、E、D、Q、T、H、P、C、K、S、N、I或V,更佳於位置186處之胺基酸係F、W、Y、E、D或K,特別佳於位置186處之胺基酸係W或Y,最佳於位置186處之胺基酸係Y; ii. 於位置280處之胺基酸不同於L,較佳於位置280處之胺基酸係E、S、K、D或A,更佳於位置280處之胺基酸係A; iii. 於位置312係處之胺基酸不同於P,較佳於位置312處之胺基酸係N、F、D、Q或K,更佳於位置312處之胺基酸係N; iv. 於位置3處之胺基酸不同於M,較佳於位置3處之胺基酸係L、Q或C,更佳於位置3處之胺基酸係Q; v. 於位置29處之胺基酸不同於N,較佳於位置29處之胺基酸係H、W或Y,更佳於位置29處之胺基酸係H或W,最佳於位置29處之胺基酸係H; vi. 於位置17處之胺基酸不同於L,較佳於位置17處之胺基酸係P或T,更佳於位置17處之胺基酸係P; vii. 於位置4處之胺基酸不同於S,較佳於位置4處之胺基酸係P或L,更佳於位置4處之胺基酸係P; viii. 於位置18處之胺基酸不同於V,較佳於位置18處之胺基酸係A、T、C或S,更佳於位置18處之胺基酸係A或C; ix. 於位置202處之胺基酸不同於A,較佳於位置202處之胺基酸係Q或N,更佳於位置202處之胺基酸係N; x. 於位置301處之胺基酸不同於D,較佳於位置301處之胺基酸係A; xi. 於位置309處之胺基酸不同於P,較佳於位置309處之胺基酸係C; xii. 於位置31處之胺基酸不同於Q,較佳於位置31處之胺基酸係W; xiii. 於位置111處之胺基酸不同於Q,較佳於位置111處之胺基酸係E; xiv. 於位置85處之胺基酸不同於W,較佳於位置85處之胺基酸係H; xv. 於位置8處之胺基酸不同於K,較佳於位置8處之胺基酸係E; xvi. 於位置79處之胺基酸不同於K,較佳於位置79處之胺基酸係S、I或W,更佳於位置79處之胺基酸係S; xvii. 於位置40處之胺基酸不同於K,較佳於位置40處之胺基酸係M。 Likewise, proteins having lipase activity are used, wherein the proteins are encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and optimally 99% identity to the amino acid sequence shown in SEQ ID No. 1, except for the fact that the amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and optimally 99% identity to the amino acid sequence shown in SEQ ID No. 1 has a modification selected from the group consisting of: i. The amino acid at position 186 is different from L, preferably the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V, more preferably the amino acid at position 186 is F, W, Y, E, D or K, particularly preferably the amino acid at position 186 is W or Y, and most preferably the amino acid at position 186 is Y; ii. The amino acid at position 280 is different from L, preferably the amino acid at position 280 is E, S, K, D or A, and more preferably the amino acid at position 280 is A; iii. The amino acid at position 312 is different from P, preferably the amino acid at position 312 is N, F, D, Q or K, and more preferably the amino acid at position 312 is N; iv. The amino acid at position 3 is different from M, preferably the amino acid at position 3 is L, Q or C, and more preferably the amino acid at position 3 is Q; v. The amino acid at position 29 is different from N, preferably the amino acid at position 29 is H, W or Y, more preferably the amino acid at position 29 is H or W, and most preferably the amino acid at position 29 is H; vi. The amino acid at position 17 is different from L, preferably the amino acid at position 17 is P or T, and more preferably the amino acid at position 17 is P; vii. The amino acid at position 4 is different from S, preferably the amino acid at position 4 is P or L, and more preferably the amino acid at position 4 is P; viii. The amino acid at position 18 is different from V, preferably the amino acid at position 18 is A, T, C or S, and more preferably the amino acid at position 18 is A or C; ix. The amino acid at position 202 is different from A, preferably the amino acid at position 202 is Q or N, and more preferably the amino acid at position 202 is N; x. The amino acid at position 301 is different from D, and preferably the amino acid at position 301 is A; xi. The amino acid at position 309 is different from P, and preferably the amino acid at position 309 is C; xii. The amino acid at position 31 is different from Q, and preferably the amino acid at position 31 is W; xiii. The amino acid at position 111 is different from Q, preferably the amino acid at position 111 is E; xiv. The amino acid at position 85 is different from W, preferably the amino acid at position 85 is H; xv. The amino acid at position 8 is different from K, preferably the amino acid at position 8 is E; xvi. The amino acid at position 79 is different from K, preferably the amino acid at position 79 is S, I or W, more preferably the amino acid at position 79 is S; xvii. The amino acid at position 40 is different from K, preferably the amino acid at position 40 is M.
胺基酸縮寫A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y之含義可於下表2中標題為「序列描述」之部分中找到。The meanings of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y can be found in the section entitled "Sequence Description" in Table 2 below.
根據本發明之另一實施例涉及具有脂肪酶活性之蛋白質,其中該等蛋白質係選自由以下組成之群: a) 包含SEQ ID No. 1中顯示之胺基酸序列之蛋白質,除於位置186處之胺基酸不同於L的事實外; b) 具有與在a)顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列之蛋白質,限制條件為於位置186處之胺基酸不同於L。 Another embodiment of the present invention relates to proteins having lipase activity, wherein the proteins are selected from the group consisting of: a) a protein comprising the amino acid sequence shown in SEQ ID No. 1, except for the fact that the amino acid at position 186 is different from L; b) a protein having an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and optimally 99% identity with the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L.
較佳地,根據a)或b)之蛋白質中於位置186處之胺基酸係F、W、Y、E、D、Q、T、H、P、C、K、S、N、I或V。更佳地,於位置186處之胺基酸係F、W、Y、E、D或K。特別佳地,於位置186處之胺基酸係W或Y。最佳地,於位置186處之胺基酸係Y。Preferably, the amino acid at position 186 in the protein according to a) or b) is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V. More preferably, the amino acid at position 186 is F, W, Y, E, D or K. Particularly preferably, the amino acid at position 186 is W or Y. Most preferably, the amino acid at position 186 is Y.
「對應於第一胺基酸序列中位置X之胺基酸」 (例如SEQ ID No. 1中之位置3)於本文中意謂若第二胺基酸序列之胺基酸編號偏離該第一胺基酸序列之胺基酸編號,則相較於該第一胺基酸序列,該第二胺基酸序列之胺基酸於該第一胺基酸序列與該第二胺基酸序列之成對序列比對中出現於該第一胺基酸序列之位置x處。"An amino acid corresponding to position x in a first amino acid sequence" (e.g., position 3 in SEQ ID No. 1) herein means that if the amino acid number of a second amino acid sequence deviates from the amino acid number of the first amino acid sequence, then the amino acid of the second amino acid sequence appears at position x of the first amino acid sequence in a pairwise sequence alignment of the first amino acid sequence and the second amino acid sequence relative to the first amino acid sequence.
結合本發明,應瞭解與序列一致性或相同序列相關之術語「一致性」意謂於整個序列長度上,第一核酸或胺基酸序列與另一(第二)核酸或胺基酸序列共有之相同胺基酸或核苷酸之數量,以百分率表示。In conjunction with the present invention, it should be understood that the term "identity" in relation to sequence identity or identical sequence means the number of identical amino acids or nucleotides shared by a first nucleic acid or amino acid sequence and another (second) nucleic acid or amino acid sequence over the entire length of the sequence, expressed as a percentage.
「序列一致性」可藉由使用如(例如)諸如GAP或BESTFIT或Emboss程式「Needle」之已知軟體中含有之全域或局部比對演算法比對兩個胺基酸或兩個核苷酸序列確定。此軟體使用Needleman及Wunsch之全域比對演算法以於其等整個長度上比對兩個序列,用於使匹配數量最大化及用於使空位數量最小化。一般而言,使用默認參數,及空位產生罰分= 10及空位擴展罰分= 0.5 (針對核苷酸比對及蛋白質比對兩者)。針對核苷酸,使用之默認評分矩陣係DNAFULL,及針對蛋白質,該默認評分矩陣係Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919)。用於序列一致性百分比之序列比對及分數可例如使用諸如可於EBI網站(ebi.ac.uk/Tools/emboss/)上獲得之EMBOSS之軟體確定。或者,序列相似性或一致性可藉由使用通常已知的演算法及輸出格式(諸如FASTA、BLAST等)於資料庫(例如EMBL、GenBank)中搜索確定,但較佳應檢索命中並成對比對以最終確定序列一致性。"Sequence identity" can be determined by aligning two amino acid or two nucleotide sequences using a global or local alignment algorithm as contained in known software such as, for example, GAP or BESTFIT or the Emboss program "Needle". This software uses the global alignment algorithm of Needleman and Wunsch to align two sequences over their entire length, for maximizing the number of matches and for minimizing the number of gaps. In general, default parameters are used, and gap creation penalty = 10 and gap expansion penalty = 0.5 (for both nucleotide and protein alignments). For nucleotides, the default scoring matrix used is DNAFULL, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignments and scores for percent sequence identity can be determined, for example, using software such as EMBOSS, available on the EBI website (ebi.ac.uk/Tools/emboss/). Alternatively, sequence similarity or identity can be determined by searching in databases (e.g., EMBL, GenBank) using commonly known algorithms and output formats (e.g., FASTA, BLAST, etc.), but preferably hits should be retrieved and paired alignments are used to ultimately determine sequence identity.
若待彼此比較之序列在長度上有所不同,則一致性應藉由確定較短序列與較長序列共有之胺基酸或核苷酸之數量之一致性百分比來確定。較佳地,一致性係在已知及公開可用之電腦程式ClustalW之幫助下確定(Thompson等人,Nucleic Acids Research 22 (1994), 4673-4680)。ClustalW可自Julie Thompson (Thompson@EMBL-Heidelberg.DE)及Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstraße 1, D 69117 Heidelberg, Germany公開獲取。ClustalW亦可自各種網站下載,諸如自IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P.163, 67404 Illkirch Cedex, France;ftp://ftp-igbmc.u-strasbg.fr/pub/)及自EBI (ftp://ftp.ebi.ac.uk/pub/software/)及自EBI之鏡像網站(European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK)獲取。If the sequences to be compared differ in length, the identity should be determined by determining the percent identity of the number of amino acids or nucleotides shared by the shorter sequence and the longer sequence. Preferably, the identity is determined with the aid of the known and publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW is publicly available from Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstraße 1, D 69117 Heidelberg, Germany. ClustalW can also be downloaded from various websites, such as from IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P.163, 67404 Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and from EBI (ftp://ftp.ebi.ac.uk/pub/software/) and from the EBI's mirror website (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).
較佳地,使用電腦程式ClustalW之1.8版本確定本發明之內文中描述之蛋白質與其他蛋白質之間的一致性。此處,必須如下設定參數:KTUPLE=1,TOPDIAG=5,WINDOW=5,PAIRGAP=3,GAPOPEN=10,GAPEXTEND=0.05,GAPDIST=8,MAXDIV=40,MATRIX=GONNET,ENDGAPS(OFF),NOPGAP,NOHGAP。Preferably, the identity between the proteins described in the context of the present invention and other proteins is determined using the computer program ClustalW version 1.8. Here, the parameters must be set as follows: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.
較佳地,使用電腦程式ClustalW之1.8版確定例如結合本發明描述之核酸分子之核苷酸序列與其他核酸分子之核苷酸序列之間的一致性。此處,必須如下設定參數: KTUPLE=2,TOPDIAGS=4,PAIRGAP=5,DNAMATRIX:IUB,GAPOPEN=10,GAPEXT=5,MAXDIV=40,TRANSITIONS:未加權。 Preferably, the computer program ClustalW version 1.8 is used to determine the identity between the nucleotide sequence of the nucleic acid molecule described in the present invention and the nucleotide sequence of other nucleic acid molecules, for example. Here, the parameters must be set as follows: KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.
「一致性」亦意謂所述核酸分子或由此編碼之蛋白質之間存在功能及/或結構等效。功能等效意謂該等核酸分子序列或該等胺基酸序列編碼具有脂肪酶活性之蛋白質。與上文描述之分子及此等分子之衍生物同源之核酸分子一般為此等分子之變體,其等係具有相同生物功能或催化相同反應(即編碼具有脂肪酶活性之蛋白質)之修飾。其等可為自然發生之變體,例如來自其他物種之序列,或突變,其中該等突變可為自然發生或係藉由靶向誘變引入。此外,該等變體可為合成產生之序列。對偶基因變體可為自然發生之變體或合成產生之變體,或藉由重組DNA技術產生之變體。然而,對本發明而言至關重要的是,此等變體編碼具有脂肪酶活性之蛋白質且包括關於根據本發明之蛋白質之目前描述之胺基酸取代(置換)、缺失或插入。"Consistency" also means that there is functional and/or structural equivalence between the nucleic acid molecules or the proteins encoded thereby. Functional equivalence means that the nucleic acid molecule sequences or the amino acid sequences encode proteins with lipase activity. Nucleic acid molecules homologous to the molecules described above and derivatives of such molecules are generally variants of such molecules, which are modifications that have the same biological function or catalyze the same reaction (i.e., encode proteins with lipase activity). They can be naturally occurring variants, such as sequences from other species, or mutations, wherein the mutations can occur naturally or be introduced by targeted mutagenesis. In addition, the variants can be synthetically produced sequences. Paragenic variants can be naturally occurring variants or synthetically produced variants, or variants produced by recombinant DNA technology. However, it is essential to the present invention that these variants encode proteins having lipase activity and comprise the presently described amino acid substitutions (replacements), deletions or insertions for the proteins according to the present invention.
衍生物之一特定類型係例如核酸分子,其透過遺傳編碼之簡併性而不同於本發明之內文中描述之核酸分子。One particular type of derivative is, for example, a nucleic acid molecule that differs from the nucleic acid molecules described in the context of the present invention by virtue of the degeneracy of the genetic code.
根據NC-IUBMB (國際生物化學與分子生物學聯合會命名委員會(Nomenclature Committee of the International Union of Biochemistry and Molecular Biology)),脂肪酶屬於水解酶之類別(EC 3)。水解酶係一類通常充當生物化學觸媒並使用水來破壞化學鍵(此通常導致較大分子分裂成較小分子)的酶。該等水解酶之群包含作用於酯鍵上之酶(EC 3.1),諸如羧酸酯水解酶(EC 3.1.1),及作為亞群,脂肪酶(EC 3.1.1.3)。已自植物、哺乳動物及微生物鑑別脂肪酶,例如假單胞菌屬( Pseudomonas)、弧菌屬( Vibrio)、不動桿菌屬( Acinetobacter)、伯克氏菌屬( Burkholderia)、產色細菌屬( Chromobacterium)、來自茄鐮孢菌( Fusarium solani)之角質酶(FSC)、南極假絲酵母A ( Candida antarctica A) (CalA)、米根黴( Rhizopus oryzae) (ROL)、疏棉嗜熱黴菌( Thermomyces lanuginosus) (TLL)、米氏假根毛黴( Rhizomucor miehei) (RML)、黑麴菌( Aspergillus Niger)、異孢鐮孢菌( Fusarium heterosporum)、尖鐮孢菌( Fusarium oxysporum)或禾稈鐮孢菌( Fusarium culmorum)。 According to the NC-IUBMB (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), lipases belong to the class of hydrolases (EC 3). Hydrolases are a class of enzymes that generally act as biochemical catalysts and use water to break chemical bonds, which usually results in the splitting of larger molecules into smaller ones. This group of hydrolases includes enzymes acting on ester bonds (EC 3.1), such as carboxylate hydrolases (EC 3.1.1), and as a subgroup, lipases (EC 3.1.1.3). Lipases have been identified from plants, mammals and microorganisms, such as Pseudomonas , Vibrio , Acinetobacter , Burkholderia , Chromobacterium , cutinase from Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger , Fusarium heterosporum , Fusarium oxysporum ) or Fusarium culmorum .
若蛋白質具有脂肪酶活性,則其可使用已知及根據先前技術描述之方法偵測。使用何種方法來偵測根據本發明之蛋白質是否具有脂肪酶活性並非關鍵的。較佳地,結合本發明於「實例」部分中給定該方法之描述。If the protein has lipase activity, it can be detected using methods known and described according to the prior art. It is not critical which method is used to detect whether the protein according to the present invention has lipase activity. Preferably, a description of the method is given in the "Examples" section in conjunction with the present invention.
相較於與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文描述之胺基酸序列,根據本發明之脂肪酶變體蛋白可具有其他胺基酸修飾(胺基酸取代、缺失或插入)。Compared to the amino acid sequence described herein above in relation to the amino acid sequence shown in SEQ ID No. 1, the lipase variant protein according to the present invention may have other amino acid modifications (amino acid substitutions, deletions or insertions).
與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可另外具有於位置79、202、280、301、3、11、17、40或111處之至少一、二、三、四、五、六或七個其他胺基酸取代。換而言之,具有脂肪酶活性之根據本發明之蛋白質係選自由以下組成之群:a)包含SEQ ID No. 1中顯示之胺基酸序列之蛋白質,除於位置186處之胺基酸不同於L且其等具有至少一、二、三、四、五、六、七個或更多個選自由以下組成之群之其他胺基酸取代的事實外:(i)於位置79處之胺基酸不同於E;(ii)於位置202處之胺基酸不同於A;(iii)於位置280處之胺基酸不同於L;(iv)於位置301處之胺基酸不同於D;(v)於位置3處之胺基酸不同於M;(vi)於位置11處之胺基酸不同於C;(vii)於位置17處之胺基酸不同於L;(viii)於位置40處之胺基酸不同於K;(ix)於位置111處之胺基酸不同於Q;及b)具有與上文在a)下直接顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列之蛋白質,限制條件為於位置186處之胺基酸不同於L且具有至少一個選自上文直接提及之(i)至(ix)群之另一胺基酸取代。較佳地,於位置79處之胺基酸係S、W或I,更佳S;較佳地,於位置202處之胺基酸係N;較佳地,於位置280處之胺基酸係A;較佳地,於位置301處之胺基酸係A;較佳地,於位置3處之胺基酸係Q;較佳地,於位置11處之胺基酸係A;較佳地,於位置17處之胺基酸係P;較佳地,於位置40處之胺基酸係M;較佳地,於位置111處之胺基酸係E。The lipase variants described herein above under point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may additionally have at least one, two, three, four, five, six or seven other amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. In other words, the protein according to the invention having lipase activity is selected from the group consisting of: a) comprising SEQ ID No. 1, except that the amino acid at position 186 is different from L and it has at least one, two, three, four, five, six, seven or more other amino acid substitutions selected from the group consisting of: (i) the amino acid at position 79 is different from E; (ii) the amino acid at position 202 is different from A; (iii) the amino acid at position 280 is different from L; (iv) the amino acid at position 301 is different from D; (v) the amino acid at position 3 is different from M; (vi) the amino acid at position 11 is different from C; (vii) the amino acid at position (viii) the amino acid at position 40 is different from L; (ix) the amino acid at position 111 is different from Q; and b) a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown directly above under a), with the proviso that the amino acid at position 186 is different from L and has at least one other amino acid substitution selected from the groups (i) to (ix) mentioned directly above. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.
此外,除其他胺基酸修飾外,相較於與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文描述之胺基酸序列,根據本發明之脂肪酶變體蛋白可具有另外胺基酸取代。此等另外胺基酸取代涉及不同於與其他胺基酸修飾相關之位置79、202、280、301、3、11、17、40或111之胺基酸序列之位置。與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可具有於位置4、8、18、29、31、42、84、85、192、217、309或312處之至少一、二、三、四、五、六、七個或更多個另外胺基酸取代。於位置4處之胺基酸不同於S,較佳於此位置處之胺基酸係P。於位置8處之胺基酸不同於K,較佳於此位置處之胺基酸係E。於位置18處之胺基酸不同於V,較佳於此位置處之胺基酸係C。於位置29處之胺基酸不同於N,較佳於此位置處之胺基酸係W或H。於位置31處之胺基酸不同於Q,較佳於此位置處之胺基酸係W。於位置42處之胺基酸不同於L,較佳於此位置處之胺基酸係D。於位置84處之胺基酸不同於N,較佳於此位置處之胺基酸係T。於位置85處之胺基酸不同於W,較佳於此位置處之胺基酸係H。於位置192處之胺基酸不同於F,較佳於此位置處之胺基酸係A或V。於位置217處之胺基酸不同於Q,較佳於此位置處之胺基酸係M。於位置309處之胺基酸不同於P,較佳於此位置處之胺基酸係C。於位置312係處之胺基酸不同於P,較佳於此位置處之胺基酸係N。In addition, in addition to other amino acid modifications, the lipase variant protein according to the present invention may have additional amino acid substitutions compared to the amino acid sequence described above in this article related to the amino acid sequence shown in SEQ ID No. 1. These additional amino acid substitutions relate to positions of amino acid sequences different from positions 79, 202, 280, 301, 3, 11, 17, 40 or 111 related to other amino acid modifications. The lipase variant described above in this article under point a) related to the amino acid sequence shown in SEQ ID No. 1 may have at least one, two, three, four, five, six, seven or more additional amino acid substitutions at positions 4, 8, 18, 29, 31, 42, 84, 85, 192, 217, 309 or 312. The amino acid at position 4 is different from S, preferably the amino acid at this position is P. The amino acid at position 8 is different from K, preferably the amino acid at this position is E. The amino acid at position 18 is different from V, preferably the amino acid at this position is C. The amino acid at position 29 is different from N, preferably the amino acid at this position is W or H. The amino acid at position 31 is different from Q, preferably the amino acid at this position is W. The amino acid at position 42 is different from L, preferably the amino acid at this position is D. The amino acid at position 84 is different from N, preferably the amino acid at this position is T. The amino acid at position 85 is different from W, preferably the amino acid at this position is H. The amino acid at position 192 is different from F, preferably the amino acid at this position is A or V. The amino acid at position 217 is different from Q, preferably the amino acid at this position is M. The amino acid at position 309 is different from P, preferably the amino acid at this position is C. The amino acid at position 312 is different from P, preferably the amino acid at this position is N.
因此,根據本發明之另一實施例涉及具有其他胺基酸修飾之根據本發明之蛋白質;較佳地,此等實施例係具有脂肪酶活性之蛋白質,其中該等蛋白質係選自由以下組成之群: 具有SEQ ID No. 1中顯示之胺基酸序列之蛋白質,除以下外: - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E,其中較佳於位置186處之胺基酸較佳為W或Y,更佳Y,及於位置79處之胺基酸較佳為S、W或I,更佳S; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置111處之胺基酸較佳為E; - 具有與在a)顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列之蛋白質,限制條件為於位置186處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y,且其中該等蛋白質具有至少一個選自上文在本文列舉之符號下直接顯示之群的另一胺基酸取代。 Therefore, another embodiment according to the present invention relates to proteins according to the present invention having other amino acid modifications; preferably, these embodiments are proteins having lipase activity, wherein the proteins are selected from the group consisting of: Proteins having the amino acid sequence shown in SEQ ID No. 1, except the following: - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and the amino acid at position 79 is preferably S, W or I, more preferably S; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 111 is preferably E; - Proteins having an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and optimally 99% identity to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, and wherein the proteins have at least one other amino acid substitution selected from the group shown directly under the symbols listed above in this text.
較佳地,與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可具有於位置79、202、280、301、3、11、17、40或111處之至少兩個其他胺基酸取代。較佳地,於位置79處之胺基酸係S、W或I,更佳S;較佳地,於位置202處之胺基酸係N;較佳地,於位置280處之胺基酸係A;較佳地,於位置301處之胺基酸係A;較佳地,於位置3處之胺基酸係Q;較佳地,於位置11處之胺基酸係A;較佳地,於位置17處之胺基酸係P;較佳地,於位置40處之胺基酸係M;較佳地,於位置111處之胺基酸係E。Preferably, the lipase variants described herein above under point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least two further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.
因此,根據本發明之另一實施例涉及具有其他胺基酸修飾之根據本發明之蛋白質;較佳地,此等實施例係具有脂肪酶活性之蛋白質,其中該等蛋白質係選自由以下組成之群:具有SEQ ID No. 1中顯示之胺基酸序列之蛋白質,除以下外: - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置280處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置280處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置301處之胺基酸不同於D,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置301處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置301處之胺基酸不同於D,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置301處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置11處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置11處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置17處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置17處之胺基酸較佳為P及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 具有與在a)顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列之蛋白質,限制條件為於位置186處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y,且其中該等蛋白質具有至少兩個選自上文在本文列舉之符號下直接顯示之群的其他胺基酸取代。 Therefore, another embodiment according to the present invention relates to proteins according to the present invention having other amino acid modifications; preferably, these embodiments are proteins having lipase activity, wherein the proteins are selected from the group consisting of: proteins having the amino acid sequence shown in SEQ ID No. 1, except for the following: - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - Proteins having an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, optimally 99% identity to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, and wherein the proteins have at least two other amino acid substitutions selected from the group shown directly under the symbols listed above in this text.
較佳地,與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可具有於位置79、202、280、301、3、11、17、40或111處之至少三個其他胺基酸取代。較佳地,於位置79處之胺基酸係S、W或I,更佳S;較佳地,於位置202處之胺基酸係N;較佳地,於位置280處之胺基酸係A;較佳地,於位置301處之胺基酸係A;較佳地,於位置3處之胺基酸係Q;較佳地,於位置11處之胺基酸係A;較佳地,於位置17處之胺基酸係P;較佳地,於位置40處之胺基酸係M;較佳地,於位置111處之胺基酸係E。Preferably, the lipase variants described herein above under point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least three further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.
因此,根據本發明之另一實施例涉及具有其他胺基酸修飾之根據本發明之蛋白質;較佳地,此等實施例係具有脂肪酶活性之蛋白質,其中該等蛋白質係選自由以下組成之群:具有SEQ ID No. 1中顯示之胺基酸序列之蛋白質,除以下外: - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A及於位置301處之胺基酸不同於D,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N及於位置301處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置202處之胺基酸不同於A及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置202處之胺基酸較佳為N及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置280處之胺基酸不同於L及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置280處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置280處之胺基酸不同於L及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置280處之胺基酸較佳為A及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置280處之胺基酸不同於L及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置280處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置280處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置280處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置280處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置280處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置301處之胺基酸不同於D及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置301處之胺基酸較佳為A及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置301處之胺基酸不同於D及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置301處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置301處之胺基酸不同於D及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y,更佳Y,及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置301處之胺基酸較佳為A及於位置40處之胺基酸較佳為M,其中特別佳於位置186處之胺基酸係Y及於位置79處之胺基酸係S及於位置301處之胺基酸係A及於位置40處之胺基酸係M; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置301處之胺基酸不同於D及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置301處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置3處之胺基酸不同於M及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置3處之胺基酸較佳為Q及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置3處之胺基酸不同於M及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置3處之胺基酸較佳為Q及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置3處之胺基酸不同於M及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置3處之胺基酸較佳為Q及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置11處之胺基酸不同於C及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置11處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置11處之胺基酸不同於C及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置11處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置17處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置17處之胺基酸較佳為P及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置79處之胺基酸不同於E及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置79處之胺基酸較佳為S、W或I,更佳S,及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置280處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置280處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置301處之胺基酸不同於D及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置301處之胺基酸較佳為A及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置301處之胺基酸不同於D及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置301處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置301處之胺基酸不同於D及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置301處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置301處之胺基酸不同於D及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置301處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置3處之胺基酸不同於M及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置3處之胺基酸較佳為Q及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置3處之胺基酸不同於M及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置3處之胺基酸較佳為Q及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置3處之胺基酸不同於M及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置3處之胺基酸較佳為Q及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置11處之胺基酸不同於C及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置11處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置11處之胺基酸不同於C及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置11處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置17處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置17處之胺基酸較佳為P及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置202處之胺基酸不同於A及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置202處之胺基酸較佳為N及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置301處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置11處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置11處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置17處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置17處之胺基酸較佳為P及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置280處之胺基酸不同於L及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置280處之胺基酸較佳為A及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置3處之胺基酸不同於M及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置3處之胺基酸較佳為Q及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置11處之胺基酸不同於C及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置11處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置11處之胺基酸不同於C及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置11處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置17處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置17處之胺基酸較佳為P及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置301處之胺基酸不同於D及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置301處之胺基酸較佳為A及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置11處之胺基酸不同於C及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置11處之胺基酸較佳為A及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置17處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置17處之胺基酸較佳為P及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置3處之胺基酸不同於M及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置3處之胺基酸較佳為Q及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K,其中較佳於位置186處之胺基酸較佳為W或Y及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M; - 於位置186處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置17處之胺基酸不同於L及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置11處之胺基酸較佳為A及於位置17處之胺基酸較佳為P及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置11處之胺基酸不同於C及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置11處之胺基酸較佳為A及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 於位置186處之胺基酸不同於L及於位置17處之胺基酸不同於L及於位置40處之胺基酸不同於K及於位置111處之胺基酸不同於Q,其中較佳於位置186處之胺基酸較佳為W或Y及於位置17處之胺基酸較佳為P及於位置40處之胺基酸較佳為M及於位置111處之胺基酸較佳為E; - 具有與在a)顯示之胺基酸序列具有至少80%,較佳85%,更佳90%,甚至更佳95%,甚至更佳96%,甚至更佳97%,特別佳98%,最佳99%一致性之胺基酸序列之蛋白質,限制條件為於位置186處之胺基酸不同於L,其中較佳於位置186處之胺基酸較佳為W或Y,更佳Y,且其中該等蛋白質具有至少三個選自上文在本文列舉之符號下直接顯示之群的其他胺基酸取代。 Therefore, another embodiment according to the present invention relates to proteins according to the present invention having other amino acid modifications; preferably, these embodiments are proteins having lipase activity, wherein the proteins are selected from the group consisting of: proteins having the amino acid sequence shown in SEQ ID No. 1, except for the following: - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M, wherein the amino acid at position 186 is Y and the amino acid at position 79 is S and the amino acid at position 301 is A and the amino acid at position 40 is M; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M; - The amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - The amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E; - Proteins having an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and wherein the proteins have at least three other amino acid substitutions selected from the group shown directly under the symbols listed above.
與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可具有於位置79、202、280、301、3、11、17、40或111處之至少四個其他胺基酸取代。較佳地,於位置79處之胺基酸係S、W或I,更佳S;較佳地,於位置202處之胺基酸係N;較佳地,於位置280處之胺基酸係A;較佳地,於位置301處之胺基酸係A;較佳地,於位置3處之胺基酸係Q;較佳地,於位置11處之胺基酸係A;較佳地,於位置17處之胺基酸係P;較佳地,於位置40處之胺基酸係M;較佳地,於位置111處之胺基酸係E。The lipase variants described herein above under point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least four further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.
與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可具有於位置79、202、280、301、3、11、17、40或111處之至少五個其他胺基酸取代。較佳地,於位置79處之胺基酸係S、W或I,更佳S;較佳地,於位置202處之胺基酸係N;較佳地,於位置280處之胺基酸係A;較佳地,於位置301處之胺基酸係A;較佳地,於位置3處之胺基酸係Q;較佳地,於位置11處之胺基酸係A;較佳地,於位置17處之胺基酸係P;較佳地,於位置40處之胺基酸係M;較佳地,於位置111處之胺基酸係E。The lipase variants described herein above under point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least five further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.
與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可具有於位置79、202、280、301、3、11、17、40或111處之至少六個其他胺基酸取代。較佳地,於位置79處之胺基酸係S、W或I,更佳S;較佳地,於位置202處之胺基酸係N;較佳地,於位置280處之胺基酸係A;較佳地,於位置301處之胺基酸係A;較佳地,於位置3處之胺基酸係Q;較佳地,於位置11處之胺基酸係A;較佳地,於位置17處之胺基酸係P;較佳地,於位置40處之胺基酸係M;較佳地,於位置111處之胺基酸係E。The lipase variants described herein above under point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least six further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.
與在SEQ ID No. 1顯示之胺基酸序列相關之本文上文在點a)下描述之脂肪酶變體可具有於位置79、202、280、301、3、11、17、40或111處之至少七個其他胺基酸取代。較佳地,於位置79處之胺基酸係S、W或I,更佳S;較佳地,於位置202處之胺基酸係N;較佳地,於位置280處之胺基酸係A;較佳地,於位置301處之胺基酸係A;較佳地,於位置3處之胺基酸係Q;較佳地,於位置11處之胺基酸係A;較佳地,於位置17處之胺基酸係P;較佳地,於位置40處之胺基酸係M;較佳地,於位置111處之胺基酸係E。The lipase variants described herein above under point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least seven further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.
根據本發明之較佳實施例係編碼具有在SEQ ID No. 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、221、223、225、227、229、231、233、235、237、239、241、243、245、247、249、251、253、255、257、259、261、263、265、267、269、271、273、275、277、279、281、283、285、287、289、291、293、295、297、299、301、303、305、307、309、311、313、315、317、319、321、323、325、327、329、331、333、335、337、339、341、343、345、347、349、351、353、355、357、359、361、363、365、367、369、371、373、375、377、379、381、383、385、387、389、391、393、395、397、399、401顯示之胺基酸序列之脂肪酶的蛋白質。According to a preferred embodiment of the present invention, the encoding sequence is as follows: 3, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 2 19, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401 are lipase proteins having the amino acid sequences shown.
根據本發明之特別佳實施例係編碼具有在SEQ ID No. 233及399顯示之胺基酸序列之脂肪酶的蛋白質。Particularly preferred embodiments according to the present invention encode a protein having the lipase having the amino acid sequence shown in SEQ ID Nos. 233 and 399.
已測試具有解脂酶或脂肪酶活性之其他蛋白質。此等其他蛋白質之胺基酸序列係由SEQ ID No. 1中之胺基酸描述之胺基酸序列之變體,其中: - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置40及79處之兩個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置40處之胺基酸係M及於位置79處之胺基酸係S; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置40及186處之兩個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置40處之胺基酸係M及於位置186處之胺基酸係Y; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置40及301處之兩個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置40處之胺基酸係M及於位置301處之胺基酸係A; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置79及186處之兩個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置79處之胺基酸係S及於位置186處之胺基酸係Y; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置79及301處之兩個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置79處之胺基酸係S及於位置301處之胺基酸係A; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置186及301處之兩個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置186處之胺基酸係Y及於位置301處之胺基酸係A。 Other proteins with lipolytic or lipase activity have been tested. The amino acid sequences of these other proteins are variants of the amino acid sequence described by the amino acids in SEQ ID No. 1, wherein: - In the case of the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 79 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 79 is S; - In the case of the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 186 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 186 is Y; - In the case of the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 301 is A; - In the case of the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 79 and 186 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S and the amino acid at position 186 is Y; - In the case of the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 79 and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S and the amino acid at position 301 is A; - In the case of the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 186 and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 186 is Y and the amino acid at position 301 is A.
已測試具有解脂酶或脂肪酶活性之又其他蛋白質。此等其他蛋白質之胺基酸序列係由SEQ ID No. 1中之胺基酸描述之胺基酸序列之變體,其中: - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置40、79及186處之三個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置40處之胺基酸係M及於位置79處之胺基酸係S及於位置186處之胺基酸係Y; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置40、79及301處之三個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置40處之胺基酸係M及於位置79處之胺基酸係S及於位置301處之胺基酸係A; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置40、186及301處之三個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置40處之胺基酸係M及於位置186處之胺基酸係Y及於位置301處之胺基酸係A; - 在SEQ ID No. 1顯示之胺基酸序列之情況下,於位置79、186及301處之三個胺基酸不同於在SEQ ID No. 1顯示之序列中之相應胺基酸位置處指示之胺基酸。於與在SEQ ID No. 1顯示之胺基酸序列相關之特定測試變體中,於位置79處之胺基酸係S及於位置186處之胺基酸係Y及於位置301處之胺基酸係A。 Still other proteins have been tested for lipolytic or lipase activity. The amino acid sequences of these other proteins are variants of the amino acid sequence described by the amino acids in SEQ ID No. 1, wherein: - In the case of the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 40, 79 and 186 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant associated with the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 186 is Y; - In the case of the amino acid sequence shown in SEQ ID No. 1, three amino acids at positions 40, 79, and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant associated with the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 301 is A; - In the case of the amino acid sequence shown in SEQ ID No. 1, three amino acids at positions 40, 186, and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant associated with the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 186 is Y, and the amino acid at position 301 is A; - In the case of the amino acid sequence shown in SEQ ID No. 1, three amino acids at positions 79, 186, and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant associated with the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S, the amino acid at position 186 is Y, and the amino acid at position 301 is A.
根據本發明之脂肪酶及脂肪酶變體具有與2,6-二甲基-1-胺基茚烷(DMAI)之立體選擇性醯化或羧化相關之高選擇性及/或高比活性且能夠產生對映體富集或實質純之[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸甲酯。[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸甲酯係用於合成具有除草作用之化合物茚嗪氟草胺之重要中間物。The lipase and lipase variants according to the present invention have high selectivity and/or high specific activity in relation to the stereoselective acylation or carboxylation of 2,6-dimethyl-1-aminoindan (DMAI) and are capable of producing enantiomerically enriched or substantially pure [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamic acid methyl ester. [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamic acid methyl ester is an important intermediate for the synthesis of the herbicidal compound indoxazolidinone.
本文中「對映體富集」意謂組合物中兩種對映體中之一者係以比另一種對映體更大之量存在,較佳該組合物中之一種對映體係以至少60%之程度存在,更佳該組合物中之一種對映體係以至少65%之程度存在,甚至更佳該組合物中之一種對映體係以至少70%之程度存在,甚至更佳該組合物中之一種對映體係以至少75%之程度存在,甚至更佳該組合物中之一種對映體係以至少80%之程度存在,特別佳該組合物中之一種對映體係以至少85%之程度存在,最佳該組合物中之一種對映體係以至少90%之程度存在或極特別佳該組合物中之一種對映體係以至少94%之程度存在。"Enantiomerically enriched" as used herein means that one of the two enantiomers is present in a composition in a greater amount than the other enantiomer, preferably one enantiomer is present in the composition to an extent of at least 60%, more preferably one enantiomer is present in the composition to an extent of at least 65%, even more preferably one enantiomer is present in the composition to an extent of at least 70%, even more preferably one enantiomer is present in the composition to an extent of at least 75%, even more preferably one enantiomer is present in the composition to an extent of at least 80%, particularly preferably one enantiomer is present in the composition to an extent of at least 85%, most preferably one enantiomer is present in the composition to an extent of at least 90% or very particularly preferably one enantiomer is present in the composition to an extent of at least 94%.
本文中「對映體實質純」意謂組合物中兩種對映體中之一者係以至少95.0%之量存在,較佳組合物中兩種對映體中之一者係以至少95.5%之量存在,更佳組合物中兩種對映體中之一者係以至少96.0%之量存在,甚至更佳組合物中兩種對映體中之一者係以至少96.5%之量存在,甚至更佳組合物中兩種對映體中之一者係以至少97.0%之量存在,甚至更佳組合物中兩種對映體中之一者係以至少98.0%之量存在,特別佳組合物中兩種對映體中之一者係以至少98.5%之量存在,最佳組合物中兩種對映體中之一者係以至少99.0%之量存在或極特別佳組合物中兩種對映體中之一者係以至少99.5%之量存在。As used herein, "enantiomerically substantially pure" means that one of the two enantiomers is present in the composition in an amount of at least 95.0%, preferably one of the two enantiomers is present in the composition in an amount of at least 95.5%, more preferably one of the two enantiomers is present in the composition in an amount of at least 96.0%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 96.5%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 97.0%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 98.0%, particularly preferably one of the two enantiomers is present in the composition in an amount of at least 98.5%, most preferably one of the two enantiomers is present in the composition in an amount of at least 99.0%, or very particularly preferably one of the two enantiomers is present in the composition in an amount of at least 99.5%.
根據本發明之另一實施例涉及編碼根據本發明之蛋白質之核酸分子。Another embodiment according to the present invention relates to a nucleic acid molecule encoding a protein according to the present invention.
根據本發明之核酸分子可為任何種類之核酸,限制條件為該核酸編碼根據本發明之蛋白質。該等核酸可為核糖核酸分子(例如RNA、mRNA)或脫氧核糖核酸分子(DNA,包括基因體DNA,其可包括或可不包括內含子並編碼DNA)。The nucleic acid molecules according to the present invention may be any type of nucleic acid, provided that the nucleic acid encodes a protein according to the present invention. Such nucleic acids may be ribonucleic acid molecules (e.g., RNA, mRNA) or deoxyribonucleic acid molecules (DNA, including genomic DNA, which may or may not include introns and encode DNA).
本發明特別關注編碼具有脂肪酶活性之蛋白質之核酸分子,包含在SEQ ID No. 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、221、223、225、227、229、231、233、235、237、239、241、243、245、247、249、251、253、255、257、259、261、263、265、267、269、271、273、275、277、279、281、283、285、287、289、291、293、295、297、299、301、303、305、307、309、311、313、315、317、319、321、323、325、327、329、331、333、335、337、339、341、343、345、347、349、351、353、355、357、359、361、363、365、367、369、371、373、375、377、379、381、383、385、387、389、391、393、395、397、399、401顯示之胺基酸序列。The present invention is particularly concerned with nucleic acid molecules encoding proteins having lipase activity, comprising the following sequences: SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 2 17, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 31 1, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401 shows the amino acid sequence.
本發明進一步涉及編碼具有脂肪酶活性之蛋白質之核酸分子,選自由以下組成之群: a) 包含在SEQ ID No. 2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、224、226、228、230、232、234、236、238、240、242、244、246、248、250、252、254、256、258、260、262、264、266、268、270、272、274、276、278、280、282、284、286、288、290、292、294、296、298、300、302、304、306、308、310、312、314、316、318、320、322、324、326、328、330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、376、378、380、382、384、386、388、390、392、394、396、398、400、402顯示之核酸序列之核酸分子; b) 與在a)顯示之核酸序列具有至少60%,較佳70%,更佳80%,甚至更佳90%,甚至更佳95%,甚至更佳96%,特別佳97%,最佳98%或極特別佳99%一致性之核酸分子。 The present invention further relates to a nucleic acid molecule encoding a protein having lipase activity, selected from the group consisting of: a) a nucleic acid molecule comprising SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218 , 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 313 4. Nucleic acid molecules with nucleic acid sequences shown in 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402; b) A nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, even more preferably 95%, even more preferably 96%, particularly preferably 97%, optimally 98% or very particularly preferably 99% identity with the nucleic acid sequence shown in a).
結合本發明,「與……雜交」意謂在習知雜交條件下,較佳在嚴格條件下雜交,如描述(例如)於Sambrook等人,(Molecular Cloning, A Laboratory Manual,第3版,(2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773)中或於Ausubel等人,(Short Protocols in Molecular Biology, John Wiley & Sons;第5版,(2002), ISBN: 0471250929)中。特別佳地,「雜交」意謂在下列條件下雜交:In conjunction with the present invention, "crossing with" means crossing under known crossing conditions, preferably under strict conditions, as described, for example, in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd edition, (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or in Ausubel et al. (Short Protocols in Molecular Biology, John Wiley &Sons; 5th edition, (2002), ISBN: 0471250929). Particularly preferably, "crossing" means crossing under the following conditions:
雜交緩衝液: 2x SSC;10x登哈特(Denhardt’s)溶液(Fikoll 400+PEG+BSA;1:1:1比率);0.1% SDS;5 mM EDTA;50 mM Na 2HPO 4;250 µg/ml鯡魚精子DNA;50 µg/ml tRNA; 或 25 M磷酸鈉緩衝液,pH 7.2;1 mM EDTA;7% SDS 雜交溫度: T = 65℃至68℃ 洗滌緩衝液: 0.1x SSC;0.1% SDS 洗滌溫度: T = 65℃至68℃。 Hybridization buffer: 2x SSC; 10x Denhardt's solution (Fikoll 400+PEG+BSA; 1:1:1 ratio); 0.1% SDS; 5 mM EDTA; 50 mM Na 2 HPO 4 ; 250 µg/ml herring sperm DNA; 50 µg/ml tRNA; or 25 M sodium phosphate buffer, pH 7.2; 1 mM EDTA; 7% SDS Hybridization temperature: T = 65°C to 68°C Wash buffer: 0.1x SSC; 0.1% SDS Wash temperature: T = 65°C to 68°C.
與編碼具有脂肪酶活性之蛋白質之核酸分子雜交的核酸分子可起源於任何有機體;因此,其等可起源於細菌、真菌、動物、人類、植物或病毒。The nucleic acid molecules hybridized to the nucleic acid molecule encoding the protein having lipase activity may originate from any organism; thus, they may originate from bacteria, fungi, animals, humans, plants or viruses.
與編碼具有脂肪酶活性之蛋白質之核酸分子雜交的核酸分子較佳起源於微生物,更佳起源於真菌或細菌,最佳起源於細菌。The nucleic acid molecule hybridized with the nucleic acid molecule encoding the protein having lipase activity is preferably derived from a microorganism, more preferably from a fungus or bacteria, and most preferably from bacteria.
與前述分子雜交之核酸分子可例如自基因體DNA或cDNA庫分離。此等核酸分子可使用本文描述之核酸分子鑑別及分離或其等可使用此等分子之部分或此等分子之反向互補體鑑別及分離,例如藉由根據標準方法雜交(參見例如Sambrook等人,Molecular Cloning, A Laboratory Manual,第3版,(2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773;Ausubel等人,Short Protocols in Molecular Biology, John Wiley & Sons;第5版,(2002), ISBN: 0471250929)或藉由在PCR之幫助下擴增。Nucleic acid molecules hybridized with the aforementioned molecules can be isolated, for example, from genomic DNA or cDNA libraries. These nucleic acid molecules can be identified and isolated using the nucleic acid molecules described herein, or they can be identified and isolated using portions of these molecules or reverse complements of these molecules, for example by hybridization according to standard methods (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd edition, (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley &Sons; 5th edition, (2002), ISBN: 0471250929) or by amplification with the help of PCR.
用作雜交探針之片段亦可為合成片段,或使用習知合成技術產生之寡核苷酸,其序列係與本發明之內文中描述之核酸分子大體上相同。當鑑別及分離與結合本發明描述之核酸序列雜交之基因時,應確定該序列及應分析由該序列編碼之蛋白質之性質以確定其等是否為具有脂肪酶活性之蛋白質。用於確定蛋白質是否存在具有脂肪酶活性之蛋白質之活性的方法為一般技術者已知。The fragments used as hybridization probes may also be synthetic fragments, or oligonucleotides produced using known synthetic techniques, whose sequences are substantially identical to the nucleic acid molecules described in the context of the present invention. When identifying and isolating genes hybridized with the nucleic acid sequences described in the present invention, the sequence should be determined and the properties of the protein encoded by the sequence should be analyzed to determine whether it is a protein with lipase activity. Methods for determining whether a protein has the activity of a protein with lipase activity are known to those of ordinary skill in the art.
與本發明之內文中描述之核酸分子雜交之分子尤其包括前述核酸分子之片段、衍生物及對偶基因變體。結合本發明,術語「衍生物」意謂此等分子之序列之一或多個位置不同於上文描述之核酸分子之序列且在很大程度上係與此等序列一致。與上文描述之核酸分子相關之差異可例如歸因於缺失、添加、取代、插入或重組。Molecules hybridized to the nucleic acid molecules described in the context of the present invention include in particular fragments, derivatives and allelic variants of the aforementioned nucleic acid molecules. In conjunction with the present invention, the term "derivative" means that the sequence of these molecules differs from the sequence of the nucleic acid molecules described above at one or more positions and is largely identical to these sequences. The differences with respect to the nucleic acid molecules described above can be due, for example, to deletions, additions, substitutions, insertions or recombination.
根據本發明之較佳核酸分子係在SEQ ID No. 2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、224、226、228、230、232、234、236、238、240、242、244、246、248、250、252、254、256、258、260、262、264、266、268、270、272、274、276、278、280、282、284、286、288、290、292、294、296、298、300、302、304、306、308、310、312、314、316、318、320、322、324、326、328、330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、376、378、380、382、384、386、388、390、392、394、396、398、400、402顯示之核酸分子。Preferred nucleic acid molecules according to the present invention are those in SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 12 2, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 311 12, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402 nucleic acid molecules.
核苷酸縮寫a、c、g、t及用於簡併核苷酸r、y、s、w、k、m、b、d、h、v、n之縮寫之含義可於下表1中標題為「序列描述」之部分中找到。由簡併核苷酸編碼之胺基酸可於下表3中標題為「序列描述」之部分中找到。The meanings of the nucleotide abbreviations a, c, g, t and the abbreviations for the degenerate nucleotides r, y, s, w, k, m, b, d, h, v, n can be found in the section entitled "Sequence Description" in Table 1 below. The amino acids encoded by the degenerate nucleotides can be found in the section entitled "Sequence Description" in Table 3 below.
本發明亦揭示包含適用於根據本發明之方法之核酸分子之重組核酸分子。The present invention also discloses recombinant nucleic acid molecules comprising nucleic acid molecules suitable for use in the methods according to the present invention.
應瞭解術語「重組核酸分子」意謂不僅含有適用於根據本發明之方法之核酸分子,且亦含有用於根據本發明之方法之重組核酸中出現之組合中非自然出現之其他序列的核酸分子。前述另外序列可為任何序列;較佳其等為功能或調節序列(啟動子、終止訊號、強化子、核糖體結合位點(rbs)、增加轉錄、轉譯或RNA穩定性之前導序列、亞細胞靶向序列等),特別佳其等為於微生物中具有活性之功能或調節序列,及極特別佳其等為於真菌中,特定言之於酵母菌中或於細菌中具有活性之調節序列。用於產生適用於根據本發明之方法之重組核酸分子之方法為一般技術者已知。此等包括遺傳方法,諸如藉由核酸分子之連接、遺傳重組或從頭合成來結合核酸分子。此等方法係(例如)描述於Sambrok等人,(Molecular Cloning, A Laboratory Manual,第3版,(2001) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. ISBN: 0879695773)中或於Ausubel等人,(Short Protocols in Molecular Biology, John Wiley & Sons;第5版,(2002), ISBN: 0471250929)中。It should be understood that the term "recombinant nucleic acid molecule" means not only nucleic acid molecules suitable for the method according to the present invention, but also nucleic acid molecules containing other sequences that do not naturally occur in the combination present in the recombinant nucleic acid used in the method according to the present invention. The aforementioned additional sequences can be any sequence; preferably, they are functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (rbs), leader sequences that increase transcription, translation or RNA stability, subcellular targeting sequences, etc.), particularly preferably, they are functional or regulatory sequences active in microorganisms, and very particularly preferably, they are regulatory sequences active in fungi, in particular in yeast or in bacteria. Methods for producing recombinant nucleic acid molecules suitable for the method according to the present invention are known to those of ordinary skill in the art. These include genetic methods, such as combining nucleic acid molecules by ligation, genetic recombination or de novo synthesis of nucleic acid molecules. Such methods are described, for example, in Sambrok et al. (Molecular Cloning, A Laboratory Manual, 3rd edition, (2001) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. ISBN: 0879695773) or in Ausubel et al. (Short Protocols in Molecular Biology, John Wiley &Sons; 5th edition, (2002), ISBN: 0471250929).
可用於根據本發明之方法之重組核酸分子包含合適之核酸分子,其係連接至於原核或真核細胞中啟動轉錄之調節序列。Recombinant nucleic acid molecules that can be used in the methods according to the present invention include suitable nucleic acid molecules that are linked to regulatory sequences that initiate transcription in prokaryotic or eukaryotic cells.
於細胞中啟動轉錄之調節序列亦稱為啟動子。有關調節序列及質體之資訊為一般技術者熟知,且係(例如)描述於由國際基因工程機器(The International Genetically Engineered Machine,iGEM)基金會(一號肯德爾廣場,Suite B6104,劍橋,MA 02139,美國)於網路(http://parts.igem.org/Catalog)上支援之標準生物學元件登記庫中。Regulatory sequences that initiate transcription in cells are also called promoters. Information about regulatory sequences and plasmids is well known to the skilled artisan and is described, for example, in the standard biological component registry supported by the International Genetically Engineered Machine (iGEM) Foundation (One Kendall Square, Suite B6104, Cambridge, MA 02139, USA) on the Internet (http://parts.igem.org/Catalog).
於原核有機體(諸如大腸桿菌)中及於真核有機體中啟動轉錄之調節序列係於文獻中廣泛描述;特定言之描述彼等與酵母菌(例如釀酒酵母(Saccharomyces cerevisiae))中之表現相關者。對用於在不同宿主有機體中表現蛋白質之不同系統的概述可例如於Methods in Enzymology 153 (1987), 383-516中及於Bitter等人,(Methods in Enzymology 153 (1987), 516-544)中或於Gomes等人,(2016, Advances in Animal and Veterinary Sciences, 4(4), 346)及Baghban等人,(2018, Current Pharmaceutical Biotechnology, 19(6))中找到。習知酵母菌啟動子為pAOX1、pHIS4、pGAL、pScADH2 (Baghban等人,2018,參見上文)。習知細菌啟動子為T5啟動子、T7啟動子、鼠李糖誘導型啟動子、阿拉伯糖誘導型啟動子、PhoA啟動子、人工trc(trp-lac)啟動子,如由Marschall等人,(2017, Appl Microbiol Biotechnol 101, 501-512)及Tegel等人,(2011, FEBS Journal 278, 729-739)描述。Regulatory sequences that initiate transcription in prokaryotic organisms such as E. coli and in eukaryotic organisms are extensively described in the literature; in particular, those described in connection with expression in yeasts such as Saccharomyces cerevisiae. An overview of different systems for expressing proteins in different host organisms can be found, for example, in Methods in Enzymology 153 (1987), 383-516 and in Bitter et al., (Methods in Enzymology 153 (1987), 516-544) or in Gomes et al., (2016, Advances in Animal and Veterinary Sciences, 4(4), 346) and Baghban et al., (2018, Current Pharmaceutical Biotechnology, 19(6)). Known yeast promoters are pAOX1, pHIS4, pGAL, pScADH2 (Baghban et al., 2018, see above). Known bacterial promoters are T5 promoter, T7 promoter, rhamnose-inducible promoter, arabinose-inducible promoter, PhoA promoter, artificial trc (trp-lac) promoter, as described by Marschall et al., (2017, Appl Microbiol Biotechnol 101, 501-512) and Tegel et al., (2011, FEBS Journal 278, 729-739).
可用於根據本發明之方法之重組核酸分子之另一實施例係包含合適核酸分子之質體之載體。Another embodiment of a recombinant nucleic acid molecule that can be used in the method according to the invention is a plasmid vector comprising a suitable nucleic acid molecule.
「載體」於分子生物學領域中係熟知的,且於本文中係包含用於將遺傳物質(DNA或RNA)轉移至靶細胞內之核酸序列或包含核酸序列之媒介物。載體可為質體,例如用於產生轉基因植物之T-DNA或二元載體、用於在宿主細胞中表現核酸序列之表現載體、能夠於不同宿主中繁殖之穿梭載體,或載體可為已經修飾以將外源遺傳物質遞送至宿主內之病毒顆粒或噬菌體。"Vectors" are well known in the art of molecular biology and herein include nucleic acid sequences or vehicles containing nucleic acid sequences used to transfer genetic material (DNA or RNA) into target cells. Vectors can be plasmids, such as T-DNA or binary vectors used to produce transgenic plants, expression vectors used to express nucleic acid sequences in host cells, shuttle vectors capable of propagation in different hosts, or vectors can be viral particles or bacteriophages that have been modified to deliver foreign genetic material into a host.
「質體」於分子生物學領域中係熟知的,且於本文中係自主自我複製,通常環狀之DNA分子,當存在於宿主細胞中時,其係自染色體DNA分離。"Plastid" is well known in the field of molecular biology and, as used herein, is an autonomous self-replicating, usually circular DNA molecule that, when present in a host cell, is separate from chromosomal DNA.
合適之核酸分子、重組核酸分子、載體或質體可用於產生用於根據本發明之方法之蛋白質,例如藉由於宿主細胞中表現合適之核酸分子。Suitable nucleic acid molecules, recombinant nucleic acid molecules, vectors or plasmids may be used to produce proteins for use in the methods according to the invention, for example by expressing suitable nucleic acid molecules in host cells.
本發明亦揭示包含或表現適用於根據本發明之方法之核酸分子或包含合適之具有脂肪酶活性之蛋白質或包含適用於根據本發明之方法之重組核酸分子或包含適用於根據本發明之方法之載體或包含適用於根據本發明之方法之質體的宿主或宿主細胞。The present invention also discloses a host or host cell comprising or expressing a nucleic acid molecule suitable for use in the method according to the present invention, or comprising a suitable protein having lipase activity, or comprising a recombinant nucleic acid molecule suitable for use in the method according to the present invention, or comprising a vector suitable for use in the method according to the present invention, or comprising a plasmid suitable for use in the method according to the present invention.
編碼具有脂肪酶活性之蛋白質之合適之核酸分子可表現於宿主細胞中,例如用於其繁殖或用於產生具有脂肪酶活性之蛋白質。針對於宿主細胞中之表現,合適之核酸分子可存在於載體或質體上或係穩定整合至特定宿主細胞之基因體內。該等合適之核酸分子亦可存在於載體中,該等載體有助於將該等核酸分子引入宿主細胞內。Suitable nucleic acid molecules encoding proteins with lipase activity can be expressed in host cells, for example for their propagation or for the production of proteins with lipase activity. For expression in host cells, suitable nucleic acid molecules can be present on vectors or plasmids or stably integrated into the genome of a particular host cell. Such suitable nucleic acid molecules can also be present in vectors that facilitate the introduction of such nucleic acid molecules into host cells.
本發明亦揭示一種適用於根據本發明之方法之宿主或宿主細胞,其包含適用於根據本發明之方法之核酸分子或包含適用於根據本發明之方法之重組核酸分子或包含適用於根據本發明之方法之載體或包含適用於根據本發明之方法之質體,各包含適用於根據本發明之方法之蛋白質。本發明亦揭示適用於根據本發明之方法之宿主或宿主細胞,其包含適用於根據本發明之方法之核酸分子或包含適用於根據本發明之方法之重組核酸分子或包含適用於根據本發明之方法之載體或包含適用於根據本發明之方法之質體,各表現適用於根據本發明之方法之蛋白質,其中該蛋白質較佳具有脂肪酶活性。The present invention also discloses a host or host cell suitable for the method according to the present invention, comprising a nucleic acid molecule suitable for the method according to the present invention, or comprising a recombinant nucleic acid molecule suitable for the method according to the present invention, or comprising a vector suitable for the method according to the present invention, or comprising a plasmid suitable for the method according to the present invention, each comprising a protein suitable for the method according to the present invention. The present invention also discloses a host or host cell suitable for the method according to the present invention, comprising a nucleic acid molecule suitable for the method according to the present invention, or comprising a recombinant nucleic acid molecule suitable for the method according to the present invention, or comprising a vector suitable for the method according to the present invention, or comprising a plasmid suitable for the method according to the present invention, each expressing a protein suitable for the method according to the present invention, wherein the protein preferably has lipase activity.
本文中應瞭解「核酸分子之表現」意謂若核酸分子係RNA或mRNA,則將該核酸分子轉譯成蛋白質,較佳轉譯成具有脂肪酶活性之蛋白質,或若該核酸分子係DNA或cDNA,則將其轉錄成mRNA (且在含有內含子之基因體DNA之情況下係經處理),較佳轉錄成編碼具有脂肪酶活性之蛋白質之mRNA,且隨後轉譯成蛋白質,較佳轉譯成具有脂肪酶活性之蛋白質。It should be understood herein that "expression of a nucleic acid molecule" means that if the nucleic acid molecule is RNA or mRNA, the nucleic acid molecule is translated into a protein, preferably into a protein having lipase activity, or if the nucleic acid molecule is DNA or cDNA, it is transcribed into mRNA (and in the case of genomic DNA containing introns, it is processed), preferably into mRNA encoding a protein having lipase activity, and subsequently translated into a protein, preferably into a protein having lipase activity.
特定核酸分子於宿主中之轉錄可藉由一般技術者已知的方法偵測,例如藉由北方墨點轉漬分析法或藉由RT-PCR偵測外源核酸分子之特定轉錄本(mRNA)。The transcription of a specific nucleic acid molecule in a host can be detected by methods known to those of ordinary skill in the art, such as detecting a specific transcript (mRNA) of an exogenous nucleic acid molecule by Northern blot analysis or by RT-PCR.
確定宿主或宿主細胞是否包含特定蛋白質或包含來源於核酸分子之表現之蛋白質可藉由一般技術者已知的方法,例如藉由免疫學方法,諸如西方墨點法分析、ELISA (酶聯免疫吸附測定)或RIA (放射性免疫測定法)進行。一般技術者熟悉用於產生與特定蛋白質特異性反應之抗體(即抗體特異性結合至特定蛋白質)之方法(參見例如Lottspeich及Zorbas (編輯者),1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4)。一些公司(Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245;GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA)為產生該等抗體提供定製服務。Determining whether a host or host cell contains a specific protein or contains a protein derived from the expression of a nucleic acid molecule can be performed by methods known to those of ordinary skill, for example by immunological methods such as Western blot analysis, ELISA (enzyme-linked immunosorbent assay) or RIA (radioimmunoassay). Those of ordinary skill are familiar with methods for generating antibodies that specifically react with a specific protein (i.e., antibodies that specifically bind to a specific protein) (see, e.g., Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Several companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) offer custom services for the generation of these antibodies.
此外,一般技術者可藉由偵測具有脂肪酶活性之蛋白質於相應宿主細胞中之(另外)活性檢查宿主或宿主細胞是否包含適用於根據本發明之方法之蛋白質。較佳地,具有另外脂肪酶活性之蛋白質於相應宿主細胞中之活性係藉由比較用於根據本發明之方法之宿主細胞中脂肪酶之活性與不包含適用於根據本發明之方法之蛋白質之宿主細胞之相應活性偵測。檢查蛋白質是否具有脂肪酶活性可使用根據先前技術已知的方法進行。Furthermore, the skilled person can check whether a host or a host cell comprises a protein suitable for the method according to the invention by detecting the (additional) activity of a protein with lipase activity in the corresponding host cell. Preferably, the activity of the protein with additional lipase activity in the corresponding host cell is detected by comparing the lipase activity in the host cell used for the method according to the invention with the corresponding activity of a host cell that does not comprise the protein suitable for the method according to the invention. Checking whether a protein has lipase activity can be performed using methods known from the prior art.
用於根據本發明之方法之宿主或宿主細胞可由一般技術者在用於有機體之遺傳修飾或轉形之已知方法的幫助下產生。Hosts or host cells used in the methods according to the present invention can be generated by a person of ordinary skill in the art with the aid of known methods for genetic modification or transformation of organisms.
因此,本發明亦揭示適用於根據本發明之方法之宿主或宿主細胞,特定言之原核或真核宿主或宿主細胞,已使用合適之核酸分子或使用合適之重組核酸分子或使用合適之載體或使用合適之質體進行基因修飾(或轉形)。較佳地,用於根據本發明之方法之經基因修飾(轉形)之宿主或宿主細胞表現具有脂肪酶活性之蛋白質;更佳地,該經基因修飾(轉形)之宿主或宿主細胞表現適用於根據本發明之方法之蛋白質。本文應瞭解「使用核酸分子進行基因修飾」或「使用核酸分子轉形」意謂核酸分子係或已藉由技術及/或非自然存在之方式引入宿主內或引入宿主細胞內,較佳藉由來自分子生物學、生物技術或基因技術領域之技術方法引入。Therefore, the present invention also discloses a host or host cell suitable for the method according to the present invention, in particular a prokaryotic or eukaryotic host or host cell, which has been genetically modified (or transformed) using a suitable nucleic acid molecule or using a suitable recombinant nucleic acid molecule or using a suitable vector or using a suitable plasmid. Preferably, the genetically modified (transformed) host or host cell used in the method according to the present invention expresses a protein having lipase activity; more preferably, the genetically modified (transformed) host or host cell expresses a protein suitable for the method according to the present invention. It should be understood herein that "genetic modification using a nucleic acid molecule" or "transformation using a nucleic acid molecule" means that the nucleic acid molecule is or has been introduced into the host or into the host cell by a technical and/or non-naturally occurring means, preferably by a technical method from the field of molecular biology, biotechnology or genetic technology.
本發明同樣揭示用於根據本發明之方法之宿主或宿主細胞之後代或子代;較佳該等後代或子代包含適用於根據本發明之方法之核酸分子或包含合適之重組核酸分子或包含合適之載體或包含合適之質體或包含合適之蛋白質,更佳該等後代或子代包含合適之核酸分子或包含合適之重組核酸分子或包含合適之載體或包含合適之質體且其等在任何情況下均表現具有脂肪酶活性之蛋白質,甚至更佳該等後代或子代包含合適之核酸分子或包含合適之重組核酸分子或包含合適之載體或包含合適之質體且其等在任何情況下均表現具有脂肪酶活性之蛋白質,其可用於根據本發明之方法中。The present invention also discloses progeny or descendants of hosts or host cells used in the methods according to the present invention; preferably, the progeny or descendants comprise nucleic acid molecules suitable for use in the methods according to the present invention, or comprise suitable recombinant nucleic acid molecules, or comprise suitable vectors, or comprise suitable plasmids, or comprise suitable proteins; more preferably, the progeny or descendants comprise suitable nucleic acid molecules, or comprise suitable recombinant nucleic acid molecules, or comprise suitable vectors, or comprise suitable plasmids, and in any case, they express proteins having lipase activity; even more preferably, the progeny or descendants comprise suitable nucleic acid molecules, or comprise suitable recombinant nucleic acid molecules, or comprise suitable vectors, or comprise suitable plasmids, and in any case, they express proteins having lipase activity, which can be used in the methods according to the present invention.
用於根據本發明之方法之宿主或宿主細胞可為來自原核有機體或真核有機體之宿主或宿主細胞。該宿主或宿主細胞可為細菌或細菌細胞(例如大腸桿菌、桿菌屬之細菌(特定言之枯草桿菌( Bacillus subtilis))、農桿菌屬(特定言之根癌農桿菌( Agrobacterium tumefaciens)或根毛農桿菌( Agrobacterium rhizogenes))、假單胞菌屬(特定言之螢光假單胞菌( Pseudomonas fluorescens))、鏈黴菌屬( Streptomyces spp)、紅球菌屬(特定言之紫紅紅球菌( Rhodococcus rhodochrous))、需鈉弧菌( Vibrio natrigens)、棒狀桿菌屬(特定言之麩胺酸棒狀桿菌( Corynebacterium glutamicum)))或真菌或真菌細胞(例如傘菌屬(特定言之洋菇( Agaricus bisporus))、麴菌屬( Aspergillus)、木黴菌屬( Trichoderma)或酵母菌(特定言之釀酒酵母( S. cerevisiae)、畢赤酵母屬(諸如巴斯德畢赤酵母( P. pastoris))),及亦植物或植物細胞或其等可為動物或動物細胞。較佳之宿主細胞係微生物細胞。在本專利申請案之內文中,假定包括所有細菌及原生生物(例如真菌,特定言之酵母菌及海藻),如定義(例如)於Schlegel 「General Microbiology」 (Georg Thieme Verlag (1985), 1-2)中。關於微生物,用於根據本發明之方法之宿主或宿主細胞較佳為細菌/細菌細胞或酵母菌/酵母菌細胞,更佳細菌/細菌細胞,甚至更佳桿菌屬物種/桿菌屬物種細胞或大腸埃希氏桿菌(Escherichia coli)/大腸埃希氏桿菌細胞,最佳大腸埃希氏桿菌/大腸埃希氏桿菌細胞。或者,假單胞菌(特定言之螢光假單胞菌)、鏈黴菌屬、紅球菌屬(特定言之紫紅紅球菌)、弧菌屬(特定言之需鈉弧菌)、棒狀桿菌屬(特定言之麩胺酸棒狀桿菌),或其他宿主或宿主細胞可用於根據本發明之方法。 The host or host cell used in the method according to the present invention may be a host or host cell from a prokaryotic organism or a eukaryotic organism. The host or host cell may be a bacterium or bacterial cell (e.g., Escherichia coli, bacteria of the genus Bacillus (specifically, Bacillus subtilis ), Agrobacterium (specifically, Agrobacterium tumefaciens or Agrobacterium rhizogenes ), Pseudomonas (specifically, Pseudomonas fluorescens ), Streptomyces spp ., Rhodococcus (specifically, Rhodococcus rhodochrous ), Vibrio natrigens , Corynebacterium (specifically, Corynebacterium glutamicum), glutamicum ))) or fungi or fungal cells (e.g. Agaricus (in particular Agaricus bisporus ), Aspergillus , Trichoderma or yeasts (in particular S. cerevisiae , Pichia (such as P. pastoris ))), and also plants or plant cells or the like may be animals or animal cells. Preferred host cells are microbial cells. In the context of the present patent application, it is assumed that all bacteria and protists (e.g. fungi, in particular yeasts and algae) are included, as defined, for example, in Schlegel "General Microbiology" (Georg Thieme Verlag (1985), 1-2). With regard to microorganisms, the host or host cell used in the method according to the present invention is preferably a bacterium/bacterial cell or a yeast/yeast cell, more preferably a bacterium/bacterial cell, and even more preferably a Bacillus species/Bacillus species cell or Escherichia coli (Escherichia coli)/Escherichia coli cells, preferably Escherichia coli/Escherichia coli cells. Alternatively, Pseudomonas (particularly Pseudomonas fluorescens), Streptomyces, Rhodococcus (particularly Rhodococcus rhodochrous), Vibrio (particularly Vibrio natriuresis), Corynebacterium (particularly Corynebacterium glutamicum), or other hosts or host cells can be used in the method according to the present invention.
用於根據本發明之方法之較佳宿主或宿主細胞包含合適之核酸分子,其特徵在於該核酸分子之密碼子已經改變使得其等適應該等密碼子於宿主中或宿主細胞中之使用頻率。Preferred hosts or host cells for use in the methods according to the invention comprise a suitable nucleic acid molecule, characterized in that the codons of the nucleic acid molecule have been altered so that they are adapted to the usage frequency of the codons in the host or in the host cell.
序列描述在整個本申請案中,根據下列IUPAC編碼使用用於核苷酸及胺基酸之縮寫: Sequence Description Throughout this application, abbreviations for nucleotides and amino acids are used according to the following IUPAC code:
表1
表2
表3
表4Table 4
與本申請案相關之序列表係以電子格式提交並藉由全文引用之方式併入本專利檔案中。「PRT」意謂「蛋白質」及「NUC」意謂「核酸」。
根據本發明之方法之步驟1可在缺乏溶劑之情況下或於此溶劑中進行。較佳係來自由以下組成之群之溶劑:甲基三級丁基醚、庚烷、甲苯、二甲苯、均三甲苯、苯甲醚、氯苯、正丁醇、異丙醇、正丙醇及乙醇及亦其混合物。特別佳係來自由以下組成之群之溶劑:甲苯、二甲苯、均三甲苯、正丁醇及乙醇及亦其混合物。同樣特別佳係在缺乏溶劑之情況下進行反應。Step 1 of the method according to the present invention can be carried out in the absence of a solvent or in such a solvent. Preferred solvents are those selected from the group consisting of methyl tert-butyl ether, heptane, toluene, xylene, mesitylene, anisole, chlorobenzene, n-butanol, isopropanol, n-propanol and ethanol and mixtures thereof. Particularly preferred solvents are those selected from the group consisting of toluene, xylene, mesitylene, n-butanol and ethanol and mixtures thereof. It is also particularly preferred to carry out the reaction in the absence of a solvent.
基於使用之混合物(I)之莫耳量計,醯化劑或羧化劑R-C(=O)R 1較佳係以0.4至25當量之量使用。特別佳地,其係以0.6至10當量之量使用。尤其佳地,其係以1至5當量之量使用。 The acylation agent or carboxylation agent RC(=O)R 1 is preferably used in an amount of 0.4 to 25 equivalents, based on the molar amount of the mixture (I) used. It is particularly preferred that it is used in an amount of 0.6 to 10 equivalents. It is particularly preferred that it is used in an amount of 1 to 5 equivalents.
根據步驟1之反應通常係在20至130℃之溫度下進行。較佳地,該反應係在70至130℃下進行。特別佳地,該反應係在80至120℃下進行。The reaction according to step 1 is usually carried out at a temperature of 20 to 130° C. Preferably, the reaction is carried out at 70 to 130° C. Particularly preferably, the reaction is carried out at 80 to 120° C.
根據本發明之方法之步驟2中規定之組分(II)之分離係藉由熟習此項技術者已知的結晶進行。The separation of component (II) specified in step 2 of the process according to the present invention is carried out by crystallization known to those skilled in the art.
根據本發明之方法之步驟3中規定之鹼通常來自由以下組成之群:氫氧化鋰、氫氧化鈉、氫氧化鉀、碳酸鋰、碳酸鈉、碳酸鉀、甲醇鋰、甲醇鈉、甲醇鉀、乙醇鋰、乙醇鈉及乙醇鉀。特別佳地,使用鹼氫氧化鋰、氫氧化鈉、氫氧化鉀、乙醇鋰、乙醇鈉及乙醇鉀。The base specified in step 3 of the method according to the present invention is usually selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, lithium methoxide, sodium methoxide, potassium methoxide, lithium ethoxide, sodium ethoxide and potassium ethoxide. Particularly preferably, lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium ethoxide, sodium ethoxide and potassium ethoxide are used.
極佳地,使用鹼氫氧化鋰、氫氧化鈉及氫氧化鉀。Most preferably, lithium hydroxide, sodium hydroxide and potassium hydroxide are used.
通常,基於使用之組分(II)之莫耳量計,鹼係以1.00至3.00當量之化學計量使用。較佳地,該鹼係以1.50至2.50當量之量使用。尤其佳地,該鹼係以1.75至2.25當量之量使用。Typically, the base is used in a stoichiometric amount of 1.00 to 3.00 equivalents based on the molar amount of component (II) used. Preferably, the base is used in an amount of 1.50 to 2.50 equivalents. Particularly preferably, the base is used in an amount of 1.75 to 2.25 equivalents.
較佳地,使用乙醇、正丙醇、異丙醇、正丁醇、異丁醇、二級丁醇、三級丁醇及1-甲氧基丙-2-醇及亦甲苯、二甲苯及藜蘆醚作為溶劑。特別佳地,使用乙醇、正丁醇、異丙醇、1-甲氧基丙-2-醇、甲苯、二甲苯及藜蘆醚。尤其佳地,使用乙醇、正丁醇及二甲苯。Preferably, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, di-butanol, tertiary butanol, 1-methoxypropan-2-ol, and also toluene, xylene, and veravoether are used as solvents. Particularly preferably, ethanol, n-butanol, isopropanol, 1-methoxypropan-2-ol, toluene, xylene, and veravoether are used. Especially preferably, ethanol, n-butanol, and xylene are used.
根據步驟3之反應通常係在60至140℃之溫度下進行。較佳地,該反應係在80至120℃下進行。特別佳地,該反應係在90至110℃下進行。The reaction according to step 3 is usually carried out at a temperature of 60 to 140° C. Preferably, the reaction is carried out at 80 to 120° C. Particularly preferably, the reaction is carried out at 90 to 110° C.
根據本發明之方法之另一優勢亦在於步驟1及步驟2中殘餘之2,6-二甲基-1-胺基茚烷(III)之混合物可在金屬催化下再次異構化以資源節約型方式形成2,6-二甲基-1-胺基茚烷(I)之四種立體異構體之混合物。Another advantage of the method according to the present invention is that the residual mixture of 2,6-dimethyl-1-aminoindanane (III) in step 1 and step 2 can be isomerized again under metal catalysis to form a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindanane (I) in a resource-saving manner.
此異構化可如下進行: 較佳地,使用鈀負載為1至20重量%之碳載鈀(Pd/C)、碳酸鈣載鈀(Pd/CaCO 3)或氧化鋁載鈀(Pd/Al 2O 3)觸媒作為金屬觸媒。同樣較佳地,以1至10莫耳%之化學計量使用IUPAC名稱為1-羥基四苯基環戊二烯基(四苯基-2,4-環戊二烯-1-酮)-μ-氫四羰基二釕(II)之Shvo觸媒。特別佳地,使用鈀負載為1至20重量%之碳載鈀(Pd/C)或氧化鋁載鈀(Pd/Al 2O 3)觸媒。尤其佳地,使用鈀負載為1至20重量%之氧化鋁載鈀(Pd/Al 2O 3)觸媒。負載型觸媒係以式(III)化合物之0.1至10重量%之量使用;優先考慮使用0.5至5重量%。所使用之觸媒的量係基於該等觸媒之乾質量計算。 This isomerization can be carried out as follows: Preferably, palladium on carbon (Pd/C), palladium on calcium carbonate (Pd/CaCO 3 ) or palladium on alumina (Pd/Al 2 O 3 ) with a palladium loading of 1 to 20 wt % is used as a metal catalyst. Also preferably, the Shvo catalyst whose IUPAC name is 1-hydroxytetraphenylcyclopentadienyl (tetraphenyl-2,4-cyclopentadien-1-one)-μ-hydrotetracarbonyldirutium (II) is used in a chemical amount of 1 to 10 mol %. Particularly preferably, palladium on carbon (Pd/C) or palladium on alumina (Pd/Al 2 O 3 ) with a palladium loading of 1 to 20 wt % is used. Particularly preferably, a palladium-supported alumina (Pd/Al 2 O 3 ) catalyst having a palladium loading of 1 to 20 wt %. The supported catalyst is used in an amount of 0.1 to 10 wt % of the compound of formula (III); preferably 0.5 to 5 wt %. The amount of the catalyst used is calculated based on the dry mass of the catalyst.
較佳地,使用吡啶、N-甲基嗎啉、嗎啉、環已胺、二正丁胺、三正丁胺、三乙胺、二異丙基乙胺、哌啶、碳酸鉀、碳酸鈉、碳酸鋰、乙醇鉀、乙醇鈉及乙醇鋰作為鹼,或不使用鹼。特別佳地,使用哌啶、嗎啉、二異丙基乙胺、三正丁胺、碳酸鉀及乙醇鈉,或不使用鹼。尤其佳地,使用哌啶及乙醇鈉,或不使用鹼。Preferably, pyridine, N-methylmorpholine, morpholine, cyclohexylamine, di-n-butylamine, tri-n-butylamine, triethylamine, diisopropylethylamine, piperidine, potassium carbonate, sodium carbonate, lithium carbonate, potassium ethoxide, sodium ethoxide, and lithium ethoxide are used as the base, or no base is used. Particularly preferably, piperidine, morpholine, diisopropylethylamine, tri-n-butylamine, potassium carbonate, and sodium ethoxide are used, or no base is used. Particularly preferably, piperidine and sodium ethoxide are used, or no base is used.
較佳地,使用甲苯、二甲苯、均三甲苯、苯甲醚、氯苯、正丁醇、異丙醇、正丙醇及乙醇作為溶劑,或反應係在缺乏溶劑之情況下進行。特別佳地,使用甲苯、二甲苯、均三甲苯、正丁醇及乙醇作為溶劑,或該反應係在缺乏溶劑之情況下進行。尤其佳地,該反應係在缺乏溶劑之情況下進行。Preferably, toluene, xylene, mesitylene, anisole, chlorobenzene, n-butanol, isopropanol, n-propanol and ethanol are used as solvents, or the reaction is carried out in the absence of a solvent. Particularly preferably, toluene, xylene, mesitylene, n-butanol and ethanol are used as solvents, or the reaction is carried out in the absence of a solvent. Particularly preferably, the reaction is carried out in the absence of a solvent.
反應較佳係在0與20 巴表壓之間進行。特別佳地,該反應係在0與10 巴表壓之間進行。尤其佳地,該反應係在1至6 巴表壓之間進行。The reaction is preferably carried out at a pressure between 0 and 20 barg. Particularly preferably, the reaction is carried out at a pressure between 0 and 10 barg. Particularly preferably, the reaction is carried out at a pressure between 1 and 6 barg.
藉由注入氣體/氣體混合物大體上達成反應壓力。較佳地,注入氫、氮或氬或氫及氮或氫及氬之混合物。特別佳地,注入氫或氮及氫之混合物。尤其佳地,注入純氫。The reaction pressure is substantially achieved by injecting a gas/gas mixture. Preferably, hydrogen, nitrogen or argon or a mixture of hydrogen and nitrogen or hydrogen and argon is injected. Particularly preferably, hydrogen or a mixture of nitrogen and hydrogen is injected. Particularly preferably, pure hydrogen is injected.
反應較佳係在80至150℃之溫度下進行。特別佳地,該反應係在100至130℃下進行。尤其佳地,該反應係在110至125℃下進行。The reaction is preferably carried out at a temperature of 80 to 150° C. Particularly preferably, the reaction is carried out at 100 to 130° C. Particularly preferably, the reaction is carried out at 110 to 125° C.
因此本發明亦進一步提供一種用於藉由(1R,2R)-、(1S,2R)-及(1S,2S)-2,6-二甲基-1-胺基茚烷之三種立體異構體之混合物的金屬催化之異構化產生2,6-二甲基-1-胺基茚烷(I)之所有四種立體異構體之混合物之方法: 下列實例更特定闡明本發明: 於去礦質水中使用47.6 g/l粒狀介質及4 ml/l甘油製備Terrific肉湯(TB)培養基並在121℃下將其滅菌20分鐘。 Therefore, the present invention further provides a method for producing a mixture of all four stereoisomers of 2,6-dimethyl-1-aminoindanane (I) by metal-catalyzed isomerization of a mixture of three stereoisomers of (1R,2R)-, (1S,2R)- and (1S,2S)-2,6-dimethyl-1-aminoindanane: The following examples more particularly illustrate the invention: Terrific broth (TB) medium was prepared using 47.6 g/l granular medium and 4 ml/l glycerol in demineralized water and sterilized at 121°C for 20 minutes.
脂肪酶之選殖 編碼如本文描述之脂肪酶及脂肪酶變體之核苷酸序列可如根據先前技術已知合成,例如如由相關服務提供者提供,諸如Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany)。簡而言之,如本文描述,基於載體pKA81a,將野生型脂肪酶之核酸序列(SEQ ID No. 2)或相關變體選殖至表現載體內。藉助於通常已知的方法將基因元件引入經修飾之pKA81a載體內。藉由將表現載體引入電轉感受態大腸埃希氏桿菌MG1655細胞內表現野生型脂肪酶及脂肪酶變體。 Cloning of lipases Nucleotide sequences encoding lipases and lipase variants as described herein can be synthesized as known in the prior art, for example as provided by relevant service providers, such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany). In brief, as described herein, the nucleic acid sequence of the wild-type lipase (SEQ ID No. 2) or the relevant variants are cloned into an expression vector based on the vector pKA81a. Gene elements are introduced into the modified pKA81a vector by means of generally known methods. Wild-type lipase and lipase variants are expressed by introducing the expression vector into electroporated Escherichia coli MG1655 cells.
酶變體之產生將核苷酸取代(置換)引入核酸親代序列內,例如以用胺基酸交換其他胺基酸。可使用許多分子生物學方法以達成此等置換。一種可用於產生根據本發明之突變核酸及相應之突變蛋白質之方法係於編碼一或多種胺基酸之密碼子處之定點誘變,該等密碼子係經預先選擇。用於達成此等定點突變之方法為一般技術者熟知且充分描述於文獻中(特定言之:由M.J. McPHERSON, IRL PRESS編輯之Directed Mutagenesis: A Practical Approach, 1991)或為可使用商業套組(例如來自Qiagen或Stratagene之QUIKCHANGE™閃電誘變套組)之方法。在定點誘變之後將核酸轉形至大腸埃希氏桿菌MG1655細胞內。 The generation of enzyme variants introduces nucleotide substitutions (replacements) into the nucleic acid parent sequence, for example, to exchange other amino acids with amino acids. Many molecular biological methods can be used to achieve these substitutions. A method that can be used to produce mutant nucleic acids and corresponding mutant proteins according to the present invention is site-directed mutagenesis at codons encoding one or more amino acids, and the codons are pre-selected. The methods for achieving these site-directed mutagenesis are well known to those of ordinary skill and are fully described in the literature (specifically: Directed Mutagenesis: A Practical Approach, 1991, edited by MJ McPHERSON, IRL PRESS) or methods that can use commercial kits (e.g., QUIKCHANGE™ lightning mutation kits from Qiagen or Stratagene). After site-directed mutagenesis, the nucleic acid was transformed into Escherichia coli MG1655 cells.
於合適之生物轉形反應中測試轉形細胞以確定產物產率及產物選擇性。合適之生物轉化反應係如下描述。如根據先前技術已知進行序列驗證。Transformed cells are tested in appropriate biotransformation reactions to determine product yield and product selectivity. Suitable biotransformation reactions are described below. Sequence verification is performed as known in the art.
藉由將一體積之40%甘油溶液添加至一體積之大腸桿菌培養物製備用各別表現質體轉形之大腸桿菌培養物之甘油儲備液。Glycerol stocks of E. coli cultures expressing respective plasmid transformations were prepared by adding one volume of 40% glycerol solution to one volume of E. coli culture.
為分離單個細菌菌落,將大腸桿菌培養物之適當稀釋液接種至含有合適濃度之卡那黴素之LB瓊脂盤上並在37℃下培養直至獲得單個菌落。To isolate single bacterial colonies, appropriate dilutions of E. coli cultures were inoculated onto LB agar plates containing an appropriate concentration of kanamycin and incubated at 37°C until single colonies were obtained.
[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸乙酯之合成 實例1: Synthesis of [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl] ethyl carbamate Example 1:
於比較實驗中,測試具有脂肪酶活性之三種不同蛋白質之適合性:野生型脂肪酶(SEQ ID No. 1)及具有SEQ ID No. 233及SEQ ID No. 399之脂肪酶變體。為此,在各情況下在氬下最初將9.25 g 2,6-二甲基茚-1-胺(82%反式及18%順式2,6-二甲基茚-1-胺之外消旋混合物)裝入具有磁力攪拌器之25 mL三頸燒瓶中並在各情況下用13.6 g碳酸二乙酯稀釋。將此溶液加熱至110℃之內部溫度及然後在各情況下一次性添加250 mg具有脂肪酶活性之蛋白質。藉助於HPLC量測關於其等反應進展監測轉化。在(1R,2S)-2,6-二甲基茚-1-胺異構體完全轉化後,藉助於添加5 mL碳酸二乙酯使反應終止,冷卻至100℃並跨玻璃料(孔隙率3)過濾此反應混合物。使用另一5 mL碳酸二乙酯以洗滌濾餅。然後使經組合之橙色濾液經受在50℃及35至10 毫巴之真空下之蒸餾。將50 mL正庚烷添加至蒸餾殘餘物並在40℃下將該溶液攪拌20分鐘。在冷卻至2℃期間,形成一種懸浮液,藉助於玻璃料(孔隙率3)過濾該懸浮液。該濾餅用5 mL正庚烷洗滌及然後在減壓下乾燥。然後針對化學純度藉助於定量 1H-NMR量測及針對立體異構體純度藉助於對掌性HPLC量測測試獲得之白色固體。於下表5中比較結果。 In a comparative experiment, the suitability of three different proteins with lipase activity was tested: the wild-type lipase (SEQ ID No. 1) and the lipase variants with SEQ ID No. 233 and SEQ ID No. 399. For this, 9.25 g of 2,6-dimethylinden-1-amine (racemic mixture of 82% trans and 18% cis-2,6-dimethylinden-1-amine) were initially charged in each case under nitrogen in a 25 mL three-necked flask with a magnetic stirrer and diluted in each case with 13.6 g of diethyl carbonate. This solution was heated to an internal temperature of 110° C. and then 250 mg of the protein with lipase activity were added in each case in one portion. The conversion was monitored by means of HPLC measurement regarding the progress of the reaction. After complete conversion of the (1R,2S)-2,6-dimethylindene-1-amine isomers, the reaction is terminated by adding 5 mL of diethyl carbonate, cooled to 100° C. and filtered through a glass frit (porosity 3). A further 5 mL of diethyl carbonate are used to wash the filter cake. The combined orange filtrates are then subjected to distillation at 50° C. and a vacuum of 35 to 10 mbar. 50 mL of n-heptane are added to the distillation residue and the solution is stirred at 40° C. for 20 minutes. During cooling to 2° C., a suspension is formed which is filtered through a glass frit (porosity 3). The filter cake is washed with 5 mL of n-heptane and then dried under reduced pressure. The obtained white solid was then tested for chemical purity by means of quantitative 1 H-NMR measurement and for stereoisomeric purity by means of chiral HPLC measurement. The results are compared in Table 5 below.
表5
實例2: 於配備內部溫度計、磁力攪拌器、惰性氣體入口及計泡器之500 mL四頸圓底燒瓶中,在氬下將80.0 g 2,6-二甲基茚-1-胺(82%反式及18%順式2,6-二甲基茚-1-胺之外消旋混合物)溶解於130.0 g碳酸二乙酯中。在攪拌下將此溶液加熱至110℃之內部溫度。此後,一次性添加1.2 g用SEQ ID No. 234轉形之大腸桿菌培養物並在攪拌下及在氬下使該反應在110℃下繼續進行。在7小時之反應時間後,藉助於HPLC量測確定(1R,2S)-2,6-二甲基茚-1-胺之幾乎完全轉化並藉助於冷卻至80℃使該反應終止及隨後跨玻璃料(孔隙率3)過濾。然後用2 x 40 mL碳酸二乙酯洗滌濾餅並使經組合之濾液經受在50℃及35至10 毫巴之真空下之蒸餾。此自初始264.2 g濾液移除總計157.4 g餾出物。然後將120 mL甲基環己烷添加至蒸餾殘餘物並將獲得之混合物冷卻至2℃,產生懸浮液。固體藉助於跨玻璃料(孔隙率3)過濾移除並用少量甲基環己烷洗滌。在真空下乾燥後,獲得37.0 g白色固體。定量 1H-NMR量測針對[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸乙酯證實85.4%之化學純度。藉助於對掌性HPLC量測,經測定立體異構體純度為98.5%。此對應於27%純[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸乙酯之產率。 1H-NMR (400 MHz;CDCl 3) δ = 7.08-7.01 (m, 3H), 4.81-4.71 (m, 2H), 4.19 (q, J = 8.0 Hz, 2H), 3.00 (dd, J = 8.0, 14.0 Hz, 1H), 2.51-2.45 (m, 1H), 2.33 (s, 3H), 2.16 (dt, J = 8.0, 14.0 Hz, 1H), 1.31-1.26 (m, 6H)。 Example 2: In a 500 mL four-necked round-bottom flask equipped with an internal thermometer, magnetic stirrer, inert gas inlet and bubble counter, 80.0 g of 2,6-dimethylinden-1-amine (racemic mixture of 82% trans and 18% cis 2,6-dimethylinden-1-amine) was dissolved in 130.0 g of diethyl carbonate under argon. The solution was heated to an internal temperature of 110° C. under stirring. Thereafter, 1.2 g of the E. coli culture transformed with SEQ ID No. 234 was added in one portion and the reaction was continued at 110° C. under stirring and under argon. After a reaction time of 7 hours, the almost complete conversion of (1R,2S)-2,6-dimethylinden-1-amine was determined by means of HPLC measurement and the reaction was terminated by cooling to 80° C. and subsequently filtered across a glass frit (porosity 3). The filter cake was then washed with 2 x 40 mL of diethyl carbonate and the combined filtrates were subjected to distillation at 50° C. and a vacuum of 35 to 10 mbar. This removed a total of 157.4 g of distillate from the initial 264.2 g of filtrate. 120 mL of methylcyclohexane were then added to the distillation residue and the resulting mixture was cooled to 2° C., resulting in a suspension. The solid was removed by filtration through a glass frit (porosity 3) and washed with a small amount of methylcyclohexane. After drying under vacuum, 37.0 g of a white solid were obtained. Quantitative 1 H-NMR measurements confirmed a chemical purity of 85.4% for ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. By means of chiral HPLC measurements, the stereoisomeric purity was determined to be 98.5%. This corresponds to a yield of 27% pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. 1 H-NMR (400 MHz; CDCl 3 ) δ = 7.08-7.01 (m, 3H), 4.81-4.71 (m, 2H), 4.19 (q, J = 8.0 Hz, 2H), 3.00 (dd, J = 8.0, 14.0 Hz, 1H), 2.51-2.45 (m, 1H), 2.33 (s, 3H), 2.16 (dt, J = 8.0, 14.0 Hz, 1H), 1.31-1.26 (m, 6H).
衍生物[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸甲酯係使用碳酸二甲酯類似製備且係以白色固體之形式獲得。 1H-NMR (400 MHz;CDCl 3) δ = 7.08-7.01 (m, 3H), 4.79-4.78 (m, 2H), 3.74 (s, 3H), 3.00 (dd, J = 8.0, 16.0 Hz, 1H), 2.51-2.45 (m, 1H), 2.32 (s, 3H), 2.20-2.10 (m, 1H), 1.27 (d, J = 8.0 Hz, 3H)。 The derivative [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamic acid methyl ester was prepared similarly using dimethyl carbonate and obtained as a white solid. 1 H-NMR (400 MHz; CDCl 3 ) δ = 7.08-7.01 (m, 3H), 4.79-4.78 (m, 2H), 3.74 (s, 3H), 3.00 (dd, J = 8.0, 16.0 Hz, 1H), 2.51-2.45 (m, 1H), 2.32 (s, 3H), 2.20-2.10 (m, 1H), 1.27 (d, J = 8.0 Hz, 3H).
實例3: 於配備內部溫度計、惰性氣體入口、計泡器及磁力攪拌器之25 mL三頸燒瓶中,在氬下將14.65 g碳酸二乙酯加熱至110℃之內部溫度。此後,一次性添加均用SEQ ID No. 234轉形之10.00 g 2,6-二甲基茚-1-胺(82%反式及18%順式2,6-二甲基茚-1-胺之外消旋混合物)及0.25 g大腸桿菌培養物並使該反應在攪拌下在110℃下在氬下繼續進行10小時直至HPLC量測指示(1R,2S)-2,6-二甲基茚-1-胺之幾乎完全轉化。隨後反應混合物用另一5 mL碳酸二乙酯稀釋及然後在100℃下藉助於玻璃料(孔隙率3)過濾。然後用5 mL溫度調整至80℃之碳酸二乙酯洗滌濾餅。然後使經組合之濾液經受在50℃及35至10 毫巴之真空下之蒸餾。此後,將50 mL正庚烷添加至蒸餾殘餘物,其產生黏稠懸浮液。將該懸浮液加熱至40℃並在此溫度下攪拌20分鐘。此後,於30分鐘內將該懸浮液冷卻至21℃。隨後,將該懸浮液進一步冷卻至2℃並藉助於玻璃料(孔隙率3)過濾。濾餅用20 mL冷卻至5℃之正庚烷洗滌及然後在40℃及10 毫巴之真空下乾燥。獲得5.1 g白色固體。定量 1H-NMR量測針對[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸乙酯證實99%之化學純度。藉助於對掌性HPLC量測,經測定立體異構體純度為98.5%。此對應於34%純[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸乙酯之產率。 Example 3: In a 25 mL three-necked flask equipped with an internal thermometer, an inert gas inlet, a bubble counter and a magnetic stirrer, 14.65 g of diethyl carbonate was heated to an internal temperature of 110° C. under argon. Thereafter, 10.00 g of 2,6-dimethylindan-1-amine transformed with SEQ ID No. 234 (racemic mixture of 82% trans and 18% cis 2,6-dimethylindan-1-amine) and 0.25 g of E. coli culture were added all at once and the reaction was continued with stirring at 110° C. under argon for 10 hours until HPLC measurement indicated almost complete conversion of (1R,2S)-2,6-dimethylindan-1-amine. The reaction mixture is then diluted with a further 5 mL of diethyl carbonate and then filtered at 100° C. via a glass frit (porosity 3). The filter cake is then washed with 5 mL of diethyl carbonate, the temperature of which is adjusted to 80° C. The combined filtrate is then subjected to distillation at 50° C. and a vacuum of 35 to 10 mbar. Thereafter, 50 mL of n-heptane are added to the distillation residue, which produces a viscous suspension. The suspension is heated to 40° C. and stirred at this temperature for 20 minutes. Thereafter, the suspension is cooled to 21° C. within 30 minutes. Subsequently, the suspension is further cooled to 2° C. and filtered via a glass frit (porosity 3). The filter cake was washed with 20 mL of n-heptane cooled to 5° C. and then dried at 40° C. and 10 mbar vacuum. 5.1 g of a white solid were obtained. Quantitative 1 H-NMR measurements confirmed a chemical purity of 99% for ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. The stereoisomeric purity was determined to be 98.5% by means of chiral HPLC measurements. This corresponds to a yield of 34% pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate.
實例4: 於600 mL高壓釜中,在氬下在110℃下將100.0 g 2,6-二甲基茚-1-胺(82%反式及18%順式2,6-二甲基茚-1-胺之外消旋混合物)、139.2 g碳酸二乙酯及2 g生物質Gö171-7-032攪拌7 h。在冷卻至80℃後,跨玻璃料進行過濾並用碳酸二乙酯洗滌濾餅。在減壓下在50℃下濃縮經組合之濾液並於冰浴中藉由添加150 mL甲基環己烷使蒸餾殘餘物結晶。固體藉由跨玻璃料過濾移除並用少量甲基環己烷洗滌。在真空下乾燥後,獲得47.6 g白色固體。定量 1H-NMR量測針對[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸乙酯證實91.9%之化學純度。藉助於對掌性HPLC量測,經測定立體異構體純度為96.1%。此對應於31.9%純[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸乙酯之產率。在減壓下在50℃下濃縮該濾液並藉由對掌性GC以R,R : S,S : S,R : R,S = 6.0 : 16.2 : 77.2 : 0之異構體比率測定2,6-二甲基茚-1-胺異構體之含量。 Example 4: In a 600 mL autoclave, 100.0 g of 2,6-dimethylinden-1-amine (racemic mixture of 82% trans and 18% cis-2,6-dimethylinden-1-amine), 139.2 g of diethyl carbonate and 2 g of biomass Gö171-7-032 were stirred at 110° C. under argon for 7 h. After cooling to 80° C., filtration was performed across a glass frit and the filter cake was washed with diethyl carbonate. The combined filtrate was concentrated at 50° C. under reduced pressure and the distillation residue was crystallized in an ice bath by adding 150 mL of methylcyclohexane. The solid was removed by filtration across a glass frit and washed with a small amount of methylcyclohexane. After drying under vacuum, 47.6 g of a white solid were obtained. Quantitative 1 H-NMR measurements confirmed a chemical purity of 91.9% for ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. By means of chiral HPLC measurements, the stereoisomeric purity was determined to be 96.1%. This corresponds to a yield of 31.9% pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. The filtrate was concentrated at 50° C. under reduced pressure and the content of 2,6-dimethylinden-1-amine isomers was determined by chiral GC at an isomer ratio of R,R : S,S : S,R : R,S = 6.0 : 16.2 : 77.2 : 0.
自[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸甲酯合成(1R,2S)-2,6-二甲基茚-1-胺 實例5: Synthesis of (1R,2S)-2,6-dimethylinden-1-amine from [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate methyl ester Example 5:
在21℃下用氬惰性化之250 mL夾套反應器中,使25 g [(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸甲酯(98%立體異構體純度,98%化學純度)懸浮於80 mL正丁醇中。將該懸浮液加熱至65℃之內部溫度且獲得澄清溶液。此後,一次性添加15 g氫氧化鉀(85%化學純度)並將該溶液進一步加熱至100℃之內部溫度。在此溫度下將該溶液進一步攪拌一小時,同時不溶性固體沈澱。藉由HPLC量測指示完全轉化。隨後,施加360 毫巴之減壓並蒸餾去除55 mL溶劑。此後,添加100 mL對二甲苯並將反應混合物冷卻至20℃。用1 x 50 mL去離子水洗滌該混合物。然後在40℃下將有機相蒸餾至5 毫巴之減壓及獲得17.8 g呈淺黃色油形式及化學純度為95%(藉由定量 1H-NMR量測測定)之(1R,2S)-2,6-二甲基茚-1-胺,其對應於95%之產率。藉助於對掌性GC量測測定該立體異構體純度為98%。 1H NMR (500 MHz, CDCl 3): δ 7.12 (s, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 3.74 (d, J = 8.5 Hz, 1H), 2.98 (dd, J = 7.5, 15.5 Hz, 1H), 2.42 (dd, J = 9.5, 15.5 Hz, 1H), 2.34 (s, 3H), 1.98 (m, 1H), 1.72 (bs, 2H), 1.24 (d, J = 6.5 Hz, 3H)。 In a 250 mL jacketed reactor inertized with hydrogen at 21° C., 25 g of methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate (98% stereoisomeric purity, 98% chemical purity) were suspended in 80 mL of n-butanol. The suspension was heated to an internal temperature of 65° C. and a clear solution was obtained. Thereafter, 15 g of potassium hydroxide (85% chemical purity) were added in one portion and the solution was further heated to an internal temperature of 100° C. The solution was further stirred at this temperature for one hour, while an insoluble solid precipitated. Complete conversion was indicated by HPLC measurement. Subsequently, a reduced pressure of 360 mbar is applied and 55 mL of solvent are distilled off. Thereafter, 100 mL of p-xylene are added and the reaction mixture is cooled to 20° C. The mixture is washed with 1 x 50 mL of deionized water. The organic phase is then distilled at 40° C. to a reduced pressure of 5 mbar and 17.8 g of (1R,2S)-2,6-dimethylinden-1-amine are obtained in the form of a light yellow oil and with a chemical purity of 95% (determined by quantitative 1 H-NMR measurement), which corresponds to a yield of 95%. The stereoisomeric purity is determined to be 98% by means of chiral GC measurement. 1 H NMR (500 MHz, CDCl 3 ): δ 7.12 (s, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 3.74 (d, J = 8.5 Hz, 1H), 2.98 (dd, J = 7.5, 15.5 Hz, 1H), 2.42 (dd, J = 9.5, 15.5 Hz, 1H), 2.34 (s, 3H), 1.98 (m, 1H), 1.72 (bs, 2H), 1.24 (d, J = 6.5 Hz, 3H).
實例6: 自[(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸甲酯合成(1R,2S)-2,6-二甲基茚-1-胺鹽酸鹽 Example 6: Synthesis of (1R,2S)-2,6-dimethylindene-1-amine hydrochloride from [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-indene-1-yl]carbamic acid methyl ester
在21℃下用氬惰性化之250 mL夾套反應器中,使50 g [(1R,2S)-2,6-二甲基-2,3-二氫-1H-茚-1-基]胺甲酸甲酯(98%立體異構體純度,98%化學純度)懸浮於160 mL正丁醇中。將該懸浮液加熱至65℃之內部溫度且獲得澄清溶液。此後,一次性添加31.1 g氫氧化鉀(85%化學純度)並將該溶液進一步加熱至100℃之內部溫度。在此溫度下將該溶液進一步攪拌一小時,同時不溶性固體沈澱。藉由HPLC量測指示完全轉化。隨後,施加360 毫巴之減壓並蒸餾去除110 mL溶劑。此後,添加200 mL對二甲苯並將反應混合物冷卻至20℃。用1 x 100 mL去離子水洗滌該混合物。然後將有機相冷卻至10℃並與22.8 g濃鹽酸(37重量%於水中)以使得溫度不升高至25℃以上的方式混合。然後在50℃下自獲得之懸浮液蒸餾去除100 mL溶劑,及藉由在20℃下藉助於玻璃料(孔隙率3)過濾移除固體。濾餅用1 x 100 mL甲苯洗滌及然後在40℃及10 毫巴減壓下乾燥並獲得40 g呈白色固體之形式及化學純度為99%(藉由定量 1H-NMR量測測定)之(1R,2S)-2,6-二甲基茚-1-胺鹽酸鹽,其對應於95%之產率。藉助於對掌性GC量測測定該立體異構體純度為98%。 1H-NMR (400 MHz;DMSO-d6) δ = 8.66 (bs, 3H), 7.45 (s, 1H), 7.14 (dd, J = 8.0, 16.0 Hz, 2H), 4.20 (d, J = 8.0 Hz, 1H), 3.18-3.12 (m, 1H), 2.50-2.40 (m, 1H), 2.30 (s, 3H), 1.22 (d, J = 8.0 Hz, 3H)。 In a 250 mL jacketed reactor inertized with hydrogen at 21° C., 50 g of methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate (98% stereoisomeric purity, 98% chemical purity) were suspended in 160 mL of n-butanol. The suspension was heated to an internal temperature of 65° C. and a clear solution was obtained. Thereafter, 31.1 g of potassium hydroxide (85% chemical purity) were added in one portion and the solution was further heated to an internal temperature of 100° C. The solution was further stirred at this temperature for one hour, while an insoluble solid precipitated. Measurement by HPLC indicated complete conversion. Subsequently, a reduced pressure of 360 mbar is applied and 110 mL of solvent are distilled off. Thereafter, 200 mL of p-xylene are added and the reaction mixture is cooled to 20° C. The mixture is washed with 1 x 100 mL of deionized water. The organic phase is then cooled to 10° C. and mixed with 22.8 g of concentrated hydrochloric acid (37% by weight in water) in such a way that the temperature does not rise above 25° C. 100 mL of solvent are then distilled off from the suspension obtained at 50° C. and the solid is removed by filtering at 20° C. with the aid of a glass frit (porosity 3). The filter cake was washed with 1 x 100 mL toluene and then dried at 40° C. and 10 mbar under reduced pressure to give 40 g of (1R,2S)-2,6-dimethylindene-1-amine hydrochloride in the form of a white solid with a chemical purity of 99% (determined by quantitative 1 H-NMR measurement), corresponding to a yield of 95%. The stereoisomeric purity was determined to be 98% by means of chiral GC measurement. 1 H-NMR (400 MHz; DMSO-d6) δ = 8.66 (bs, 3H), 7.45 (s, 1H), 7.14 (dd, J = 8.0, 16.0 Hz, 2H), 4.20 (d, J = 8.0 Hz, 1H), 3.18-3.12 (m, 1H), 2.50-2.40 (m, 1H), 2.30 (s, 3H), 1.22 (d, J = 8.0 Hz, 3H).
使2,6-二甲基茚-1-胺混合物(III)異構化以形成2,6-二甲基茚-1-胺混合物(I) 實例7: Isomerization of the 2,6-dimethylinden-1-amine mixture (III) to form the 2,6-dimethylinden-1-amine mixture (I) Example 7:
於25 ml高壓釜中,使10 g獲得之2,6-二甲基茚-1-胺混合物(III) (來自實例4之濃縮濾液)與396 mg鈀觸媒(5% Pd/Al 2O 3)混合。用5 巴氬將該高壓釜吹掃三次,接著注入3 巴氫並在120℃下在3 巴氫下攪拌10 h。在冷卻至室溫並使該高壓釜排氣後,獲得之混合物之2,6-二甲基茚-1-胺異構體之含量係藉由對掌性GC以R,R : S,S : S,R : R,S = 9.2 : 8.9 : 43.0 : 39.0之異構體比率測定。 In a 25 ml autoclave, 10 g of the obtained 2,6-dimethylinden-1-amine mixture (III) (the concentrated filtrate from Example 4) was mixed with 396 mg of a palladium catalyst (5% Pd/Al 2 O 3 ). The autoclave was purged three times with 5 bar of hydrogen, then injected with 3 bar of hydrogen and stirred at 120° C. under 3 bar of hydrogen for 10 h. After cooling to room temperature and venting the autoclave, the content of 2,6-dimethylinden-1-amine isomers in the obtained mixture was determined by chiral GC with an isomer ratio of R,R : S,S : S,R : R,S = 9.2 : 8.9 : 43.0 : 39.0.
使(1R,2S)-2,6-二甲基茚-1-胺異構化為2,6-二甲基茚-1-胺(82%反式及18%順式異構體之外消旋混合物) 實例8至27: Isomerization of (1R,2S)-2,6-dimethylindene-1-amine to 2,6-dimethylindene-1-amine (racemic mixture of 82% trans and 18% cis isomers) Examples 8 to 27:
於6 mL惠頓(Wheaton)螺旋蓋小瓶中裝入1.0 g (1R,2S)-2,6-二甲基茚-1-胺、39.6 mg鈀觸媒(5% Pd/Al 2O 3)、1.47 g碳酸二乙酯及10至20莫耳%鹼。然後,用螺旋蓋封閉小瓶並以400 rpm在90至110℃下振盪6小時。此後接著冷卻至21℃並藉助於對掌性GC分析及對掌性HPLC量測進行分析。將結果製表。活性定義自異構體比率0 : 0 : 0 : 100一直至平衡比率9 : 9 : 41 : 41之異構化過程且主要藉由1R,2S異構體自100%至41%之降解過程來量測。因此,由不大於59%降解之量測比例確定活性。化學選擇性描述所需組分(所有DMAI反應物異構體及所有DMAI胺甲酸酯產物異構體)相對於異構化中形成之所有組分(包括次要組分)的總和之程度。相關資料係顯示於下表6中。 1.0 g (1R,2S)-2,6-dimethylinden-1-amine, 39.6 mg palladium catalyst (5% Pd/Al 2 O 3 ), 1.47 g diethyl carbonate and 10 to 20 mol % base were placed in a 6 mL Wheaton screw cap vial. The vial was then sealed with a screw cap and shaken at 400 rpm at 90 to 110° C. for 6 hours. It was then cooled to 21° C. and analyzed by chiral GC analysis and chiral HPLC measurement. The results were tabulated. Activity is defined as the isomerization process from an isomer ratio of 0:0:0:100 to an equilibrium ratio of 9:9:41:41 and is measured primarily by the degradation of the 1R,2S isomer from 100% to 41%. Thus, activity is determined by a measured ratio of no more than 59% degradation. Chemical selectivity describes the extent of the desired component (all DMAI reactant isomers and all DMAI carbamate product isomers) relative to the sum of all components (including minor components) formed in the isomerization. The relevant data are shown in Table 6 below.
表6
TW202412627A_112129495_SEQL.xmlTW202412627A_112129495_SEQL.xml
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