CN113481179A - 一种脱卤酶和制备方法及在制备阿托伐他汀中间体中的应用 - Google Patents
一种脱卤酶和制备方法及在制备阿托伐他汀中间体中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及生物制药技术领域,具体领域为一种制备阿托伐他汀中间体的方法。
背景技术
阿托伐他汀钙为羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂(结构式如下)。
阿托伐他汀钙主要通过抑制HMGCoA还原酶的合成,从而抑制体内胆固醇的合成,降低血清低密度脂蛋白胆固醇、甘油三酯的含量。由于阿托伐他汀钙抑制了细胞合成胆固醇,干扰了脂蛋白的生成,使血清总胆固醇水平下降,能有效降低血清甘油三酯水平,还能升高血清高密度脂蛋白胆固醇水平。能降低某些纯合子家族性高胆固醇血症患者的血浆低密度脂蛋白胆固醇水平,而这一类型的人群,对其他降脂药治疗很少有应答。临床用于原发性高胆固醇血症、混合型高脂血症、高甘油三酯血症、纯合子家族性高胆固醇血症及防治动脉粥样硬化。他汀类药物是20世纪80年代后期开发的羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂类降血脂药。
1978年,默沙东在土曲霉中分离得到具有降血脂作用的HMG-CoA还原酶抑制剂洛伐他汀。Parke-Davis公司(Warner-Lambert的分公司,后被Pfizer公司收购)在默沙东的前期研究成果的基础上,设计合成了2-苯基吡咯衍生物,于1997年开发了第五个上市的他汀类药物——阿托伐他汀。
该制剂于1996年在美国获批,并于1999年进入中国市场,商品名为立普妥,规格为10mg、20mg和40mg。
原料药全球年销售量超过600吨,预计2020年全年需求量超过700吨。阿托伐他汀原料药有着巨大的需求,其重要中间体化合物I的制备主要通过大肠杆菌进行制备:US2006009970提供了一种脱卤上氰基的酶突变体的制备方法,并通过大肠杆菌进行表达,获得了高效催化效果的脱卤酶,可用于制备化合物但是在该化合物I制备的反应中,酶最适温度相对较高,同时该反应中需要使用NaCN,该反应对人员操作要求较高。
因此,开发一种稳定性更强的脱卤酶,寻找一种绿色环保、经济化的制备方法成为了本领域亟需解决的技术难题之一。
发明内容
本发明的目的在于提供一种脱卤酶和制备方法及在制备阿托伐他汀中间体中的应用,利用生物催化进行脱卤、上-CN基团。
为实现上述目的,本发明提供如下技术方案:
一种脱卤酶,其核苷酸序列如SEQ ID No:2所示。
其中,所述脱卤酶的表达细胞为枯草芽孢杆菌。
进一步的,所述脱卤酶的表达细胞优选为Bacillus subtilis WB800N。
其中,所述脱卤酶的外源表达载体为pHT01。
其中,所述外源表达载体pHT01的外源序列如SEQ ID No:1所示,包括脱卤酶序列和优化后的Shine-Dalgarno序列。
其中,所述脱卤酶序列如SEQ ID No:2所示;所述优化后的Shine-Dalgarno序列如SEQ ID No:3所示。
本发明所述脱卤酶的制备方法:将核苷酸序列如SEQ ID No:1所示的基因经过DNA序列合成后,进行PCR扩增,然后导入脱卤酶表达载体pHT01的BamHI和ZraI酶切位点获得重组表达载体,编号Re_vector001;
将重组表达载体Re_vector001电转入脱卤酶的表达细胞中,得到表达工程菌,经过抗生素抗性平板涂布筛选后,获得克隆菌株,经检验重组成功后,对获得的菌株进行活化后发酵培养,离心收集菌体,洗涤后获得Bacillus subtilis WB800N细胞,超声波破碎,纯化粗酶液冻干获得脱卤酶。
本发明所述脱卤酶在生物催化制备阿托伐他汀中间体中的应用,具体为:
以化合物II为底物,在脱卤酶和缓冲液的存在下,进行生物催化反应生成阿托伐他汀中间体,即化合物I;合成路线如下:
其中,所述化合物II与所述脱卤酶的质量比为1:0.01~1。
其中,所述缓冲液为PBS缓冲液,其浓度为0.2mM,pH为6.0~8.5。
与现有技术相比,本发明的有益效果是:
本发明的脱卤酶稳定性强,催化效率高,达到了经济化生产标准,且常温反应即可,操作简单便捷,对环境友好。本发明中添加了优化的Shine-Dalgarno序列,外源基因的表达量大幅度增加。
与大肠杆菌表达细胞相比,本发明的产物中无细菌内毒素残留,且相对于大肠杆菌,枯草芽孢表达过程中极少产生对人体有害物质,因此本发明使用枯草芽孢杆菌生产作为医药中间体制备所需的生物催化剂,是一种安全、绿色环保且经济化的制备方法。
附图说明
图1为重组Re_vector001质粒的构建示意图。
图2为重组Re_vector002质粒的构建示意图。
图3为GC检测结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一、Bacillus subtilis WB800N细胞构建
将核苷酸序列如SEQ ID No:1所示的基因,经过DNA序列合成后,进行PCR扩增,引物如下(序列如SEQ ID No:4-5所示):
F:CGCGGATCCTAAGGAGGAAAAAAAAATG;
R:GACGTCAGGCAAATAGCCCCCCGT。
PCR扩增条件:98℃3min,95℃30s,57℃90s、72℃90s,35个循环;
PCR扩增体系:模板1.5μL,上下游引物各1.5μL,灭菌的双蒸水20.5μL,PrimerSTARMix 25μL;
扩增后,导入脱卤酶表达载体pHT01的BamHI和ZraI酶切位点,获得重组表达载体编号Re_vector001。
之后,将重组表达载体(如图1所示)转入脱卤酶表达细胞(Bacillus subtilisWB800N)中,得到表达工程菌,挑取阳性转化子并测序鉴定其核苷酸序列如SEQ ID No.1所示后,获得Bacillus subtilis WB800N全细胞菌株。
其中,脱卤酶表达载体pHT01购自湖南丰晖生物科技有限公司。Bacillussubtilis WB800N购自北京百奥莱博科技有限公司,货号BTN12-118y。
二、脱卤酶的制备
将获得的Bacillus subtilis WB800N全细胞菌株,接种到含有抗生素氨苄抗性的LB液体培养基中,于37℃过夜培养,得到种子培养液。将种子培养液按照1-2%比例接种至TB液体发酵培养基中。然后置于37℃下培养至OD600值为0.6~0.8,加入终浓度为0.5mol/L的IPTG,置于25℃继续培养16h后,12000rmp,5℃下离心收集菌体,采用pH值为7.0的0.2M的PBS缓冲液将收集后菌株进行洗涤并重悬,离心后收集,超声波破碎后获得粗酶液,纯化后冻干获得脱卤酶冻干粉1.02g。
实施例2不含有Shine-Dalgarno序列的外源表达基因载体的构建
将核苷酸序列如SEQ ID No:2所示的脱卤酶,经过DNA序列合成后,进行PCR扩增,引物如下(序列如SEQ ID No:6-7所示):
F:CGCGGATCC ATGCGCATCGCATTAGTAA;
R:GACGTCAGGCAAATAGCCCCCCGT。
PCR扩增条件:98℃3min,95℃30s,56℃90s、72℃90s,34个循环;
PCR扩增体系:模板1.5μL,上下游引物各1.5μL,灭菌的双蒸水20.5μL,PrimerSTARMix 25μL;扩增后,参照实施例1的方法构建重组质粒Re_vector002(如图2所示),并参照实施例1的方法进行脱卤酶制备,同等条件下,获得脱卤酶干粉0.665g。
通过实施例1与实施例2的对比可以看出,添加了优化的Shine-Dalgarno序列,外源基因的表达量大幅度增加。
实施例3化合物I的制备
于250ml三口瓶中投入30%氰化钠溶液29.4g,加入84mL pH7.0 0.2M PBS缓冲液,加入化合物II 20g,实施例1制备的脱卤酶冻干粉1g,30%硫酸调节pH控制在7.0左右,220rpm搅拌,室温过夜反应。TLC点板确定基本法反应完全,取样送检。反应结果经GC检测(如图3及下表所示),转化率约为93%,过柱纯化后产品纯度约为99.14%。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 江苏阿尔法药业有限公司
<120> 一种脱卤酶和制备方法及在制备阿托伐他汀中间体中的应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 751
<212> DNA
<213> 外源表达载体中的外源序列(Artificial Sequence)
<400> 1
taaggaggaa aaaaaaatgc gcatcgcatt agtaacccat gcaagacatt ttgccggacc 60
tgccgccgtt gaagctttaa ctcgtgacgg atatacggtt gtttgccacg atgcaagctt 120
tgccgatgcg gctgaaagac aaagatttga gtcggaaaac ccgggcacta tcgcactcgc 180
ggaacaaaaa ccggaacgcc ttgtggatgc gacgctgcaa tacggtgaag cgatcgacac 240
gatcgtatca aatgactata ttcctcggcc aatgaaccgg ttaccgattg aaggaacttc 300
agaagctgat atccggcaaa tgttcgaagc gctgtctatt tttcctattc tgcttctgca 360
gtcagcaatt gctccattac gcgccgcagg aggagcttct gtaattttta tcacgagcag 420
cgtcggaaaa aaacctcttg cgtacaatcc gctttatgga ccagcgagag cagctacagt 480
cgcattggta gaatctgcgg cgaaaacact gagccgtgat ggtatcttgt tatacgcgat 540
tggaccgaac ttttttaaca acccgacata ttttccaacg tccgattggg agaacgatcc 600
tgaattgaga gatcgcgtgg aaagagatgt gcctttaggc cgtttaggcc ggccggacga 660
aatgggagct ctgatcacgt tcttagcctc aagacgcgct gcaccgattg tgggccaatt 720
tttcgcgttt acggggggct atttgcctta g 751
<210> 2
<211> 735
<212> DNA
<213> 脱卤酶序列(Artificial Sequence)
<400> 2
atgcgcatcg cattagtaac ccatgcaaga cattttgccg gacctgccgc cgttgaagct 60
ttaactcgtg acggatatac ggttgtttgc cacgatgcaa gctttgccga tgcggctgaa 120
agacaaagat ttgagtcgga aaacccgggc actatcgcac tcgcggaaca aaaaccggaa 180
cgccttgtgg atgcgacgct gcaatacggt gaagcgatcg acacgatcgt atcaaatgac 240
tatattcctc ggccaatgaa ccggttaccg attgaaggaa cttcagaagc tgatatccgg 300
caaatgttcg aagcgctgtc tatttttcct attctgcttc tgcagtcagc aattgctcca 360
ttacgcgccg caggaggagc ttctgtaatt tttatcacga gcagcgtcgg aaaaaaacct 420
cttgcgtaca atccgcttta tggaccagcg agagcagcta cagtcgcatt ggtagaatct 480
gcggcgaaaa cactgagccg tgatggtatc ttgttatacg cgattggacc gaactttttt 540
aacaacccga catattttcc aacgtccgat tgggagaacg atcctgaatt gagagatcgc 600
gtggaaagag atgtgccttt aggccgttta ggccggccgg acgaaatggg agctctgatc 660
acgttcttag cctcaagacg cgctgcaccg attgtgggcc aatttttcgc gtttacgggg 720
ggctatttgc cttag 735
<210> 3
<211> 16
<212> DNA
<213> Shine-Dalgarno序列(Artificial Sequence)
<400> 3
taaggaggaa aaaaaa 16
<210> 4
<211> 28
<212> DNA
<213> 引物F(Artificial Sequence)
<400> 4
cgcggatcct aaggaggaaa aaaaaatg 28
<210> 5
<211> 24
<212> DNA
<213> 引物R(Artificial Sequence)
<400> 5
gacgtcaggc aaatagcccc ccgt 24
<210> 6
<211> 28
<212> DNA
<213> 引物F2(Artificial Sequence)
<400> 6
cgcggatcca tgcgcatcgc attagtaa 28
<210> 7
<211> 24
<212> DNA
<213> 引物R2(Artificial Sequence)
<400> 7
gacgtcaggc aaatagcccc ccgt 24
Claims (10)
1.一种脱卤酶,其特征在于:其核苷酸序列如SEQ ID No:2所示。
2.根据权利要求1所述的脱卤酶,其特征在于:所述脱卤酶的表达细胞为枯草芽孢杆菌。
3.根据权利要求2所述的脱卤酶,其特征在于:所述脱卤酶的表达细胞为Bacillussubtilis WB800N。
4.根据权利要求1所述的脱卤酶,其特征在于:所述脱卤酶的外源表达载体为pHT01。
5.根据权利要求4所述的脱卤酶,其特征在于:所述外源表达载体pHT01的外源序列如SEQ ID No:1所示,包括脱卤酶序列和优化后的Shine-Dalgarno序列。
6.根据权利要求5所述的脱卤酶,其特征在于:所述脱卤酶序列如SEQ ID No:2所示;所述优化后的Shine-Dalgarno序列如SEQ ID No:3所示。
7.权利要求1-6任一所述脱卤酶的制备方法,其特征在于:将核苷酸序列如SEQ ID No:1所示的基因经过DNA序列合成后,进行PCR扩增,然后导入脱卤酶表达载体pHT01的BamHI和ZraI酶切位点获得重组表达载体,编号Re_vector001;
将重组表达载体Re_vector001电转入脱卤酶的表达细胞中,得到表达工程菌,经过抗生素抗性平板涂布筛选后,获得克隆菌株,经检验重组成功后,对获得的菌株进行活化后发酵培养,离心收集菌体,洗涤后获得Bacillus subtilis WB800N细胞,超声波破碎,纯化粗酶液冻干获得脱卤酶。
9.根据权利要求8所述的脱卤酶在生物催化制备阿托伐他汀中间体中的应用,其特征在于:所述化合物II与所述脱卤酶的质量比为1:0.01~1。
10.根据权利要求8所述的脱卤酶在生物催化制备阿托伐他汀中间体中的应用,其特征在于:所述缓冲液为PBS缓冲液,其浓度为0.2mM,pH为6.0~8.5。
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