CN111979207A - 一种醛酮还原酶及不对称还原制备手性度洛西汀中间体的方法 - Google Patents
一种醛酮还原酶及不对称还原制备手性度洛西汀中间体的方法 Download PDFInfo
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Abstract
本发明公开了一种醛酮还原酶及不对称还原制备手性度洛西汀中间体的方法,属于生物法不对称合成手性化合物技术领域。本发明筛选得到醛酮还原酶来源于巨大芽孢杆菌BM1‑1(Bacillus megaterium BM1‑1),经过菌株培养,基因克隆,构建工程菌,醛酮还原酶表达与纯化,在加入NADPH和辅助底物情况下,醛酮还原酶催化催化底物3‑(二甲氨基)‑1‑(2‑噻吩基)‑1‑丙酮(DKTP)生产(S)‑3‑(二甲氨基)‑1‑(2‑噻吩基)‑1‑丙醇(S‑DHTP)。本发明确定了基于该酶催化DKTP不对称还原的反应体系和反应条件。本发明操作方便,设备简单,在生物催化制备手性度洛西汀中间体领域具有较好的工业应用前景,对于今后生物催化专用酶源的开发和手性化合物合成方法的研究具有较为重要的意义。
Description
技术领域
本发明涉及一种醛酮还原酶酶催化3-(二甲氨基)-1-(2-噻吩基)-1-丙酮立体选择性制备(S)-3-(二甲氨基)-1-(2-噻吩基)-1-丙醇的方法。
背景技术
在自然界中,有一种极其重要的对称结构,这种结构指一个物体与其镜像不重合,就像人的左手与右手一样,这种结构就称为手性结构。在立体化学中,对手性化合物的研究愈来愈受到人们的关注。在许多的手性化合物中,手性醇较为特别,其手性碳原子上连有活泼的羟基官能团,基于此结构,又可以被其他的官能团取代,从而衍生出其他多种旋光性化合物,例如芳香醇,邻二醇等。手性醇及其衍生物是许多手性药物合成的重要中间体,因此,其在医药领域具有极其重要的价值。
(S)-3-(二甲氨基)-1-(2-噻吩基)-1-丙醇(S-DHTP)是一种手性醇,广泛应用于抗抑郁药物度洛西汀的合成。度洛西汀(Duloxetine)作为一种世界范围内的一线药物,主要用于抑郁症和焦虑症的治疗,并且能够缓解抑郁症引发的肌肉疼痛及躯体疼痛。度洛西汀作为一种手性药物,其发挥药效性能的是S型异构体。和其他抗抑郁药相比,度洛西汀安全性更好,副作用更少,具有更为优越的安全性和更少的成瘾依赖性。目前,度洛西汀合成工艺中,最直接有效的手性中间体为S-DHTP。作为合成度洛西汀的重要中间体,S-DHTP的合成方法主要有化学法和生物法,目前工业上以化学合成法为主。然而化学法合成S-DHTP往往需要高温高压,副反应多且工艺路线长,更重要的是,化学合成法会造成污染的环境,不符合绿色化学的要求。相比之下,生物法合成条件温和,环境友好,绿色节能,在合成S-DHTP方面具有更好的潜能。此前,Pankaj Soni等人从土壤中分离的酵母菌株(Candidatropicalis)能够全细胞催化将DKTP催化还原生成S-DHTP,虽然能够获得大于80%的转化率和高于99%的对映体过量值。然而,由于全细胞限制了酶与底物的相互接触,使得反应时间长且底物耐受性较低,大大的限制了其工业应用。因此,利用酶法制备S-DHTP越来越受到关注。因此,筛选含有催化不对称还原前手性酮高活性高选择性的醛酮还原酶酶是实现S-DHTP有效制备的关键。
发明内容
本发明的目的之一,在于一种编码醛酮还原酶,其序列如SEQ ID NO:1所示。
SEQ ID NO:1
MKYHTIANTDLHVSNLIMGNMRLTELSLQEAEKLIRTAMEEEINFFDHADIYGPDYVGQCEEYFANAIQMNPSLREKMVIQSKCGINAAENYYDFSKKHIIDSVNGSLERLKTDYLDLLLLHRPDPLMDPEEVAEAFNELQNSGKVRHFGVSNHNPAQIQLLEKYINQKLVVNQVQFSIAHTPIIDSGISLNMGIDQAVNRDSSVLEYCRLNDITLQAWSPFQNGFFEGPFLGDKEKFGKLNEVIDRIASKYSVTNTAIAVAWITTHPANIQVVLGTTNIQRMKDASKGSEIKLTRQEWYDIYKAAGNMVP
本发明的目的之二,在于提供编码所述的醛酮还原酶的序列,其如SEQ ID NO:2所示。
SEQ ID NO:2:
ATGAAGTATCATACAATTGCTAATACTGACTTACACGTATCAAATTTAATTATGGGAAACATGCGTTTAACAGAGCTTTCTTTACAAGAAGCAGAGAAGCTAATCCGCACAGCGATGGAAGAAGAAATTAACTTTTTTGATCATGCTGATATTTACGGTCCTGATTATGTAGGGCAATGCGAAGAATATTTTGCAAACGCTATACAAATGAACCCAAGTCTTCGTGAAAAAATGGTGATTCAAAGCAAATGCGGCATTAACGCAGCGGAAAATTACTATGACTTTTCAAAGAAGCATATCATAGACTCTGTTAACGGCAGCTTAGAGCGTTTAAAAACAGATTACTTAGATCTTTTATTACTGCATCGTCCAGATCCACTAATGGACCCTGAAGAAGTAGCAGAAGCATTTAACGAACTGCAAAACAGCGGAAAGGTACGTCACTTTGGTGTATCGAATCACAACCCAGCGCAAATTCAGCTGCTTGAAAAATATATTAACCAAAAGCTTGTCGTAAACCAAGTGCAGTTCAGCATCGCACATACGCCAATCATTGATTCTGGTATTTCGTTAAACATGGGAATTGATCAAGCTGTTAACCGCGACAGCAGCGTGCTTGAATATTGCCGTTTGAACGATATCACGCTTCAAGCATGGTCGCCGTTCCAAAACGGATTTTTTGAAGGTCCTTTCCTAGGTGATAAAGAGAAATTTGGAAAGCTAAACGAAGTAATTGATCGCATCGCAAGTAAATATAGCGTGACGAATACAGCGATTGCCGTTGCGTGGATTACAACGCATCCTGCAAATATTCAAGTAGTACTTGGTACGACAAATATTCAGCGTATGAAAGATGCAAGCAAAGGTTCAGAAATCAAGTTAACACGCCAAGAATGGTACGATATTTATAAAGCCGCTGGCAACATGGTGCCTTAA
本发明的目的之三,在于提供所述的醛酮还原酶在制备手性度洛西汀中间体中的应用。
本发明的目的之四,在于提供一种醛酮还原酶不对称还原制备手性度洛西汀中间体的方法,其包括以下步骤:
1)获取前述的醛酮还原酶;
2)在含有步骤1)的酶溶液中,加入NADPH作为辅酶,加入3-(二甲氨基)-1-(2-噻吩基)-1-丙酮(DKTP)作为底物,催化不对称还原得到产物(S)-3-(二甲氨基)-1-(2-噻吩基)-1-丙醇(S-DHTP)。
在本发明的优选实施例中,步骤1)包括以下步骤:
a.基因组DNA的提取:将菌种接入LB液体培养基中培养,抽提细菌基因组DNA;所述的细菌为巨大芽孢杆菌YC4-R4(Bacillus megaterium BM1-1);
b.基因克隆、表达:以基因组DNA为模板扩增目的基因,并装载到pET28a载体上,转入大肠杆菌DE3中,培养DE3,并使用IPTG诱导表达;
c.蛋白纯化:将步骤2)被诱导表达的DE3超声破碎,产物纯化获得该醛酮还原酶。
在本发明的优选实施例中,所述的巨大芽孢杆菌(Bacillus megaterium BM1-1)的保藏号为CCTCC NO:M 2018595。
在本发明的优选实施例中,在步骤a)中,所述基因组DNA的提取方法为:所述的菌种接种量为1%-10%;所述LB培养基组成:5g酵母粉;10g蛋白胨;10g NaCl;pH值为7.0,用去离子水配制;所述的培养条件为:起始pH 7.0,装液量体积分数为5%-15%,培养温度28℃,摇床转速200rpm,培养时间12-48h,Ezup柱式细菌基因组DNA抽提试剂盒抽提总DNA。
在本发明的优选实施例中,在步骤b)中所述基因克隆及表达的制备方法为:以巨大芽孢杆菌基因组DNA为模板,合成该目的基因的引物,并通过PCR得到如SEQ ID NO:2所示的基因序列。然后将该DNA片段亚克隆到pET-28a的原始载体中以构建重组表达载体,其最终被转化到大肠杆菌BL21(DE3)中。将大肠杆菌BL21(DE3)培养在200mL的含有100μg/mL卡那霉素的LB培养基(pH 6.5)中,并于37℃,200r/min进行培养。当600nm处的光密度(OD600)达到0.6-0.8时,将温度变为18℃,然后加入IPTG,使其终浓度为0.1mM,并继续培养14h。
在本发明的优选实施例中,在步骤c)中所述的纯化方法为:所述的LB培养基的组成为:通过离心(5000rpm,3h)收集细胞,并在4℃下用PBS洗涤3次;随后将细胞沉淀物悬浮在PBS中,并通过超声破碎;随后,将破碎的细胞碎片在12,000r/min,4min,4℃的条件下离心除去沉淀物;然后,用装有10mL Ni-IDA柱(美国GE Healthcare)的AKTA Prime系统纯化带有组氨酸标签的酶。
在本发明的优选实施例中,在步骤2)中酶催化反应,将0.05g DKTP溶解在5mLAKR3-2-9酶溶液中,并添加NADPH(终浓度为0.5mM;然后,将其置于37℃下,并在200r/min的振荡器中反应24小时;反应完成后,将反应后的样品在4℃,10000r/min的条件下离心10min,取上清液。
在本发明的优选实施例中,还包括如下步骤,将酶催化底物DKTP,使用高效液相色谱法对产物进行定量分析。色谱柱为Chiralcel OD-H柱(250mm×4.6mm),紫外检测波长241nm,流动相为正己烷:乙醇(9:1v/v),流动相流速为1.0mL/min。R-DHTP及S-DHTP的保留时间分别为:4.908min和5.212min。S-DHTP的产率和光学纯度可以根据R-DHTP及S-DHTP的出峰面积算出。
本发明筛选获得含有依赖NADPH的醛酮还原酶不同以往报道的新酶。本发明还考察了底物浓度、pH、温度等影响,优化了该酶不对称还原制备S-DHTP的反应体系和反应条件。
本发明所述的醛酮还原酶催化DKTP不对称还原制备S-DHTP,是利用醛酮还原酶的高度对映体选择性,催化DKTP的不对称还原,获得S-DHTP。其NADPH为辅酶的化学反应式如下:
所述酶为来源于一种海洋菌巨大芽孢杆菌(Bacillus megaterium BM1-1)的醛酮还原酶,该菌株为筛选自厦门附近海域。该菌株已进行了保藏,其保藏地点为中国武汉,机构为中国典型培养物保藏中心,保藏日期为2018年09月05日,保藏号为CCTCC NO:M2018595。
本发明的有益效果如下:
本发明筛选获得含有依赖NADPH的醛酮还原酶,该新酶不同以往报道的酶。本发明还考察了底物浓度、pH、温度等影响,优化了该酶不对称还原制备S-DHTP的反应体系和反应条件。
所获产物手性S-DHTP的光学纯度达到90%以上。本发明操作方便,具有产品光学纯度高、收率高,设备简单等优点,在生物催化制备S-DHTP领域具有较好的应用前景。
附图说明
图1为不同底物DKTP浓度下的S-DHTP的催化产率及光学纯度图。
具体实施方式
下面通过实施例对本发明做详细说明
实施例1
(1)重组工程菌的制备:
提取巨大芽孢杆菌(Bacillus megaterium BM1-1)的DNA,提取DNA所使用的的试剂盒为Ezup柱式细菌基因组DNA抽提试剂盒。
以提取得到的巨大芽孢杆菌(Bacillus megaterium BM1-1)基因组为模板进行DNA扩增,得到如附录1所示的醛酮还原酶基因序列。其中PCR反应体系为:13μL H2O,1μL上游引物(引物序列为F1:5’GGAATTCCATATGATGCAATATCGAAAGCTTGGAAC3’),1μL下游引物(引物序列为R1:5’CCGCTCGAGTTAATACAGTGAATTCACGGTATTC3’),1μL总DNA,4μL PrimeSTAR MaxPremix;PCR反应条件为:94℃预变性5min,94℃变性10s,54℃复性10s,72℃延伸6s,循环30次,72℃延伸10min;用NdeI和XhoI分别双酶切醛酮还原酶基因及pET28a质粒,用T4 DNA连接酶连接后转化大肠杆菌DH5α,然后提取质粒转化大肠杆菌BL21(DE3)构建醛酮还原酶基因的E.coli工程菌。将醛酮还原酶的工程菌接入LB(含卡纳霉素)培养基中培养
(2)酶液制备:将转入重组表达载体的大肠杆菌BL21(DE3)培养在200mL的含有100μg/mL卡那霉素的LB培养基(pH 6.5)中,并于37℃,200r/min进行培养。当600nm处的光密度(OD 600)达到0.6-0.8时,将温度变为18℃,然后加入IPTG,使其终浓度为0.1mM,并继续培养14h。然后通过离心(5000rpm,3h)收集细胞,并在4℃下用PBS洗涤3次。随后将细胞沉淀物悬浮在PBS中,并通过超声破碎。随后,将破碎的细胞碎片在12000r/min,4min,4℃的条件下离心除去沉淀物。然后,用装有10mL Ni-IDA柱(美国GE Healthcare)的AKTA Prime系统纯化带有组氨酸标签的酶。最后,通过12%SDS-PAGE对酶的表达和纯化结果进行表征,并通过Bradford Protein Assay Kit确定纯蛋白的浓度。
(3)酶催化反应:将2.5mg DKTP溶解在5mL AKR3-2-9酶溶液中,并添加NADPH(终浓度为0.5mM)。然后,将其置于37℃下,并在200r/min的振荡器中反应24小时。
(4)分析检测:产物S-DHTP的浓度和对映体过量值用高效液相色谱测定。反应完成后,将反应后的样品在4℃,10000r/min的条件下离心10min,取上清液。将酶催化底物DKTP,使用高效液相色谱法对产物进行定量分析。S-DHTP的产率为40%,光学纯度大于92%。
实施例2
步骤(1)至步骤(2)同实施例1,步骤(3)如下
(3)酶催化反应:将5mg DKTP溶解在5mL AKR3-2-9酶溶液中,并添加NADPH(终浓度为0.5mM)。然后,将其置于37℃下,并在200r/min的振荡器中反应24小时。
(4)分析检测:分析检测:产物S-DHTP的浓度和对映体过量值用高效液相色谱测定。反应完成后,将反应后的样品在4℃,10000r/min的条件下离心10min,取上清液。将酶催化底物DKTP,使用高效液相色谱法对产物进行定量分析。S-DHTP的产率为31%,光学纯度大于92%。
实施例3
步骤(1)至步骤(2)同实施例1,步骤(3)如下
(3)酶催化反应:将10mg DKTP溶解在5mL AKR3-2-9酶溶液中,并添加NADPH(终浓度为0.5mM)。然后,将其置于37℃下,并在200r/min的振荡器中反应24小时。
(4)分析检测:分析检测:产物S-DHTP的浓度和对映体过量值用高效液相色谱测定。反应完成后,将反应后的样品在4℃,10000r/min的条件下离心10min,取上清液。将酶催化底物DKTP,使用高效液相色谱法对产物进行定量分析。S-DHTP的产率为13%,光学纯度大于92%。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
序列表
<110> 华侨大学
<120> 一种醛酮还原酶及不对称还原制备手性度洛西汀中间体的方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 323
<212> PRT
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 1
Met Lys Tyr His Thr Ile Ala Asn Thr Asp Leu His Val Ser Asn Leu
1 5 10 15
Ile Met Gly Asn Met Arg Leu Thr Glu Leu Ser Leu Gln Glu Ala Glu
20 25 30
Lys Leu Ile Arg Thr Ala Met Glu Glu Glu Ile Asn Phe Phe Asp His
35 40 45
Ala Asp Ile Tyr Gly Pro Asp Tyr Val Gly Gln Cys Glu Glu Tyr Phe
50 55 60
Ala Asn Ala Ile Gln Met Asn Pro Ser Leu Arg Glu Lys Met Val Ile
65 70 75 80
Gln Ser Lys Cys Gly Ile Asn Ala Ala Glu Asn Tyr Tyr Asp Phe Ser
85 90 95
Lys Lys His Ile Ile Asp Ser Val Asn Gly Ser Leu Glu Arg Leu Lys
100 105 110
Thr Asp Tyr Leu Asp Leu Leu Leu Leu His Arg Pro Asp Pro Leu Met
115 120 125
Asp Pro Glu Glu Val Ala Glu Ala Phe Asn Glu Leu Gln Asn Ser Gly
130 135 140
Lys Val Arg His Phe Gly Val Ser Asn His Asn Pro Ala Gln Ile Gln
145 150 155 160
Leu Leu Glu Lys Tyr Ile Asn Gln Lys Leu Val Val Asn Gln Val Gln
165 170 175
Phe Ser Ile Ala His Thr Pro Ile Ile Asp Ser Gly Ile Ser Leu Asn
180 185 190
Met Gly Ile Asp Gln Ala Val Asn Arg Asp Ser Ser Val Leu Glu Tyr
195 200 205
Cys Arg Leu Asn Asp Ile Thr Leu Gln Ala Trp Ser Pro Phe Gln Asn
210 215 220
Gly Phe Phe Glu Gly Pro Phe Leu Gly Asp Lys Glu Lys Phe Gly Lys
225 230 235 240
Leu Asn Glu Val Ile Asp Arg Ile Ala Ser Lys Tyr Ser Val Thr Asn
245 250 255
Thr Ala Ile Ala Val Ala Trp Ile Thr Thr His Pro Ala Asn Ile Gln
260 265 270
Val Val Leu Gly Thr Thr Asn Ile Gln Arg Met Lys Asp Ala Ser Lys
275 280 285
Gly Ser Glu Ile Lys Leu Thr Arg Gln Glu Trp Tyr Asp Ile Tyr Lys
290 295 300
Ala Ala Gly Asn Met Val Pro Ala Cys Asp Glu Phe Gly His Ile Lys
305 310 315 320
Asx Xaa Glx
<210> 2
<211> 936
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 2
atgaagtatc atacaattgc taatactgac ttacacgtat caaatttaat tatgggaaac 60
atgcgtttaa cagagctttc tttacaagaa gcagagaagc taatccgcac agcgatggaa 120
gaagaaatta acttttttga tcatgctgat atttacggtc ctgattatgt agggcaatgc 180
gaagaatatt ttgcaaacgc tatacaaatg aacccaagtc ttcgtgaaaa aatggtgatt 240
caaagcaaat gcggcattaa cgcagcggaa aattactatg acttttcaaa gaagcatatc 300
atagactctg ttaacggcag cttagagcgt ttaaaaacag attacttaga tcttttatta 360
ctgcatcgtc cagatccact aatggaccct gaagaagtag cagaagcatt taacgaactg 420
caaaacagcg gaaaggtacg tcactttggt gtatcgaatc acaacccagc gcaaattcag 480
ctgcttgaaa aatatattaa ccaaaagctt gtcgtaaacc aagtgcagtt cagcatcgca 540
catacgccaa tcattgattc tggtatttcg ttaaacatgg gaattgatca agctgttaac 600
cgcgacagca gcgtgcttga atattgccgt ttgaacgata tcacgcttca agcatggtcg 660
ccgttccaaa acggattttt tgaaggtcct ttcctaggtg ataaagagaa atttggaaag 720
ctaaacgaag taattgatcg catcgcaagt aaatatagcg tgacgaatac agcgattgcc 780
gttgcgtgga ttacaacgca tcctgcaaat attcaagtag tacttggtac gacaaatatt 840
cagcgtatga aagatgcaag caaaggttca gaaatcaagt taacacgcca agaatggtac 900
gatatttata aagccgctgg caacatggtg ccttaa 936
<210> 3
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggaattccat atgatgcaat atcgaaagct tggaac 36
<210> 4
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccgctcgagt taatacagtg aattcacggt attc 34
Claims (10)
1.醛酮还原酶,其序列如SEQ ID NO:1所示。
2.如权利要求1所述的醛酮还原酶在制备手性度洛西汀中间体中的应用。
3.编码醛酮还原酶的序列,其序列如SEQ ID NO:2所示。
4.一种醛酮还原酶不对称还原制备手性度洛西汀中间体的方法,包括以下步骤:
1)获取权利要求1所述的醛酮还原酶;
2)在含有步骤1)的酶溶液中,加入NADPH作为辅酶,加入3-(二甲氨基)-1-(2-噻吩基)-1-丙酮(DKTP)作为底物,催化不对称还原得到产物(S)-3-(二甲氨基)-1-(2-噻吩基)-1-丙醇(S-DHTP)。
5.如权利要求4所述的一种醛酮还原酶不对称还原制备手性度洛西汀中间体的方法,其特征在于,步骤1)包括以下步骤:
a.基因组DNA的提取:将菌种接入LB液体培养基中培养,抽提细菌基因组DNA;所述的细菌为巨大芽孢杆菌YC4-R4(Bacillus megaterium BM1-1);
b.基因克隆、表达:以基因组DNA为模板扩增目的基因,并装载到pET28a载体上,转入大肠杆菌DE3中,培养DE3,并使用IPTG诱导表达;
c.蛋白纯化:将步骤b)被诱导表达的DE3超声破碎,产物纯化获得该醛酮还原酶。
6.如权利要求5所述的醛酮还原酶不对称催化制备手性度洛西汀中间体的方法,其特征在于,所述的巨大芽孢杆菌(Bacillus megaterium BM1-1)的保藏号为CCTCC NO:M2018595。
7.如权利要求5所述的醛酮还原酶不对称催化制备手性度洛西汀中间体的方法,其特征在于在步骤a)中,所述基因组DNA的提取方法为:所述的菌种接种量为1%-10%;所述LB培养基组成:5g酵母粉;10g蛋白胨;10g NaCl;pH值为7.0,用去离子水配制;所述的培养条件为:起始pH 7.0,装液量体积分数为5%-15%,培养温度28℃,摇床转速200rpm,培养时间12-48h,Ezup柱式细菌基因组DNA抽提试剂盒抽提总DNA。
8.如权利要求5所述的醛酮还原酶不对称催化制备手性度洛西汀中间体的方法,其特征在于在步骤b)中所述基因克隆及表达的制备方法为:以巨大芽孢杆菌基因组DNA为模板,合成该目的基因的引物,并通过PCR得到如SEQ ID NO:2所示的基因序列;然后将该DNA片段亚克隆到pET-28a的原始载体中以构建重组表达载体,其最终被转化到大肠杆菌BL21(DE3)中;将大肠杆菌BL21(DE3)培养在200mL的含有100μg/mL卡那霉素的LB培养基(pH 6.5)中,并于37℃,200r/min进行培养;当600nm处的光密度(OD 600)达到0.6-0.8时,将温度变为18℃,然后加入IPTG,使其终浓度为0.1mM,并继续培养14h。
9.如权利要求5所述的醛酮还原酶不对称催化制备手性度洛西汀中间体的方法,其特征在于在步骤c)中所述的纯化方法为:所述的LB培养基通过离心收集细胞,并在4℃下用PBS洗涤3次;随后将细胞沉淀物悬浮在PBS中,并通过超声破碎;随后,将破碎的细胞碎片在12,000r/min,4min,4℃的条件下离心除去沉淀物;然后,用装有10mL Ni-IDA柱的AKTAPrime系统纯化带有组氨酸标签的酶。
10.如权利要求4所述的醛酮还原酶不对称催化制备手性度洛西汀中间体的方法,其特征在于在步骤2)中酶催化反应,将0.05g DKTP溶解在5mL AKR3-2-9酶溶液中,并添加NADPH(终浓度为0.5mM;然后,将其置于37℃下,并在200r/min的振荡器中反应24小时;反应完成后,将反应后的样品在4℃,10000r/min的条件下离心10min,取上清液。
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