CN114085820A - 来源于Candida viswanathii的酮基泛解酸内酯还原酶 - Google Patents
来源于Candida viswanathii的酮基泛解酸内酯还原酶 Download PDFInfo
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- CN114085820A CN114085820A CN202111408399.3A CN202111408399A CN114085820A CN 114085820 A CN114085820 A CN 114085820A CN 202111408399 A CN202111408399 A CN 202111408399A CN 114085820 A CN114085820 A CN 114085820A
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- Prior art keywords
- ketopantolactone
- reductase
- pantolactone
- reaction
- glucose dehydrogenase
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Abstract
本发明属于生物催化技术领域,具体涉及一种来源于Candida viswanathii的酮基泛解酸内酯还原酶。本发明首次提供了一种来源于Candida viswanathii的酮基泛解酸内酯还原酶,可用于不对称合成D‑泛酸钙的关键中间体D‑泛解酸内酯,底物转化率达到99%,产物光学纯度(e.e.)达到99%,D‑泛解酸内酯的时空得率达到520g L‑1d‑1,具有良好的应用前景。
Description
技术领域
本发明涉及生物催化技术领域,具体涉及一种来源于Candida viswanathii的酮基泛解酸内酯还原酶、其编码多核苷酸、重组载体、重组细胞,以及该酮基泛解酸内酯还原酶的制备方法和在制备D-泛解酸内酯中的应用。
背景技术
D-泛酸又称维生素B5,其主要的主要的商品化形式为D-泛酸钙。D-泛酸钙广泛应用于动物饲料、食品添加剂及医药原料药,其中动物饲料占比最大,达到75%,由饲料行业主导的下游需求较为刚性。
D-泛解酸内酯是合成D-泛酸钙的关键中间体。目前,工业生产中D-泛解酸内酯主要是由异丁醛-甲醛-氰化钠法首先合成DL-泛解酸内酯,然后由D-泛解酸内酯水解酶选择性水解其中的D-泛解酸内酯得到D-泛解酸,通过溶剂萃取分离D-泛解酸与L-泛解酸内酯,分离后的D-泛解酸通过内酯化转化为D-泛解酸内酯以供合成D-泛酸钙,而L-泛解酸内酯则经化学法消旋后重新参与水解。D-泛解酸内酯水解酶催化的微生物酶法拆分工艺成熟,但存在工艺路线繁琐、萃取溶剂用量大、酸碱用量高等问题。因此,开发工艺简单、环保高效的生物酶法不对称还原路线具有重要意义。
以DL-泛解酸内酯为底物,通过氧化还原酶法不对称合成D-泛解酸内酯涉及两步酶催化反应,第一步是由L-泛解酸内酯脱氢酶专一性地将底物中的L-泛解酸内酯氧化为酮基泛解酸内酯,第二步是由酮基泛解酸内酯还原酶将酮基泛解酸内酯对映选择性地还原为D-泛解酸内酯,从而实现L-泛解酸内酯的构型翻转。该路线以混旋的泛解酸内酯为底物,在温和的反应条件下,通过多酶一锅反应,绿色高效地实现D-泛解酸内酯的制备。
酮基泛解酸内酯还原酶是氧化还原酶法不对称合成D-泛解酸内酯工艺路线的关键酶,其催化活性和对映选择性对酶催化反应效率及产品的光学纯度具有重要影响。目前,公开报道的酮基泛解酸内酯还原酶及其基因资源数量较少,限制了其酶学性质研究、构效关系研究及其在高效不对称合成D-泛解酸内酯中的应用。Kataoka等于2003年报道了来自Candida parapsilosis IFO 0708的两种共轭聚酮还原酶CPR-C1和CPR-C2(AppliedMicrobiology and Biotechnology,2004,64:359–366),其在大肠杆菌中异源表达水平较低,重组细胞比活力仅0.251U/mg,导致其酶催化反应效率较低。2018年以来,仅公开报道了3个酮基泛解酸内酯还原酶用于D-泛解酸内酯的不对称合成,包括CorCPR(Journal ofBiotechnology 291,2019,26–34)、CduCPR(Enzyme and Microbial Technology 126,2019,77–85)以及CalCPR(CN110452861 B),其中,以CduCPR的催化反应效率最高,但其时空得率也仅为307.5g L-1d-1。
综上所述,在现有的酮基泛解酸内酯还原酶参与的氧化还原酶法不对称合成D-泛解酸内酯技术路线中,仍然存在关键酶酮基泛解酸内酯还原酶来源匮乏、催化活力不够高、不对称还原反应时空得率较低等问题,限制了其在工业规模的应用。
发明内容
本发明目的在于提供一种来源于维斯假丝酵母(Candida viswanathii)的酮基泛解酸内酯还原酶,旨在通过提供该新的酮基泛解酸内酯还原酶,提高对酮基泛解酸内酯的催化活性和时空得率。
为了实现上述发明目的,第一方面,本发明提供了一种来源于Candidaviswanathii的酮基泛解酸内酯还原酶,其氨基酸序列如SEQ ID NO:1所示。
第二方面,本发明提供了编码所述酮基泛解酸内酯还原酶的多核苷酸,其核苷酸序列如SEQ ID NO:2所示。
第三方面,本发明提供了一种重组载体,其包括编码所述酮基泛解酸内酯还原酶的多核苷酸。
在一些实施方案中,所述重组载体还包括编码葡萄糖脱氢酶的多核苷酸。
在一些优选实施方案中,所述葡萄糖脱氢酶来源于巨大芽孢杆菌(Bacillusmegaterium),其氨基酸序列如SEQ ID NO:3所示,编码所述葡萄糖脱氢酶的多核苷酸的核苷酸序列如SEQ ID NO:4所示。
第四方面,本发明提供了一种重组细胞,其包括上述的重组载体。
在一些实施方案中,所述重组细胞诱导产生本发明所述的来源于Candidaviswanathii的酮基泛解酸内酯还原酶。
在一些实施方案中,所述重组细胞还诱导产生葡萄糖脱氢酶。
在一些优选实施方案中,所述葡萄糖脱氢酶来源于Bacillus megaterium,其氨基酸序列如SEQ ID NO:3所示,编码所述葡萄糖脱氢酶的多核苷酸的核苷酸序列如SEQ IDNO:4所示。
第五方面,本发明提供了一种酮基泛解酸内酯还原酶的制备方法,包括:
对所述的重组细胞进行诱导培养,得到培养物;
从所述培养物中分离所述酮基泛解酸内酯还原酶。
在一些实施方案中,所述制备方法还包括从所述培养物中分离葡萄糖脱氢酶的步骤。
第六方面,本发明提供了本发明所述的酮基泛解酸内酯还原酶、本发明所述的多核苷酸、本发明所述的重组载体和/或本发明所述的重组细胞在制备D-泛解酸内酯中的应用。
第七方面,本发明提供了一种D-泛解酸内酯的制备方法,包括:以本发明所述的酮基泛解酸内酯还原酶、本发明所述的重组细胞和/或本发明所述制备方法制备得到的酮基泛解酸内酯还原酶作为催化剂,对酮基泛解酸内酯进行催化反应,得到D-泛解酸内酯。
在一些实施方案中,所述催化反应的反应pH为6-8。
在一些实施方案中,所述催化反应的反应温度为30℃-50℃。
在一些实施方案中,所述催化反应在磷酸盐缓冲液中进行。
在一些实施方案中,所述催化反应中,所述酮基泛解酸内酯的累积终浓度为1mM-400mM。
在一些实施方案中,所述催化反应中,所述催化剂与所述酮基泛解酸内酯的质量比为(0.01-0.1):1。
本发明首次提供了一种来源于Candida viswanathii的酮基泛解酸内酯还原酶,其对酮基泛解酸内酯具有优良的催化活性和对映选择性。本发明提供的酮基泛解酸内酯还原酶用于不对称合成D-泛酸钙的关键中间体D-泛解酸内酯,底物转化率达到99%,产物光学纯度(e.e.)达到99%,D-泛解酸内酯的时空得率达到520g L-1d-1。相对于目前工业上普遍采用的酶解拆分法而言,利用本发明酮基泛解酸内酯还原酶生产D-泛解酸内酯,不仅简化了生产工艺,还避免了大量有机溶剂萃取剂以及强酸碱的使用,是一种具有广泛应用前景的生物催化技术。
附图说明
图1为本发明酮基泛解酸内酯不对称合成D-泛解酸内酯的原理图,其中,CviCPR表示来源于Candida viswanathii的酮基泛解酸内酯还原酶;
图2为本发明实施例1中对基因组数据挖掘得到的候选酶的进化关系分析图,其中,CviCPR表示来源于Candida viswanathii的酮基泛解酸内酯还原酶;
图3为本发明实施例4中D-泛解酸内酯的气相色谱图;
图4为本发明实施例4中基因工程菌催化转化酮基泛解酸内酯生成D-泛解酸内酯的反应进程曲线图。
具体实施方式
本发明实施例提供了一种来源于Candida viswanathii的酮基泛解酸内酯还原酶,其氨基酸序列如SEQ ID NO:1所示。
本发明的发明人通过基因组数据挖掘手段,在美国国立生物技术信息中心(NCBI)数据库海量的基因数据中进行基于一级序列比对的高通量筛选,初步获得具有潜在催化性能的20个候选基因,然后结合实验验证筛选,进一步得到具有较优催化性能的来源于Candida viswanathii的酮基泛解酸内酯还原酶。该来源于Candida viswanathii的酮基泛解酸内酯还原酶对酮基泛解酸内酯具有优良的催化活性和对映选择性。在不对称合成D-泛解酸内酯时,底物转化率达到99%,产物光学纯度(e.e.)达到99%,D-泛解酸内酯的时空得率达到520g L-1d-1。
本发明实施例提供的来源于Candida viswanathii的酮基泛解酸内酯还原酶可以是天然、重组或合成的活性多肽。该活性多肽可以是天然纯化的产物、化学合成的产物,或是使用重组技术从原核宿主(例如大肠杆菌)或真核宿主(例如酵母、高等植物)中产生的产物。
在一些优选实施方案中,所述来源于Candida viswanathii的酮基泛解酸内酯还原酶采用如下方法获得:
(11)以目前公开报道的催化性能最优的来源于Candida dubliniensis CD36的酮基泛解酸内酯还原酶CduCPR的序列为探针,在美国国立生物技术信息中心(NCBI)数据库中进行BLAST(Basic Local Alignment Search Tool),基于一级序列比对选取具有潜在酮基泛解酸内酯还原活性的新酶资源(通常认为氨基酸序列一致性超过30%即具备与探针相似催化活性的潜力);
(12)对步骤(11)所获得的新酶资源进行序列分析,筛选具有特征指纹序列(GXGTX)的候选酶;
(13)对步骤(12)所获得的候选酶进行进化关系分析,筛选与探针进化关系较近的候选酶做进一步功能筛选(以酶蛋白的氨基酸序列作为反映进化关系的指标,选取与探针氨基酸序列一致性大于50%的酶作为与探针进化关系较近的候选酶);
(14)根据与探针进化关系较近的候选酶的基因序列设计引物,以候选酶所在野生菌的基因组为模板,进行基因克隆,获取编码候选酶的基因片段;
(15)通过基因工程技术,将步骤(14)所得候选酶的基因片段在大肠杆菌工程菌中异源表达;
(16)将所述工程菌涂布于平板,挑选单克隆,送测序,选择其中的阳性克隆转接试管培养;
(17)将获取的菌液离心得到湿菌体,加入10mL磷酸钾缓冲液(100mM,pH 6.5)和10%(w/v)的底物酮基泛解酸内酯,在30℃、200rpm条件下反应12h。加入乙酸乙酯萃取反应液,取样分析酮基泛解酸内酯的转化率及D-泛解酸内酯的光学纯度。
(18)当D-泛解酸内酯的光学纯度为99%以上,且酮基泛解酸内酯的转化率高于使用所述探针时,所述候选酶为目标酶。
其中,步骤(18)中,以D-泛解酸内酯的光学纯度作为第一指标,且需在99%以上,这是因为产物光学纯度偏低将会对终产品D-泛酸钙的纯化产生不利影响;以酮基泛解酸内酯的转化率(%)作为第二指标,相同条件下,当候选酶对酮基泛解酸内酯的转化率越高,意味着该候选酶的催化活性越好。
来源于Candida viswanathii的酮基泛解酸内酯还原酶的氨基酸序列(SEQ IDNO:1,CviCPR):
MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYLIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEAFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL
相应地,编码该来源于Candida viswanathii的酮基泛解酸内酯还原酶的多核苷酸的核苷酸序列如SEQ ID NO:2所示。
本发明实施例提供的所述多核苷酸通常可以通过PCR扩增或人工合成的方法获得。
编码来源于Candida viswanathii的酮基泛解酸内酯还原酶的多核苷酸的核苷酸序列(SEQ ID NO:2):
atgccttcacaaactcaccctgtcaaattaaccagagaatttaagaccaagtcgggcgcagacgtctcaattgctaccggaacaggcaccaagtggaaaaaagactccaaggacgacgacatcaaccaggaattggtcgaccagatcttgcttgcgctcaagtctgggttcagacacattgacaccgctgaagtgtacaacacgcaggctgaagtcggcgaggccgtcaagcaactgggcatcccaagagaagacctctggatcaccaccaagtacaacccgggctggagaaccatcaagtcgagcagcgccagtccaaatgcgtctattgacaaggcattgcagcagttgggtaccgattacattgacttgtacttgatccaccagcctttcttcacggaggaaagcacccatgggttctcgttggaagacacctggaagatcttgattgactacaagaagcagggcaagatcagagaaattggcgtttctaacttcgctgaggaacacatcgagagattgaagaagatcctggacccagagttctaccccgtggtcaaccagatcgagagccatccattccttcaagaccagtccaagggcatcaccgcatactcgcaagccaacggcatcttagtcgaagcattctcgccattgacgcctgtttcaagagtcgacaagaacgcattgactggttacttggaggaattgtccaagaagtacaacaagaccggtggccagatcttgctcagatggacgttgcagagaggtgtgttgccaattactacctcggccaaagaagagagaatcaaagaggcattggatgtttttgactttgagttgaccaaagaggagtttgacaagatcaccgaaatcggtcaggccaacccacaccgtgtcttcttccatgaggagttcaaggatttgtaa
本发明实施例还提供了一种重组载体,其包括编码所述酮基泛解酸内酯还原酶的多核苷酸。具体地,所述重组载体包括克隆载体和表达载体,所述克隆载体用于复制相关序列,所述表达载体用于表达相关基因。
在一些实施方案中,所述重组载体为包括编码所述酮基泛解酸内酯还原酶的多核苷酸的pACYCDuet-1。
在一些优选实施方案中,所述重组载体还包括编码葡萄糖脱氢酶的多核苷酸。其优势是所得重组载体可同时表达所述酮基泛解酸内酯还原酶和所述葡萄糖脱氢酶。而且,在酮基泛解酸内酯还原酶催化酮基泛解酸的不对称还原制备D-泛解酸内酯的过程中,所述葡萄糖脱氢酶催化NADP+还原为NADPH,NADPH为酮基泛解酸内酯还原酶催化的不对称还原反应提供还原力,从而避免了外源添加昂贵的辅因子,有效降低生产成本。更优选地,所述葡萄糖脱氢酶来源于Bacillus megaterium,其氨基酸序列如SEQ ID NO:3所示,编码所述葡萄糖脱氢酶的多核苷酸的核苷酸序列如SEQ ID NO:4所示。
来源于Bacillus megaterium的葡萄糖脱氢酶的氨基酸序列(SEQ ID NO:3,BmGDH):
MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDVMINNAGLENPVSSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTQYPSFQAGRG
编码来源于Bacillus megaterium的葡萄糖脱氢酶的核苷酸序列(SEQ ID NO:4):
atgtataaagatttagaaggaaaagtagtggtcataacaggttcatctacaggtttgggaaaatcaatggcgattcgttttgcgacagaaaaagctaaagtagttgtgaattatcgttctaaggaagacgaagctaacagcgttttagaagaaattaaaaaagttggcggagaggctattgccgttaaaggtgacgtaacagttgagtctgatgtaatcaatttagttcaatctgcaattaaagaatttggaaagttagacgtcatgattaataacgcaggactagaaaatccggtttcatctcatgaaatgtctttaagcgattggaataaagtaattgatacgaacttaacgggagctttcttaggtagccgtgaagcgattaaatattttgttgaaaatgatattaagggaacagttattaacatgtcgagtgttcacgagaaaattccttggccattatttgttcattatgcagcaagtaaaggcggtatgaagcttatgactgaaacactggcattagaatacgctccaaaaggtattcgtgtaaataacattggaccgggagcgattaatacaccgattaacgctgagaaatttgctgatcctgagcagcgtgcagatgtagaaagcatgattccaatgggatacatcggagagccggaagaaattgcagcagttgctgcatggctagcttcttcagaggcgagttatgtaacaggaattacgctctttgctgacggcggtatgacacagtacccatcattccaagcaggacgcggataa
在一些优选实施方案中,所述重组载体的构建方法如下:
以pACYCDuet-1为载体质粒,将来源于Candida viswanathii的酮基泛解酸内酯还原酶CviCPR基因插入第一组多克隆位点的NcoI和NotI酶切位点之间,将来源于Bacillusmegaterium的葡萄糖脱氢酶BmGDH基因插入第二组多克隆位点的BglII与PacI酶切位点之间,得到重组载体pACYCDuet-1-CviCPR-BmGDH。
本发明实施例还提供了一种重组细胞,其包括上述的重组载体。
在一些优选实施方案中,所述重组细胞诱导产生本发明所述的来源于Candidaviswanathii的酮基泛解酸内酯还原酶。进一步地,所述重组细胞还诱导产生葡萄糖脱氢酶。更进一步地,所述葡萄糖脱氢酶来源于Bacillus megaterium,其氨基酸序列如SEQ IDNO:3所示,编码所述葡萄糖脱氢酶的多核苷酸的核苷酸序列如SEQ ID NO:4所示。
在一些优选实施方案中,所述重组细胞的构建方法如下:
将所述重组载体转化到宿主细胞中,经诱导,得到表达来源于Candidaviswanathii的酮基泛解酸内酯还原酶的重组细胞。
进一步地,所述重组载体为重组载体pACYCDuet-1-CviCPR-BmGDH,所述宿主细胞为原核生物细胞或真核生物细胞,例如大肠杆菌、酵母等,优选基因工程菌大肠杆菌E.coliBL21(DE3)。将所述重组载体pACYCDuet-1-CviCPR-BmGDH转化到E.coli BL21(DE3)中,经诱导,得到同时表达来源于Candida viswanathii的酮基泛解酸内酯还原酶和来源于Bacillus megaterium的葡萄糖脱氢酶的工程菌(重组细胞)。
本发明实施例还提供了一种酮基泛解酸内酯还原酶的制备方法,包括:
(21)对所述的重组细胞进行诱导培养,得到培养物;
(22)从所述培养物中分离所述酮基泛解酸内酯还原酶。
在一些优选实施方案中,所述步骤(22)还包括从所述培养物中分离葡萄糖脱氢酶。其中,对重组细胞进行诱导培养的方法、从培养物中分离酮基泛解酸内酯还原酶的方法,以及从所述培养物中分离葡萄糖脱氢酶的方法均为本领域的常规方法。
本发明实施例提供的来源于Candida viswanathii的酮基泛解酸内酯还原酶、本发明所述的多核苷酸、本发明所述的重组载体和/或本发明所述的重组细胞可以用于制备D-泛解酸内酯。具体地,上述来源于Candida viswanathii的酮基泛解酸内酯还原酶、本发明所述的多核苷酸、本发明所述的重组载体和/或本发明所述的重组细胞用于催化D-泛解酸内酯的不对称合成。
本发明实施例还提供了一种D-泛解酸内酯的制备方法,包括:以本发明所述来源于Candida viswanathii的酮基泛解酸内酯还原酶、本发明所述的重组细胞和/或本发明所述制备方法制备得到的酮基泛解酸内酯还原酶作为催化剂,对酮基泛解酸内酯进行催化反应,得到D-泛解酸内酯。其中,所述D-泛解酸内酯的制备方法涉及的反应原理如图1所示,酮基泛解酸内酯为底物,葡萄糖为辅底物。
在一些优选实施方案中,所述D-泛解酸内酯的制备方法如下:
将所述表达来源于Candida viswanathii的酮基泛解酸内酯还原酶和来源于Bacillus megaterium的葡萄糖脱氢酶的重组细胞与底物酮基泛解酸内酯接触,进行不对称还原反应,然后从反应产物中收集所生成的D-泛解酸内酯。
底物上载量(即反应体系中的底物浓度)是表征酶催化反应的关键绩效指标。在其它条件一致的情况下,更高的底物上载量意味着酶催化反应更为高效,有利于获得更高的产物滴度(即产物浓度)与更高的时空得率。然而,过高的底物浓度则会导致尚未完全转化的酮基泛解酸内酯的积累,其自发水解既造成了原料浪费,也会对产品纯度产生不利影响。因此,在一些优选实施方案中,底物酮基泛解酸内酯的累积终浓度为1~400mM。
在一些优选实施方案中,葡萄糖作为反应辅底物,起到再生辅因子NADPH并推动主反应正向移动的作用,其添加量为底物酮基泛解酸内酯的1.1~1.5倍当量,即440~600mM,优选440mM。该添加量可以在保证反应效果的同时,产生较少的副产物葡萄糖酸钠,为后续分离提纯减轻负担。
在一些优选实施方案中,基于底物酮基泛解酸内酯质量的催化剂的使用量为0.01~0.1克细胞/克酮基泛解酸内酯。
在一些优选实施方案中,所述反应在磷酸盐缓冲液中进行。
在一些优选实施方案中,反应温度为30~50℃。
在一些优选实施方案中,反应体系的pH为6.0~8.0,可通过滴加碱液(例如碳酸钠)调控pH。
在一些优选实施方案中,所述催化剂包括下列形式中的任一种:
(i)将表达来源于Candida viswanathii的酮基泛解酸内酯还原酶和来源于Bacillus megaterium的葡萄糖脱氢酶的重组工程菌,通过离心或过滤收集到的湿菌体;
(ii)采用研磨或匀浆等方法将湿菌体破碎,再用水或缓冲液抽提所得到的无细胞抽提液;
(iii)采用适当的方法,包括但不限于亲和层析等,处理上述无细胞抽提液得到的纯酶;
(iv)采用适当的方法,包括但不限于载体吸附、戊二醛交联或凝胶包埋等,处理含有上述活性酶组分的物料(即,所述重组工程菌、所述湿菌体、所述无细胞抽提液和/或所述纯酶)所得到的固定化细胞或固定化酶。
底物酮基泛解酸内酯极易水解,为了减少底物在催化过程中的自发水解,底物和辅底物的添加优选采用流加模式,从而不断提升反应体系中的酮基泛解酸内酯累积浓度,同时避免高浓度酮基泛解酸内酯累积导致的自发水解及其对催化剂活性的抑制作用。
为使本发明上述实施细节和操作能清楚地被本领域技术人员理解,以及本发明实施例酮基泛解酸内酯还原酶及其制备方法和应用的进步性能显著的体现,以下通过实施例来举例说明上述技术方案。
下面实施例中未注明具体条件的实验方法,通常按照分子生物学领域常规的实验方法进行,包括但不限于M.R.格林所著《分子克隆实验指南》[Molecular Cloning:ALaboratory Manual]、Robert·F·Weaver所著《分子生物学》[Molecular Biology]等所述的实验方法,或按照试剂盒及仪器设备厂商所建议的实验方法。实施例中使用的试剂和生物材料如无特殊说明均可从商业途径获得。
实施例1酮基泛解酸内酯还原酶的筛选
目前公开报道能够催化酮基泛解酸内酯不对称还原生成D-泛解酸内酯的催化性能最优的还原酶是来源于Candida dubliniensis CD36的CduCPR,以其GenBank登录号CCG25060.1在美国国立生物技术信息中心(NCBI)数据库中检索获得该酶的氨基酸序列。以如上获得的CduCPR的氨基酸序列为探针,在NCBI数据库中进行BLAST(Basic LocalAlignment Search Tool),基于一级序列比对选取具有潜在酮基泛解酸内酯还原活性的新酶资源。使用BioEdit对如上获得的新酶资源进行序列分析,筛选具有D-泛解酸内酯还原酶特征指纹序列(GXGTX)的候选酶。使用MEGA对如上获得的新酶进行进化关系分析,筛选与探针CduCPR进化关系较近的候选酶(图2)。根据候选酶的基因序列设计PCR引物,以候选酶所在野生菌的基因组为模板,进行基因克隆,获取编码候选酶的基因片段。通过基因工程技术,将候选酶的基因片段在大肠杆菌工程菌中异源表达。将工程菌涂布于平板,挑选单克隆,经测序,选择其中的阳性克隆转接试管培养。将获取的菌液离心得到湿菌体,加入10mL磷酸钾缓冲液(100mM,pH 6.5)和10%(w/v)的底物酮基泛解酸内酯,在30℃、200rpm条件下反应12h。加入乙酸乙酯萃取反应液,取样分析酮基泛解酸内酯的转化率及D-泛解酸内酯的光学纯度,结果见表1。
表1酮基泛解酸内酯还原酶功能筛选结果
酶 | 来源 | 转化率(%) | e.e.(%) | 产物构型 |
CviCPR | Candida viswanathii | >99 | >99 | R |
CmaCPR | Candida maltosa Xu316 | >99 | >99 | R |
CduCPR | Candida dubliniensis CD36 | 60 | 99 | R |
CorCPR | Candida orthopsilosis Co 90-125 | 58 | 99 | R |
SpaCPR-1 | Spathaspora passalidarum NRRL Y-27907 | 57 | 78 | R |
SstCPR | Scheffersomyces stipitis CBS 6054 | 50 | 89 | R |
MafCPR | Metschnikowia aff.pulcherrima | 48 | 88 | S |
HbuCPR-1 | Hyphopichia burtonii NRRL Y-1933 | 47 | 98 | R |
SpaCPR-2 | Spathaspora passalidarum NRRL Y-27907 | 42 | 99 | R |
SliCPR | Sugiyamaella lignohabitans | 40 | 57 | R |
WciCPR-1 | Wickerhamomyces ciferrii | 37 | 87 | R |
WciCPR-2 | Wickerhamomyces ciferrii | 37 | 78 | R |
HbuCPR-2 | Hyphopichia burtonii NRRL Y-1933 | 35 | 88 | S |
HguCPR | Hanseniaspora guilliermondii | 31 | 90 | R |
AseCPR | Aspergillus sergii | 29 | 74 | R |
TmaCPR | Talaromyces marneffei ATCC 18224 | 29 | 65 | R |
CjaCPR | Cyberlindnera jadinii NRRL Y-1542 | 22 | 90 | S |
MbiCPR | Metschnikowia bicuspidata | 22 | 95 | S |
TtoCPR | Trichophyton tonsurans CBS 112818 | 18 | 95 | S |
CpoCPR | Coccidioides posadasii str.Silveira | 15 | 96 | S |
TruCPR | Trichophyton rubrum CBS 118892 | 10 | 61 | R |
通过表1可以看出,来源于Candida viswanathii的酮基泛解酸内酯还原酶CviCPR能够高效且立体专一性地不对称还原底物酮基泛解酸内酯生成D-泛解酸内酯,其催化反应的转化率及光学纯度显著优于探针CduCPR。
实施例2酮基泛解酸内酯还原酶CviCPR重组质粒的构建
培养Candida viswanathii野生菌,通过基因组抽提试剂盒获得其基因组。以Candida viswanathii基因组为聚合酶链式反应(PCR)的模板,设计引物(见表2),通过基因克隆获得编码酮基泛解酸内酯还原酶CviCPR的基因。以同样方法克隆得到来源于Bacillusmegaterium的葡萄糖脱氢酶BmGDH。
表2酮基泛解酸内酯还原酶及葡萄糖脱氢酶的基因克隆引物
引物名称 | 序列编号 | 引物序列(5’→3’) |
CviCPR-FP | SEQ ID NO:5 | atgccttcacaaactcaccctgtcaaatta |
CviCPR-RP | SEQ ID NO:6 | ttacaaatccttgaactcctcatggaagaa |
BmGDH-FP | SEQ ID NO:7 | atgtataaagatttagaaggaaaagtagtg |
BmGDH-RP | SEQ ID NO:8 | ttatccgcgtcctgcttggaatgatgggta |
以pACYCDuet-1为载体质粒,将如上获得的来源于Candida viswanathii的酮基泛解酸内酯还原酶CviCPR基因插入第一组多克隆位点的NcoI和NotI酶切位点之间,将如上获取的来源于Bacillus megaterium的葡萄糖脱氢酶BmGDH基因插入第二组多克隆位点的BglII于PacI酶切位点之间,得到共表达酮基泛解酸内酯还原酶CviCPR和葡萄糖脱氢酶BmGDH的重组质粒pACYCDuet-1-CviCPR-BmGDH。
实施例3酮基泛解酸内酯还原酶CviCPR异源表达工程菌的构建
以大肠杆菌E.coli BL21(DE3)为表达宿主,采用化学法将获取的重组表达质粒pACYCDuet-1-CviCPR-BmGDH转化到大肠杆菌E.coli BL21(DE3)中,经诱导,得到同时表达来源于Candida viswanathii的酮基泛解酸内酯还原酶和来源于Bacillus megaterium的葡萄糖脱氢酶的工程菌。
实施例4基因工程菌E.coli催化酮基泛解酸内酯合成D-泛解酸内酯
将获得的共表达酮基泛解酸内酯还原酶CviCPR和葡萄糖脱氢酶BmGDH的基因工程菌E.coli加入到磷酸钾缓冲液(100mM,pH6.5)中,使湿细胞浓度达5.1g/L,通过连续流加含有底物酮基泛解酸内酯和葡萄糖的缓冲液进行反应,反应温度为30℃,200rpm,通过在线监测pH反馈调节Na2CO3的滴加,控制pH在6.5左右。反应过程中间隔取样,使用乙酸乙酯萃取反应液,经稀释,气相分析底物酮基泛解酸内酯的浓度及产物D-泛解酸内酯的光学纯度,据此得到的反应进程曲线(图4)。
底物酮基泛解酸内酯与产物D-泛解酸内酯,及其同分异构体L-泛解酸内酯的气相色谱(SHIMADZU GC2030)分析检测方法如下:色谱柱为BGB-174(30m×250μm×0.25μm),FID检测器(250℃),氮气作载气(30mL/min),氢气40mL/min,进样量为1μL,分流比为30:1,进样口250℃,柱温为175℃维持8min。在该分析条件下,酮基泛解酸内酯、D-泛解酸内酯,L-泛解酸内酯的保留时间分别为6.397min,5.983min,6.116min。检测结果如图3所示。
反应2.4h结束,底物酮基泛解酸内酯的累积加入浓度达到400mM。结合图3和图4可知,底物酮基泛解酸内酯保留时间6.397min位置没有检测到色谱峰,可认为底物酮基泛解酸内酯基本完全转化为D-泛解酸内酯,底物转化率≥99%,且D-泛解酸内酯的光学纯度(e.e.)达到99%,经计算,D-泛解酸内酯的时空得率为520g L-1d-1。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
序列表
<110> 万华化学集团股份有限公司
<120> 来源于Candida viswanathii的酮基泛解酸内酯还原酶
<130> DSP1F213647ZX
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 309
<212> PRT
<213> 维斯假丝酵母(Candida viswanathii)
<400> 1
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<213> 维斯假丝酵母(Candida viswanathii)
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atgccttcac aaactcaccc tgtcaaatta accagagaat ttaagaccaa gtcgggcgca 60
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atcccaagag aagacctctg gatcaccacc aagtacaacc cgggctggag aaccatcaag 300
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ggcatcaccg catactcgca agccaacggc atcttagtcg aagcattctc gccattgacg 660
cctgtttcaa gagtcgacaa gaacgcattg actggttact tggaggaatt gtccaagaag 720
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attactacct cggccaaaga agagagaatc aaagaggcat tggatgtttt tgactttgag 840
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<213> 巨大芽孢杆菌(Bacillus megaterium)
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50 55 60
Asp Val Thr Val Glu Ser Asp Val Ile Asn Leu Val Gln Ser Ala Ile
65 70 75 80
Lys Glu Phe Gly Lys Leu Asp Val Met Ile Asn Asn Ala Gly Leu Glu
85 90 95
Asn Pro Val Ser Ser His Glu Met Ser Leu Ser Asp Trp Asn Lys Val
100 105 110
Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Thr Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Glu Gln Arg Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Ala Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
<210> 4
<211> 786
<212> DNA
<213> 巨大芽孢杆菌(Bacillus megaterium)
<400> 4
atgtataaag atttagaagg aaaagtagtg gtcataacag gttcatctac aggtttggga 60
aaatcaatgg cgattcgttt tgcgacagaa aaagctaaag tagttgtgaa ttatcgttct 120
aaggaagacg aagctaacag cgttttagaa gaaattaaaa aagttggcgg agaggctatt 180
gccgttaaag gtgacgtaac agttgagtct gatgtaatca atttagttca atctgcaatt 240
aaagaatttg gaaagttaga cgtcatgatt aataacgcag gactagaaaa tccggtttca 300
tctcatgaaa tgtctttaag cgattggaat aaagtaattg atacgaactt aacgggagct 360
ttcttaggta gccgtgaagc gattaaatat tttgttgaaa atgatattaa gggaacagtt 420
attaacatgt cgagtgttca cgagaaaatt ccttggccat tatttgttca ttatgcagca 480
agtaaaggcg gtatgaagct tatgactgaa acactggcat tagaatacgc tccaaaaggt 540
attcgtgtaa ataacattgg accgggagcg attaatacac cgattaacgc tgagaaattt 600
gctgatcctg agcagcgtgc agatgtagaa agcatgattc caatgggata catcggagag 660
ccggaagaaa ttgcagcagt tgctgcatgg ctagcttctt cagaggcgag ttatgtaaca 720
ggaattacgc tctttgctga cggcggtatg acacagtacc catcattcca agcaggacgc 780
ggataa 786
<210> 5
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> CviCPR-FP
<400> 5
atgccttcac aaactcaccc tgtcaaatta 30
<210> 6
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> CviCPR-RP
<400> 6
ttacaaatcc ttgaactcct catggaagaa 30
<210> 7
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> BmGDH-FP
<400> 7
atgtataaag atttagaagg aaaagtagtg 30
<210> 8
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> BmGDH-RP
<400> 8
ttatccgcgt cctgcttgga atgatgggta 30
Claims (10)
1.一种来源于Candida viswanathii的酮基泛解酸内酯还原酶,其特征在于,所述酮基泛解酸内酯还原酶的氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述酮基泛解酸内酯还原酶的多核苷酸,其特征在于,所述多核苷酸的核苷酸序列如SEQ ID NO:2所示。
3.一种重组载体,其特征在于,包括权利要求2所述的多核苷酸;优选地,所述重组载体还包括编码葡萄糖脱氢酶的多核苷酸;更优选地,所述葡萄糖脱氢酶来源于Bacillusmegaterium,其氨基酸序列如SEQ ID NO:3所示,编码所述葡萄糖脱氢酶的多核苷酸的核苷酸序列如SEQ ID NO:4所示。
4.一种重组细胞,其特征在于,所述重组细胞包括权利要求3所述的重组载体;优选地,所述重组细胞诱导产生权利要求1所述的酮基泛解酸内酯还原酶;更优选地,所述重组细胞还诱导产生葡萄糖脱氢酶。
5.一种酮基泛解酸内酯还原酶的制备方法,其特征在于,包括:
对权利要求4所述的重组细胞进行诱导培养,得到培养物;
从所述培养物中分离所述酮基泛解酸内酯还原酶。
6.根据权利要求5所述的制备方法,其特征在于,所述制备方法还包括从所述培养物中分离葡萄糖脱氢酶的步骤。
7.权利要求1所述的酮基泛解酸内酯还原酶、权利要求2所述的多核苷酸、权利要求3所述的重组载体和/或权利要求4所述的重组细胞在制备D-泛解酸内酯中的应用。
8.一种D-泛解酸内酯的制备方法,其特征在于,包括:以权利要求1所述的酮基泛解酸内酯还原酶、权利要求4所述的重组细胞和/或权利要求5-6所述制备方法制备得到的酮基泛解酸内酯还原酶作为催化剂,对酮基泛解酸内酯进行催化反应,得到D-泛解酸内酯。
9.根据权利要求8所述的制备方法,其特征在于,所述催化反应的反应pH为6-8;和/或
所述催化反应的反应温度为30℃-50℃。
10.根据权利要求8或9所述的制备方法,其特征在于,所述催化反应在磷酸盐缓冲液中进行;和/或
所述催化反应中,所述酮基泛解酸内酯的累积终浓度为1mM-400mM;和/或
所述催化反应中,所述催化剂与所述酮基泛解酸内酯的质量比为(0.01-0.1):1。
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