CN112410312A - 一种环己酮单加氧酶及其应用 - Google Patents
一种环己酮单加氧酶及其应用 Download PDFInfo
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- CN112410312A CN112410312A CN202011353623.9A CN202011353623A CN112410312A CN 112410312 A CN112410312 A CN 112410312A CN 202011353623 A CN202011353623 A CN 202011353623A CN 112410312 A CN112410312 A CN 112410312A
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- Prior art keywords
- ala
- cyclohexanone monooxygenase
- gly
- thr
- reaction
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 44
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 15
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims description 8
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- HTMQZWFSTJVJEQ-UHFFFAOYSA-N benzylsulfinylmethylbenzene Chemical compound C=1C=CC=CC=1CS(=O)CC1=CC=CC=C1 HTMQZWFSTJVJEQ-UHFFFAOYSA-N 0.000 claims description 3
- KIQQUVJOLVCZKG-UHFFFAOYSA-N 1-chloro-4-methylsulfanylbenzene Chemical compound CSC1=CC=C(Cl)C=C1 KIQQUVJOLVCZKG-UHFFFAOYSA-N 0.000 claims description 2
- DQNSKXYRRRCKGH-UHFFFAOYSA-N 1-methoxy-4-methylsulfanylbenzene Chemical compound COC1=CC=C(SC)C=C1 DQNSKXYRRRCKGH-UHFFFAOYSA-N 0.000 claims description 2
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- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
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Abstract
本发明公开了一种环己酮单加氧酶及其应用，属于生物工程技术领域。本发明的环己酮单加氧酶来源于嗜甲基拟无枝酸菌(Amycolatopsismethanolica)，可作为催化剂用于硫醚的高效合成。能够给以环酮、线性酮等硫醚为底物，能耐受高浓度底物、催化活性高、立体选择性强、适用的反应条件温和、对环境友好，具有很好的应用开发前景。
Description
技术领域
本发明涉及一种环己酮单加氧酶及其应用，属于生物工程技术领域。
背景技术
环己酮单加氧酶(CHMO)是一种FAD和NADPH依赖型Baeyer-Villiger单加氧酶，在众多BVMO中CHMO的相关研究最为广泛。环己酮单加氧酶除了催化酮类底物在羰基碳原子邻近插入一个氧原子生成相应的酯或内酯，还能够催化包括氮、硼、硒、磷和硫等杂原子的氧化以及环氧化反应。这类酶在立体选择性催化氧化硫醚不对称氧化上得到了很好的应用。
光学活性亚砜作为一种重要的手性化合物，在不对称合成中的应用日渐增多。手性亚砜具有广泛而重要的应用价值，它的用途大致可以分为三类手性辅助试剂和手性中间体，手性配体和手性催化剂以及手性药物。
苯甲硫醚体积相对较小，一些野生型CHMO对这种底物本身就有很强的活性和立体选择性。例如，CHMOTm在转化这种底物时主要生成R构型产物(ee＝97％)，CHMOAcinet和FDH(甲酸脱氢酶)共表达时，显著提高了细胞对NADPH的再生能力，获得高立体选择性的S构型亚砜产物(ee＝99％)，可以用于脱硫过程中使生成和。Holland等人发现具有二苯并噻吩单加氧酶可以用于催化硫醚不对称氧化，生成相应的亚砜和砜，产物主要为R构型(《Biotransformation of sulfides by Rhodocoeccuserythropolis》，公开于2003年)。但是，除了催化氧化蛋氨酸及衍生物得到产物的光学纯度在以上，对其他硫醚氧化所得产物的ee值都不高。用产环己酮单加氧酶的菌株作为催化剂催化氧化硫醚时，其立体选择性比其胞内分离得到的环己酮单加氧酶要低。对于底物苯基烷基硫醚而言，生成的产物为构型而当苯环的对位有取代基时，产物的构型就变为了S构型，生成亚砜的ee值最高也只有中等水平。
ε-己内酯(ε-CL)是一种用途广泛的化学中间体，主要用于制备在医药、合成革、汽车涂料、鞋底料以及胶黏剂等领域具有广泛用途的高性能聚合物材料聚己内酯(PCL)。此外还可用作手术缝合线、生物降解塑料袋等。目前，ε-己内酯的合成工艺主要有环己酮和非环己酮两条工艺路线，其中采用环己酮合成ε-己内酯是国内外普遍采用的工艺路线。根据所用氧化剂的不同，它又可分为过氧酸氧化法、双氧水氧化法和氧气/空气氧化法等。在ε-己内酯的众多制备方法中，生物氧化法绿色氧化环己酮制备ε-己内酯越来越受到人们的关注。
采用生物酶氧化环已酮合成ε-己内酯，其主要困难之一，是用酶催化反应时，需要昂贵的辅酶循环系统，制约了该法的工业发展。相对来说，此类方法目前研究得还比较少。如果用微生物细胞为催化剂则可能存在以下不足：(1)细胞内酶系复杂，可能使产物降解；(2)、底物、产物扩散限制严重，底物要通过细胞壁，进入胞内反应，产物要通过细胞壁，离开细胞，大大降低了细胞催化反应速度。一般而言，与游离状态时相比，酶在细胞内的反应速度至少降低5倍。(3)、氧传递限制，细胞要用氧，反应也要用氧，氧负荷显著增加，加剧了氧传递限制；(4)、底物浓度低，因为细胞较酶更敏感，底物、产物可能对细胞抑制，这就要求反应液中底物、产物浓度必须较低(例如底物、产物浓度小于lg/L)，而且与离体酶相比，细胞催化时无菌条件要求高，使成本显著增加。另外，采用生物氧化法反应条件温和，但是技术成本高、稳定性差、效率低，而且使用的某些菌株是第二类病原体，具有较高的环境风险。
发明内容
本发明要解决的技术问题是针对已经报道的环己酮单加氧酶的对底物硫醚的低催化活性，热稳定性差等问题，提供了一种环己酮单加氧酶，其具有良好的可溶性，对硫醚表现出高立体选择性，并对环己酮和苯甲硫醚具有高的催化活性。
本发明的第一个目的是提供环己酮单加氧酶在催化硫醚中的应用，所述环己酮单加氧酶的氨基酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，编码所述环己酮单加氧酶的基因的核苷酸序列如SEQ ID NO.2所示。
在本发明的一种实施方式中，所述硫醚包括苯甲硫醚、对氯-苯甲硫醚、对甲氧基-苯甲硫醚。
在本发明的一种实施方式中，当底物为苯甲硫醚时，催化得到R-苯甲亚砜。
本发明的第二个目的是提供一种制备R-苯甲亚砜的方法，利用氨基酸序列如SEQID NO.1所示的环己酮单加氧酶为催化剂，以苯甲硫醚为底物，生产R-苯甲亚砜。
在本发明的一种实施方式中，苯甲硫醚在反应体系中的浓度为30-100mmol·L-1。
在本发明的一种实施方式中，所述环己酮单加氧酶的添加量为量为20-100kU·L-1。
在本发明的一种实施方式中，反应温度为25-35℃。
在本发明的一种实施方式中，反应pH为8.0-9.0。
在本发明的一种实施方式中，反应体系中还需加入葡萄糖脱氢酶。
在本发明的一种实施方式中，葡萄糖脱氢酶的添加量为10-80kU·L-1。
在本发明的一种实施方式中，反应体系中还含有1-5％甲醇。
在本发明的一种实施方式中，当产物不再生成时反应结束，反应时间为8-30h。
本发明的第三个目的是提供一种制备ε-内酯的方法，利用氨基酸序列如SEQ IDNO.1所示的环己酮单加氧酶为催化剂，以环己酮为底物，生产ε-内酯。
在本发明的一种实施方式中，环己酮在反应体系中的浓度为50-150mmol·L-1。
在本发明的一种实施方式中，所述环己酮单加氧酶的量为30-100kU·L-1。
在本发明的一种实施方式中，反应温度为25-35℃。
在本发明的一种实施方式中，反应pH为8.0-9.0。
在本发明的一种实施方式中，反应体系中还需加入10-100kU·L-1葡萄糖脱氢酶。
在本发明的一种实施方式中，当产物不再生成时反应结束，反应时间为4-25h。
本发明还提供所述环己酮单加氧酶在制备含有苯甲亚砜或以苯甲亚砜为中间产物的产品中的应用。
有益效果：本发明提供了一种环己酮单加氧酶可作为催化剂应用于备光学纯R-苯甲亚砜，其可溶性好，催化效率高(转化率>99％)、立体选择性强(e.e.>99.9％)、适用的反应条件温和、环境友好。本发明的环己酮单加氧酶催化效果佳，底物适用性广，能以苯甲硫醚、环酮、线性酮为底物，能耐受高浓度的底物、反应时间短，反应彻底、转化率高，产物纯度高，具有很好的应用开发前景。
附图说明
图1为基因Amchmo的PCR扩增电泳图谱；M，Marker；1，基因Amchmo。
图2为pET28a-Amchmo重组质粒物理图谱。
图3为重组环己酮单加氧酶的蛋白电泳图；M，Marker；泳道1、2、3分别为重组基因工程菌BL21(DE3)/pET28a-Amchmo诱导后上清、沉淀及纯酶。
图4为环己酮单加氧酶的选择性分析液相检测图。
具体实施方式
(1)酶的分离纯化
悬浮重组细胞于A液(20mmol·L-1磷酸钠，500mmol·L-1NaCl，20mmol·L-1咪唑，pH7.4)中，超声波破碎离心后获得粗酶液。纯化所使用的柱子为亲和柱HisTrap FF crude(镍柱)，利用重组蛋白上的组氨酸标签进行亲和结合来完成。首先使用A液将镍柱平衡，粗酶液上样，继续使用A液将穿透峰洗脱下来，待平衡后用B液(20mmol·L-1磷酸钠，500mmol·L-1NaCl，1000mmol·L-1咪唑，pH 7.4)进行梯度洗脱，将结合到镍柱上的重组蛋白洗脱下来，获得重组环己酮单加氧酶。对纯化后的蛋白进行酶活测定(苯甲硫醚为底物)及SDS-PAGE分析(图3)。由图3可知，镍柱纯化后，在59kDa左右显示单条带，且杂蛋白较少，说明镍柱纯化效果较好。之后使用HiTrap Desalting脱盐柱(GE Healthcare)将纯化后的环己酮单加氧酶置换到Tris-HCl(100mmol·L-1，pH 9.0)缓冲液中，进行下一步酶学性质分析。
(2)酶活力测定
测定环己酮单加氧酶对底物苯甲硫醚的酶活力。
测定体系为：适量酶液、5mmol·L-1苯甲硫醚。于30℃静置反应15min。反应结束后，取样进行液相检测。液相检测条件：色谱柱为手性OD-H色谱柱(250mm×4.6mm×5μm)，流动相为正己烷：异丙酮(90：10)，流速为0.2～1mL·min-1，检测波长为254nm。
酶活力单位定义(U)：
在30℃下，环己酮单加氧酶催化底物苯甲硫醚生成1μmol的苯甲亚砜所需要的酶量，定义为一个酶活力单位(U)。
实施例1：重组大肠杆菌BL21(DE3)/pET28a-Amchmo的构建及培养
以核苷酸序列如SEQ ID NO.2所示的基因序列为模板，利用上游引物和下游引物(F和R)，克隆目的基因，体系如下(μL)：10×PCR Mix 10，上游引物0.2，下游引物0.2，基因组0.2，DNA聚合酶0.2，ddH2O 9.2。PCR程序为：95℃预变性10min，95℃裂解30s，55℃退火30s，72℃延伸1min 30s，循环30次，72℃延伸10min。PCR产物经琼脂糖凝胶电泳纯化，并利用琼脂糖胶回收试剂盒回收1500～2000bp区间的条带(图1)，即环己酮单加氧酶基因。所得环己酮单加氧酶基因命名为Amchmo，所编码的蛋白质序列如SEQ ID NO.1所示。
F：TGGGTCGCGGATCCTCAG ACGGCCGCCG(SEQ ID NO.3)，
R：TCGCGGATCCTCAGACGGCCGCCGTCGC(SEQ ID NO.4)。
用限制性内切酶NdeI和BamHI将质粒pET28a和Amchmo于37℃水浴中过夜双酶切，次日经琼脂糖凝胶电泳纯化并利用琼脂糖回收试剂盒回收目标片段。37℃下，使用T4 DNA连接酶将基因Amchmo与酶切过的质粒pET28进行连接，即得重组表达载体pET28a-Amchmo(图2)。将构建好的重组表达载体pET28a-Amchmo热转入大肠杆菌BL21(DE3)感受态中，涂布含卡那霉素抗性LB固体平板，过夜培养后进行菌落PCR验证，阳性克隆子即为重组大肠杆菌BL21(DE3)/pET28a-Amchmo。挑取阳性克隆子于LB培养基中过夜培养，次日按1mL/100mL转接量转接入新鲜LB培养中，培养至OD600达到0.6～0.8时，加入0.2mmol·L-1IPTG，30℃诱导培养6小时后，4℃、8000r/min离心10min收集菌体。将收集好的菌体悬浮于磷酸钾缓冲液(100mmol·L-1，pH 8.0)中，超声破碎，并通过SDS-PAGE分析蛋白的表达情况。
由图3可知，目的蛋白全部都在上清中，说明重组酶在大肠杆菌中成功得到可溶表达。
实施例2：底物谱分析
将大肠杆菌表达的酶进行分离纯化，测定环己酮单加氧酶(AmCHMO)催化不同的硫醚的酶活力，以苯甲硫醚为底物测得的酶活为100％对照，其他底物测得的酶活力以二者的百分比计算。测定结果如表1所示。
表1 AmCHMO的底物谱
表1显示，AmCHMO底物谱很广泛，能够催化苯甲硫醚，环酮及线性酮。
实施例3：环己酮单加氧酶的性质
(1)酶的最适pH
配制100mmol·L-1不同pH的缓冲液：磷酸缓冲液(pH 6.0～8.0)、Tris-HCl(8.0～9.0)、甘氨酸-NaOH缓冲液(pH 9.0～11.0)。然后以苯甲硫醚为底物，测定AmCHMO在不同pH缓冲液的相对酶活力。AmCHMO的最适反应pH为Tris-HCl，8.0-9.0，酶活力为0.64-0.95U·mg-1。在pH为6.0～7.0的磷酸缓冲液中，酶活降至20％以下。
(2)酶的最适温度
分别以苯甲硫醚为底物，测定AmCHMO在不同温度(20～55℃)下、反应15min的酶活，测得的最高酶活定为100％，其他温度下测得的酶活以相对于最高活力的百分比计算。结果显示AmCHMO的最适反应温度为35℃，为0.21～0.56U·mg–1。
表2 AmCHMO最适酶活
(3)酶的热稳定性
以苯甲硫醚为底物，分别测定AmCHMO在不同温度(30℃和40℃)下的热稳定性，测得的0小时酶的初始活力定为100％，其他时间段测得的酶活以相对于0小时酶的初始活力的百分比计算。结果显示AmCHMO在30℃下26小时后活力降低为初始活力的50％以下。
表3 AmCHMO的热稳定性
(4)动力学参数分析
测定AmCHMO对底物苯甲硫醚的动力学参数。
酶活测定体系列举如下：Tris-HCl缓冲液(100mmol·L-1，pH 9.0)，苯甲硫醚(0～5mmol·L-1)。通过计算比酶活来表征反应速率，从而计算动力学参数。测定的AmCHMO对底物苯甲硫醚的动力学参数分别为Km为0.089mmol·L-1，Vmax为2.296μmol·min–1·mg–1。
(5)金属离子对酶活的影响
将终浓度为1mmol·L-1的氯化盐形式的金属离子加入到纯酶液中，于30℃下温育15min后，在Tris-HCl缓冲液(100mmol·L-1，pH 9.0)中以苯甲硫醚为底物测定其残余酶活。同等条件下，不加任何金属离子测得的酶活力定为100％，加入金属离子测得的酶活力以对照的百分比计算。结果表2所示。
表4金属离子对环己酮单加氧酶酶活力的影响
由表2可知，当加入EDTA时环己酮单加氧酶的活力没有受到抑制，因此该酶为非金属离子依赖的酶，但是Fe2+，Mn2+和Co2+可以抑制环己酮单加氧酶的活力。
(6)酶的立体选择性分析
测定AmCHMO催化底物苯甲硫醚的选择性。反应体系(10mL)为：适量纯化的酶液、5mmol·L-1苯甲硫醚。于30℃静置反应15min。反应结束后，取样进行液相检测。检测条件：手性OD-H色谱柱(250mm×4.6mm×5μm)，检测波长为254nm，流动相为正己烷：异丙醇(90：10)，流速为0.2～1mL/min。由图4可知，该酶具有非常好的立体选择性，仅有R-苯甲亚砜生成。
实施例4：环己酮单加氧酶应用于R-苯甲亚砜的制备
取2～500U所得的重组环己酮单加氧酶和2～500U的葡萄糖脱氢酶(GDH)(购自诺唯赞公司，5U/g)于Tris-HCl缓冲液(pH 9.0，100mmol·L-1)中，加入5％甲醇，30～100mmol·L-1苯甲硫醚(表5)，反应液总体积为10mL。将反应置于30℃下，取样检测转化过程，条件如下：手性OD-H色谱柱(250mm×4.6mm×5μm)，检测波长为254nm，流动相为正己烷：异丙醇(90：10)，流速为0.2～1mL/min。
表5环己酮单加氧酶应用于制备R-苯甲亚砜
实施例5：环己酮单加氧酶应用于ε-内酯的制备
取2～500U所得的重组环己酮单加氧酶和2～500U的葡萄糖脱氢酶(GDH，购自诺唯赞公司，5U/g)于Tris-HCl缓冲液(pH 9，100mmol·L-1)中，加入5％甲醇，在反应体系中浓度为50～150mmol·L-1的环己酮(表6)，反应液总体积为10mL。将反应置于30℃下，取样检测转化过程，条件如下：岛津气相GC-2014，非手性柱：AT-SE-54(30m×0.25mm×0.33μm)，分析程序如下：100℃保持1min，然后以10℃/min的速度升至180℃，最后在180℃保持3min。对反应过程实时取样检测，当检测到产物不再增加时，反应结束。结果如表5所示，Baeyer-Villiger单加氧酶在高底物浓度下能够保持很好的催化性能，转化率能保持在95％以上。
表6环己酮单加氧酶制备ε-内酯
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种环己酮单加氧酶及其应用
<130> BAA201282A
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 541
<212> PRT
<213> Amycolatopsis methanolica
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Gly Ala Tyr Arg Ala Lys Leu Arg Glu Val Ala Asp Ala Gly Tyr Thr
515 520 525
Gly Phe Glu Leu Thr Arg Glu Asn Ala Thr Ala Ala Val
530 535 540
<210> 2
<211> 1626
<212> DNA
<213> Amycolatopsis methanolica
<400> 2
atgagcacca cccatacccc ggacgtcgac gcgatcgtca tcggcgccgg attcggcggc 60
atctacatgc tgcacaagct gcgcaacgaa ctcggcctgt ccgtcacggc cttcgagaag 120
ggcggcggcg tcggcggcac ctggtacttc aaccggtacc cgggtgccaa gtccgacacc 180
gagggcttcg tctaccgcta ctccttcgac aaggatctgc tgcgggagtg gaactggacg 240
acccgctacc tggagcaggc ggacgtgctc gcctacctcg agcacgtcgt cgagcgtttc 300
gacctcggcc gcgacatccg gctgaacacc gaggtgaccg gcgcggtctt cgacgaggag 360
agcgacctgt ggacggtcac caccgccact ggggagacca ccacggcgcg ctacctggtc 420
aacgcgctcg gcctgctggc caagagcaac atccccgaca tcccgggccg ggacggcttc 480
gccggccgcc tggtgcacac caacgcctgg ccggacgacc tggacatcac gggcaagcgg 540
gtcggggtga tcggcaccgg gtcgaccggc acccagttca tcatcgcggc cgcgaagacg 600
gcgagccacc tcaccgtctt ccagcgttcg ccgcagtatt gcgtgccgtc cggcaacggc 660
ccggtggacc agaccgaagt ggacggcacc aaggagaact tcgacgccat ctgggaccag 720
gtccgcaact ccgtcgtcgc gttcgggttc gaggagagcg gcgtcgaggc gatgagcgtg 780
tccgaagagg aacgtcgcaa ggtgttccag gaagcctggg acaagggcaa cggcttccgg 840
ttcatgttcg gcacgttctg cgacatcgcc acgaacccgg aagcgaacgc ggccgccgcg 900
gcgttcatcc gtgccaagat cgccgagatc gtcgacgacc cggagaccgc gcgcaagctc 960
accccgaccg acctctacgc caagcgcccg ctgtgcaacg agggctacta cgagacctac 1020
aaccgggaca acgtcgagct ggtttcgatc aaggagaacc cgatccgcga gatcaccccg 1080
gccggcgtgc gcaccgccga cgggaccgag cacccactcg acgtcctggt gttcgcgacc 1140
gggttcgacg cggtcgacgg caactaccgg gcgatggacc tgcgcggccg cggtgggcgg 1200
cacatcagcg agcactggac cggcgggccg accagctacc tcggcgtgtc cacagccggt 1260
ttcccgaaca tgttcatgat cctcggcccg aacggcccct tcaccaacct gccgccgagc 1320
atcgaaaccc aggtcgactg gatcggcgag ctgatccgcc acgccgagcg aaccggggtg 1380
cgcacggtcg agccgaccgc ggccgcggag gaggcgtgga cggccacgtg cgcggagatc 1440
gcggacatga ccttgttccc gaaggccgat tcgtggatct tcggggcgaa catcccgggg 1500
aagcgcaacg cagtgatgtt ctacctcgcg gggctcggcg cctaccgggc gaagctgcgt 1560
gaggtcgccg acgctggata caccggcttc gagctgaccc gggagaacgc gacggcggcc 1620
gtctga 1626
<210> 3
<211> 28
<212> DNA
<213> 人工序列
<400> 3
tgggtcgcgg atcctcagac ggccgccg 28
<210> 4
<211> 28
<212> DNA
<213> 人工序列
<400> 4
tcgcggatcc tcagacggcc gccgtcgc 28
Claims (10)
1.环己酮单加氧酶在催化硫醚中的应用，其特征在于，所述环己酮单加氧酶的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述硫醚包括苯甲硫醚、对氯-苯甲硫醚、对甲氧基-苯甲硫醚。
3.根据权利要求1所述的应用，其特征在于，当底物为苯甲硫醚时，催化得到R-苯甲亚砜。
4.一种制备R-苯甲亚砜的方法，其特征在于，利用氨基酸序列如SEQ ID NO.1所示的环己酮单加氧酶为催化剂，以苯甲硫醚为底物，生产R-苯甲亚砜。
5.根据权利要求4所述的方法，其特征在于，苯甲硫醚在反应体系中的浓度为30-100mmol·L-1。
6.根据权利要求4所述的方法，其特征在于，所述环己酮单加氧酶的添加量为20-100kU·L-1。
7.根据权利要求6所述的方法，其特征在于，反应温度为25-35℃，反应pH为8.0-9.0。
8.根据权利要求4所述的方法，其特征在于，反应体系中还需加入葡萄糖脱氢酶。
9.根据权利要求4所述的方法，其特征在于，反应体系中还含有甲醇。
10.氨基酸序列如SEQ ID NO.1所示的环己酮单加氧酶在制备含有苯甲亚砜或以苯甲亚砜为中间产物的产品中的应用。
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