CN113025588A - 一种能够催化合成哌啶类化合物的醇脱氢酶KpADH突变体 - Google Patents
一种能够催化合成哌啶类化合物的醇脱氢酶KpADH突变体 Download PDFInfo
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- CN113025588A CN113025588A CN202110340244.4A CN202110340244A CN113025588A CN 113025588 A CN113025588 A CN 113025588A CN 202110340244 A CN202110340244 A CN 202110340244A CN 113025588 A CN113025588 A CN 113025588A
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Abstract
本发明公开了一种能够催化合成哌啶类化合物的醇脱氢酶KpADH突变体,属于基因工程和酶工程技术领域。本发明的KpADH突变体是以来源于克鲁维多孢酵母Kluyveromycespolyspora的重组醇脱氢酶KpADH为基础进行突变的,构建的醇脱氢酶KpADH突变体对于催化氧化吡咯烷、哌啶类、醇类的性能均有显著提升,在以rac‑NBHP为底物时,能在6小时内就使得转化率达到99.9%且能够以高浓度rac‑NBHP为底物,转化生产NBPO,较野生型的催化性能显著提升,这将使其具有更高的工业应用价值,尤其是在制备药物依鲁替尼中的应用。
Description
技术领域
本发明涉及一种能够催化合成哌啶类化合物的醇脱氢酶KpADH突变体,属于基因工程和酶工程技术领域。
背景技术
依鲁替尼(ibrutinib,原研商品名为Imbruvica)是一种布鲁顿酪氨酸激酶(BTK)抑制剂类抗淋巴癌新药。与其他药物相比,ibrutinib对BTK具有高效特异性、不可逆性及靶向性高的特点。其具有光学活性的前体物质N-丙烯酰基哌啶环,化学合成步骤复杂,成本高,收率低等。因此,从价格较低的底物3-羟基吡啶出发,将化学合成与生物酶催化相结合,首先突破前体化合物N-Boc-3-哌啶酮的合成瓶颈,继而通过偶联KpADH催化的不对称还原反应实现(S)-N-Boc-3-羟基哌啶((S)-NBHP)的从头合成。该化学生物催化方法,成本大大降低、底物专一性高、反应条件温和、环境友好等优势,更具有研究和应用价值。
醇脱氢酶KpADH(Kluveromyces polyspora)分属于醇脱氢酶(alcoholdehydrogenase,ADH)和醛酮还原酶超家族(Aldo-keto Reductase superfamily,AKR),它能够使用辅酶NAD(P)+和还原剂(通常为葡萄糖,甲酸酯或异丙醇)引入手性分子。自然界中广泛存在于动植物,细菌等组织中。醇脱氢酶可依据肽链的长短和对金属的依赖性划分为不依赖金属离子的短链脱氢酶超家族(Short-chain Dehydrogenase/Reductase,SDR)和依赖Zn2+的中链脱氢酶超家族(Medium-chain Dehydrogenase/Reductase,MDR)。近年来,有许多关于醇脱氢酶制备手性苄醇的报道。KpADH是已知的、对大位阻醇底物具有氧化能力的酶,在工业中具有广泛的用途,并为未来工业生物催化提供广泛的应用前景。
虽然KpADH有着巨大的应用和研究价值,但是从野生菌中筛选出的KpADH特异性催化NBHP的活力低,使之无法进一步将该酶应用于工业生产中,大大降低了开发和运用。
发明内容
鉴于目前野生型KpADH的活力低、无法适应工业化生产的需求的问题,本发明将来自克鲁维多孢酵母Kluyveromyces polyspora的重组醇脱氢酶KpADH进行异源表达和定点突变改造,使改造后的KpADH氧化NBHP的能力得到改善,对KpADH的工业化应用及推广具有重要意义。
本发明提供了醇脱氢酶突变体,所述醇脱氢酶突变体是以氨基酸序列如SEQ IDNO.1所示的醇脱氢酶为亲本,将亲本第128位、131位、161位、165位、196位一个或多个位点突变。
在一种实施方式中,将第128位的丙氨酸突变为半胱氨酸或缬氨酸,将第161位的苯丙氨酸突变成色氨酸,将第196位的丝氨酸突变成甘氨酸。
在一种实施方式中,所述突变体能够特异性催化NBHP合成N-Boc-3-哌啶酮(NBPO),继而实现药物依鲁替尼中间体(S)-NBHP从头合成的KpADH突变体。
在一种实施方式中,所述突变体为在SEQIDNO:1的氨基酸序列的基础上将第128位置的丙氨酸突变成半胱氨酸,命名为M1。
在一种实施方式中,所述突变体为在SEQIDNO:1的氨基酸序列的基础上将第128位的丙氨酸突变成缬氨酸,命名为M2。
在一种实施方式中,所述突变体为在SEQIDNO.1的氨基酸序列的基础上将第161位的苯丙氨酸突变成色氨酸,命名为M3。
在一种实施方式中,所述突变体为在SEQIDNO.1的氨基酸序列的基础上将第196位的丝氨酸突变成甘氨酸,命名为M4。
在一种实施方式中,所述突变体为在SEQIDNO.1的氨基酸序列的基础上将第128位的丙氨酸突变成半胱氨酸,同时第196位的丝氨酸突变成甘氨酸命名为M5。
在一种实施方式中,所述突变体为在SEQIDNO:1的氨基酸序列的基础上将第128位的丙氨酸突变成缬氨酸,同时第161位的苯丙氨酸突变成色氨酸命名为M6。
在一种实施方式中,所述突变体为在SEQIDNO.1的氨基酸序列的基础上将第196位的丝氨酸突变成甘氨酸,同时第128位的丙氨酸突变成缬氨酸命名为M7。
在一种实施方式中,所述突变体为在SEQIDNO.1的氨基酸序列的基础上将第161位的苯丙氨酸突变成色氨酸,同时第128位的丙氨酸突变成半胱氨酸命名为M8。
本发明提供了编码所述醇脱氢酶突变体的基因。
本发明提供了携带所述基因的重组质粒。
在一种实施方式中,所述重组质粒以pET系列载体为出发载体。
本发明提供了表达所述突变体,或携带所述重组质粒的微生物细胞。
在一种实施方式中,所述微生物细胞以大肠杆菌为宿主。
本发明提供了一种生产哌啶酮类化合物的方法,所述方法是以哌啶类化合物为底物,以所述突变体为催化剂。
在一种实施方式中,所述哌啶类化合物的浓度为10-60mM。
在一种实施方式中,所述哌啶类化合物包括rac-N-叔丁氧羰基-3-羟基哌啶、N-叔丁氧羰基-4-羟基哌啶、3-羟基哌啶。
在一种实施方式中,在pH7.0-8.0下反应。
本发明提供了所述突变体,或所述基因,或所述重组质粒,或所述微生物细胞在生产手性醇类化合物中的应用。
本发明提供了所述突变体,或所述基因,或所述重组质粒,或所述微生物细胞在生产酮类化合物中的应用。
在一种实施方式中,所述酮类化合物以外消旋醇为底物转化得到。
在一种实施方式中,所述酮类化合物包括哌啶酮类化合物。
本发明取得的有益效果:
本发明的KpADH突变体是以来源于克鲁维多孢酵母Kluyveromycespolyspora的重组醇脱氢酶KpADH为基础进行突变的,构建的醇脱氢酶KpADH突变体对于催化氧化吡咯烷、哌啶类、醇类的性能均有显著提升,在以rac-NBHP为底物时,能在6小时内就使得转化率达到99.9%且能够以高浓度rac-NBHP为底物,转化生产NBPO,较野生型的催化性能显著提升,这将使其具有更高的工业应用价值,尤其是在制备药物依鲁替尼中的应用。
附图说明
图1为野生型和醇脱氢酶突变体M1~M7的全质粒PCR核酸电泳图。
具体实施方式
醇脱氢酶酶活检测方法:
测活原理:根据NAD(P)H在340nm下有特征性吸收峰,醇脱氢酶在发生氧化或还原的反应过程中会产生或消耗NAD(P)H。因此,采用NAD(P)H在340nm下的变化,可间接计算出酶的活力。
总反应体系为200μL,包括:20mM NAD(P)+,100mM底物rac-NBHP,磷酸钠缓冲液(PBS,l00 mM,pH 7.0),充分混匀,30℃保温2min,加入适量的酶液,检测340nm下光吸收值的变化。
酶活计算公式:U=EW×V×103/(6200×L)
式中,EW:1min内340nm处吸光度的改变值;V:反应液的总体积(mL);6220:摩尔消光系数(L·mol-1·cm-1);L:光程长度(cm)。
酶活定义如下:
一个酶活力单位(1U)定义为在上述测活条件下每分钟催化1μmol NAD(P)+生成NAD(P)H所需的酶量。
实施例1:醇脱氢酶突变体基因和重组表达转化体的构建
采用全质粒PCR方法对位于128位、131位、161位、196位的氨基酸残基进行定点突变,构建迭代组合突变体。将核苷酸序列如SEQ ID NO.2所示的醇脱氢酶KpADH的编码基因连接至pET28a载体的多克隆酶切位点;根据醇脱氢酶KpADH的序列(氨基酸序列如SEQ IDNO.1所示),其引物设计如下(均按5’-3’方向描述,下划线代表突变位点):
M1(以pET28a-KpADH重组质粒为模板):
A128C-F:ACTGCTTCTTATTGTTCAATT,
A128C-R:GGTCATAATTGAACAATAAGA;
M2(以pET28a-KpADH重组质粒为模板):
A128V-F:ACTGCTTCTTATGTTTCAATT,
A128V-R:GGTCATAATTGAAACATAAGA;
M3(以pET28a-KpADH重组质粒为模板):
F161W-F:TATGAAAATGTCTGGACTGCT,
F161W-R:ACAATAAGCAGTCCAGACATT;
M4(以pET28a-KpADH重组质粒为模板):
S196G-F:ACTATCCACCCAGGTTTCGTT,
S196G-R:TCCGAAAACGAAACCTGGGTG;
M5(以M1重组质粒为模板):
S196G-F:ACTATCCACCCAGGTTTCGTT,
S196G-R:TCCGAAAACGAAACCTGGGTG;
M6(以M2重组质粒为模板):
F161V-F:TATGAAAATGTCGTTACTGCT,
F161V-R:ACAATAAGCAGTAACGACATT;
M7(以M3重组质粒为模板):
A128V-F:ACTGCTTCTTATGTTTCAATT,
A128V-R:GGTCATAATTGAAACATAAGA;
M8(以M4重组质粒为模板):
A128C-F:ACTGCTTCTTATTGTTCAATT,
A128C-R:GGTCATAATTGAACAATAAGA。
PCR反应体系为:PCR反应体系(20μL)包括KOD酶(2.5U/mL)0.4μL,模板(5-20ng)0.3μL,dNTP 2μL,10×reactionbuffer 2μL,上下游引物各0.3μL,ddH2O补足至20μL。
PCR的扩增程序为:95℃预变性6min,98℃变性10s,50℃退火30s,68℃延伸3.5min,循环20次,68℃延伸10min,4℃保存。通过1%琼脂糖核酸凝胶电泳验证扩增成功后,使用Quick Cut Dpn I酶,37℃保温1h,用热转化法将10μL消化后PCR反应液转入100μLE.coliBL21(DE3)感受态细胞,并均匀涂布于含50μg/mL卡那霉素的LB固体平板上,37℃的恒温培养12h。图1为野生型和醇脱氢酶突变体M1~M7的全质粒PCR核酸电泳图,由图可知,PCR产物在10000bp和3000bp之间(条带大小为5000bp)。
实施例2:醇脱氢酶及其突变体的表达与纯化
将保藏的WT KpADH和实施例2中的突变体的甘油菌接种至含50μg/mL卡那霉素的LB培养基中,37℃、120rpm摇床培养8h,将活化的种子液按1%(v/v)的接种量转接至30mLLB培养基(含50μg/mL卡那霉素)中。当OD600达到0.8时,加入0.2mMIPTG进行诱导,诱导温度为25℃,诱导8h后,8000rpm离心10min获得高效表达重组醇脱氢酶突变体的菌体,将收集的菌体悬浮于磷酸钾缓冲液(100mM,pH 6.0)中,超声破碎。
将细胞裂解液以4℃、8000rpm离心20min。
纯化所使用的柱子为镍亲和柱HisTrap FF crude,利用重组蛋白上的组氨酸标签进行亲和层析来完成。梯度洗脱步骤:(1)样品处理:收集超声离心后的细胞破碎液,用0.22μm膜过滤除杂;(2)镍柱处理:将用于层析柱保存的20%乙醇自然流出,先用10mL超纯水冲洗,再用10mL抽滤过的PBS缓冲液(100mM,pH 7.0)冲洗以平衡柱子;(3)上样:将预处理的样品缓慢倒入层析柱中,收集流出液再次过柱;(4)洗脱:依次用含有不同咪唑浓度(20mM、50mM、100mM、150mM、300mM)的PBS缓冲液(100mM,pH 7.0)洗脱,每个浓度洗脱5mL,分别收集不同咪唑浓度洗脱时的透过液测定酶活。测活发现洗脱过程中咪唑浓度达到300mM时KpADH及其突变体可以完全洗脱下来。收集咪唑300mM时洗脱下的样品进行SDS-PAGE分析目的条带。
实施例3:醇脱氢酶突变体的底物谱活力分析
以实施例2得到的醇脱氢酶突变体为催化剂,以潜手性羟基化合物为底物,展示各突变体对于氧化潜手性羟基化合物的能力:所考察的潜手性羟基化合物包括N-叔丁氧羰基-3-羟基吡咯烷(N-Boc-3-hydroxypyrrolidine),rac-N-叔丁氧羰基-3-羟基哌啶(rac-N-Boc-3-hydroxypiperi-dine,rac-NBHP),N-叔丁氧羰基-4-羟基哌啶(N-Boc-4-hydrox-ypiperidine),1-苯基-乙醇(1-Phenyl-ethanol),4-氯苯乙醇(4'-Chlorophenethyl-ethanol),(4-氯苯基)-吡啶-2-基-甲醇((4-Chloro-phenyl)-pyridin-2-y1-methanol),3-羟基哌啶(3-Pipe-ridinol)。
表1醇脱氢酶突变体的底物谱活力
由表1可知,与WT相比,M8对rac-N-叔丁氧羰基-3-羟基哌啶(rac-NBHP)有最高活力。
实施例4:醇脱氢酶突变体氧化rac-NBHP制备NBPO
10mL的生物催化体系:100mM磷酸钾缓冲液(pH 7.0),加入实例2获得的醇脱氢酶突变体M8和野生KpADH 10g/L,rac-NBHP 10mM,20mM和50mM。在30℃和200rpm条件下反应,恒定pH7.5。
反应过程中的转化率分析结果见表2和表3,野生型脱氢酶能够在较低的底物浓度下获得较高的转化率,但是其转化能力有限。当rac-NBHP浓度为20mM时,野生型KpADH和突变体M8仅需6h就能达到99.9%的转化率,而野生型需要反应12h分别需要12h和24h以达到接近99.9%的转化率。
表2野生型醇脱氢酶KpADH氧化rac-NBHP
表3醇脱氢酶突变体M8氧化rac-NBHP
由此可知,醇脱氢酶突变体酶M8在rac-NBHP的转化方面具有非常好的性能。
对比例1
将氨基酸序列如SEQ ID NO.1所示的醇脱氢酶的127位突变为亮氨酸、128位突变为苏氨酸、131位突变为组氨酸、165位突变为酪氨酸、苯丙氨酸、脯氨酸,结果显示对底物的催化比活力小于野生菌。
将氨基酸序列如SEQ ID NO.1所示的醇脱氢酶的131位突变为组氨酸、161位突变为色氨酸、165位突变为丝氨酸,结果显示对底物的催化比活力提高不多。
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> 一种能够催化合成哌啶类化合物的醇脱氢酶KpADH突变体
<130> BAA210207A
<160> 2
<170> PatentIn version 3.3
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<213> Kluyveromyces polyspora
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atgagcgtat taattagtgg tgcttctgga tacattgcca aacatatcgt cagagttctt 60
ttggaacaaa attacaaagt aattggtact gttagaagtc aagacaaagc tgataagtta 120
ttgaaacaat ataataatcc taatttgtct tatgaaattg tacctgaaat agcaaactta 180
gatgcttttg atgacatttt taagaaacat ggtaaggaaa taaaatatgt cattcatgca 240
gcttcaccag tgaacttcgg cgcaaaagat ttggaaaaag atttagttat tcctgccatt 300
aatggtacca agaatatgtt cgaagctatt aaaaagtatg ccccagatac tgtcgaacgt 360
gttgtaatga ctgcttctta tgcttcaatt atgaccccac atagacaaaa tgatccaact 420
ttaactttag atgaagaaac ttggaatcca gtaactgaag aaaatgctta tgaaaatgtc 480
ttcactgctt attgtgcttc aaaaactttt gctgaaaagg aagcttggaa gttcgttaaa 540
gaaaatagtg atgctgttaa gtttaaacta accactatcc acccatcctt cgttttcgga 600
cctcagaact ttgatgaaga cgtcactaag aaactaaatg aaacttgtga aattatcaat 660
ggtttattac atgctccatt tgacaccaaa gtcgaaaaga ctcacttcag tcaattcatt 720
gatgttcgtg atgtcgccaa aactcacgtt ttgggtttcc aaaaagatga attaatcaac 780
caaagattgt tattatgtaa cggcgccttc tctcaacaag atattgttaa tgtatttaat 840
gaagatttcc cagagttaaa aggccaattc ccaccagaag ataaggacac cgatttaaac 900
aaaggtgtaa caggttgtaa aattgataat gaaaagacta aaaaattatt agcatttgaa 960
tttactcctt tccataaaac aattcatgac actgtctatc aa 1002
Claims (10)
1.醇脱氢酶突变体,其特征在于,所述醇脱氢酶突变体是以氨基酸序列如SEQ ID NO.1所示的醇脱氢酶为亲本,将亲本第128位、131位、161位、165位、196位一个或多个位点突变。
2.根据权利要求1所述的醇脱氢酶突变体,其特征在于,将第128位的丙氨酸突变为半胱氨酸或缬氨酸,将第161位的苯丙氨酸突变成色氨酸,将第196位的丝氨酸突变成甘氨酸。
3.编码权利要求1或2所述醇脱氢酶突变体的基因。
4.携带权利要求3所述基因的重组质粒。
5.表达权利要求1或2所述突变体,或携带权利要求4所述重组质粒的微生物细胞。
6.一种生产哌啶酮类化合物的方法,其特征在于,以哌啶类化合物为底物,以权利要求1或2所述突变体为催化剂。
7.根据权利要求6所述的方法,其特征在于,所述哌啶类化合物的浓度为10-60mM。
8.根据权利要求6所述的方法,其特征在于,所述哌啶类化合物包括rac-N-叔丁氧羰基-3-羟基哌啶、N-叔丁氧羰基-4-羟基哌啶、3-羟基哌啶。
9.根据权利要求8所述的方法,其特征在于,在pH7.0-8.0下反应。
10.权利要求1或2所述突变体,或权利要求3所述基因,或权利要求4或5所述重组质粒,或权利要求6所述微生物细胞在生产手性醇类化合物和哌啶酮类化合物中的应用。
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