CN113278599A - 酿酒酵母羰基还原酶及其在制备光学活性烷基内酯中的应用 - Google Patents
酿酒酵母羰基还原酶及其在制备光学活性烷基内酯中的应用 Download PDFInfo
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- CN113278599A CN113278599A CN202110554333.9A CN202110554333A CN113278599A CN 113278599 A CN113278599 A CN 113278599A CN 202110554333 A CN202110554333 A CN 202110554333A CN 113278599 A CN113278599 A CN 113278599A
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Abstract
本发明涉及酿酒酵母羰基还原酶及其在制备光学活性烷基内酯中的应用。具体包括来源于酿酒酵母的羰基还原酶OdCR2,其基因、含有该基因的重组表达载体和重组表达转化体,以及利用该重组羰基还原酶或重组表达转化体作为催化剂催化长链γ‑/δ‑酮酸不对称还原,进而制备光学活性长链烷基内酯的方法。与现有技术相比,本发明使用羰基还原酶催化制备手性烷基内酯,具有工艺简单、产品光学纯度高和环境友好的显著优势,有很好的工业应用前景。本发明提供的羰基还原酶OdCR2,可立体选择性催化γ‑/δ‑长链酮酸的不对称还原,生成相应的光学活性长链γ‑/δ‑羟基酸,进而通过化学法环化生成γ‑/δ‑长链烷基内酯,反应条件温和,转化率高,产品光学纯度好,具有很好的工业应用前景。
Description
技术领域
本发明属于生物工程技术领域,尤其是涉及一种来源于酿酒酵母(Saccharomycescerevisiae)的羰基还原酶,其基因、含有该基因的重组表达载体和重组表达转化体,以及利用该重组羰基还原酶或重组表达转化体作为催化剂催化长链γ-/δ-酮酸不对称还原,进而制备光学活性长链烷基内酯的方法。
背景技术
光学活性的长链γ、δ-烷基内酯通常具有不同的水果香味,是一类重要的香料食品添加剂。比如(R)-γ-癸内酯具有桃子、杏子和草莓的香气,其最早从桃子中提取,而(R)-δ-癸内酯具有椰奶香味。这两种内酯作为重要的食品添加剂,被广泛应用于冰激凌、人造奶油和糖果等食品的制作中。
对具有前手性的γ-或δ-酮酸进行不对称还原,对转化获得的光学活性γ-或δ-羟基酸进行内酯化是制备光学活性γ/δ-烷基内酯的一种直接的方法。1962年Muys等首次报道用酿酒酵母(Saccharomyces cerevisiae)静息细胞作为催化剂,催化γ-或δ-长链酮酸(碳链长度8~12)的生物转化制备(R)-γ-/δ-内酯和,细胞上载量10%(w/v),底物酮酸上载量0.05~0.1%(w/v),另外加入5~10%的葡萄糖(w/v),转化48h,内酯的收率最高达85%(Nature,1962,194:995–996)。1987年,Utaka等使用酿酒酵母静息细胞催化δ-羰基癸酸酯转化生成(R)-癸内酯,底物浓度24mM,转化2天,产物收率低于20%,光学纯度高于98%(J Org Chem,1987,52:4363–4368)。2001年Forzato等用埃切毕赤酵母(Pichiaetchellsii)整细胞催化γ-酮酸(酯)转化制备γ-内酯,以γ-羰基壬酸作为底物,底物浓度2.5g/L,转化6天,产物(R)-γ-壬内酯的转化率和光学纯度分别为37%和90%,而以γ-羰基壬酸乙酯作为底物,在相同时间和底物浓度下,产物(R)-γ-壬内酯的转化率和光学纯度达到90%和99%(Tetrahedron:Asymmetry,2001,12:1039-1046)。2015年,苏州百福安酶技术有限公司通过筛选,发现近平滑假丝酵母CGMCC2.3539的整细胞以及无细胞提取物可以催化4-羰基癸酸和5-羰基癸酸的不对称还原,生成相应的(R)-羟基癸酸,底物浓度最高为50g/L,转化率99%,ee值高于99%,对获得的(R)-羟基癸酸进行化学环化即可获得(R)-癸内酯(CN 104651425A)。
综上所述,一些野生型酵母菌整细胞能催化γ-/δ-酮酸转化制备γ-/δ-烷基内酯,但是使用野生型酵母细胞催化存在许多缺陷,如酶的表达量少,底物传质阻力大,催化活力低,难以满足工业应用要求。1963年Francke对酿酒酵母中催化γ-/δ-酮酸还原的酶进行分离,发现催化该反应的酶位于酵母的线粒体中(Nature,1963,197:384–385)。尽管如此,迄今为止,并未获得酿酒酵母中催化γ-/δ-酮酸还原酶的基因序列,因此无法进行高效的异源重组表达,也难以通过蛋白质工程的手段对该酶进行进化以提高酶的催化活性。
发明内容
本发明所要解决的技术问题是,针对目前生物法制备光学活性手性烷基内酯的技术中γ/δ-酮酸还原酶的基因序列未见公布的现状,提供一种来源于酿酒酵母(Saccharomyces cerevisiae)的催化活性高、选择性强的酮酸还原酶。本发明还进一步提供包含该羰基还原酶或其突变体基因的重组表达载体和重组表达转化体,所述重组羰基还原酶在不对称转化长链羰基酸以制备相应光学活性长链烷基内酯的应用。
本发明的目的可以通过以下技术方案来实现:
本发明的技术方案之一:提供一种羰基还原酶,其是如下(a)或(b)的蛋白质:
蛋白质(a):由SEQ ID No.2所示氨基酸序列组成的蛋白质;
蛋白质(b):SEQ ID No.2所示氨基酸序列中经过取代、缺失或添加几个氨基酸且可以催化γ-/δ-酮酸不对称还原制备光学活性γ-/δ-羟基酸的由(a)衍生,同时催化活性高于(a)的蛋白质。
进一步地,本发明所述羰基还原酶来源于一种酿酒酵母Saccharomycescerevisiae,记为S288C,该酿酒酵母(Saccharomyces cerevisiae)S288C保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.22135,保藏时间为2021年04月06日,保藏地点为:北京市朝阳区北辰西路1号院3号。
本发明还提供所述羰基还原酶的获得方法:
通过对自然界微生物以及实验室保藏微生物菌株的大规模筛选,发现酿酒酵母S288C可以催化γ/δ-羰基癸酸还原生成相应的(R)-羟基癸酸。通过鸟枪法克隆获得了酿酒酵母S288C中催化相应反应的羰基还原酶,命名为羰基还原酶OdCR2,该酶是NADPH依赖型的。所述羰基还原酶的氨基酸序列如SEQ ID No.2所示。
本发明中提及的鸟枪法克隆采用本领域常规的生物技术手段即可实现。
本发明所述羰基还原酶具有以下性能:可以催化γ-/δ-酮酸不对称还原制备光学活性γ-/δ-羟基酸。
本发明的技术方案之二:提供一种分离的核酸,所述核酸编码所述羰基还原酶。
在本发明的一个实施方式中,提供羰基还原酶基因,其核苷酸序列如SEQ ID No.1所示,全长为894个核苷酸碱基。其编码序列(CDS)从第1个碱基起至第894个碱基止,起始密码子为ATG,终止密码子为TAA,无内含子。该基因编码的蛋白质的氨基酸序列如SEQ IDNo.2所示。
本发明还提供所述羰基还原酶OdCR2的编码DNA的来源包括:通过基因克隆技术获得所述羰基还原酶OdCR2的编码DNA,或者通过人工全序列合成的方法得到所述羰基还原酶的编码DNA。
在本发明的一个实施方式中,本发明所述羰基还原酶基因来源于酿酒酵母S288C。其编码DNA的具体制备方法包括:以酿酒酵母S288C的基因组DNA为模板,采用本领域常规技术方法(如聚合酶链式反应,PCR),获得编码所述羰基还原酶OdCR2的完整DNA序列。
其中涉及的合成引物,优选的如SEQ ID No.3(上游引物)和SEQ ID No.4(下游引物)所示:
上游引物:5’-CCG GAATTC ATGTCGGAGTTACAGTCACAACC-3’,下划线所示序列为限制性内切酶EcoR I的酶切位点;
下游引物:5’-CCG CTCGAG CTAATCGTCCTTATTCTTCTGTTTCG-3’,下划线所示序列为限制性内切酶Xho I的酶切位点。
本发明的技术方案之三:提供包含所述羰基还原酶基因核酸序列的重组表达载体。所述重组表达载体可通过本领域常规方法将所述羰基还原酶基因克隆到各种表达载体上构建而成。
所述的表达载体较佳的包括本领域常规的各种质粒载体,优选的为pET28a质粒。
较佳的,可通过下述方法制得本发明所述的重组表达载体:将通过PCR扩增所得的羰基还原酶OdCR2的基因序列DNA片段用限制性内切酶EcoR I和Xho I双酶切,同时将空载质粒pET28a用限制性内切酶EcoR I和Xho I双酶切,回收上述酶切后的羰基还原酶OdCR2的基因DNA片段以及pET28a质粒,利用T4 DNA连接酶连接,构建获得包含所述羰基还原酶OdCR2基因的重组表达载体pET28a-OdCR2。
本发明的技术方案之四:提供包含所述羰基还原酶OdCR2重组表达载体的重组表达转化体。
所述重组表达转化体可通过将上述重组表达载体转化至宿主细胞中制得。
在本发明的一些实施方式中,所述宿主细胞为本领域常规的宿主细胞,只要能满足重组表达载体可稳定地自行复制,并且其所携带的羰基还原酶OdCR2的基因可被有效表达即可。
在本发明的一些实施方式中,所述宿主细胞优选为大肠杆菌,更优选的为:大肠杆菌E.coli BL21(DE3)或大肠杆菌E.coli DH5α。
将所述重组表达载体转化至大肠杆菌E.coli BL21(DE3)中,即可获得本发明优选的基因工程菌株。例如,将重组表达载体pET28a-OdCR2转化至大肠杆菌E.coli BL21(DE3)中,获得重组大肠杆菌E.coli BL21(DE3)/pET28a-OdCR2。
本发明的技术方案之五:提供一种羰基还原酶催化剂,选自以下形式中的任意一种:
(1)培养所述的重组表达转化体,分离含有所述羰基还原酶的转化体细胞;
(2)培养所述的重组表达转化体,分离含有所述羰基还原酶的转化体细胞,对含有所述羰基还原酶的转化体细胞进行破碎,获得的细胞破碎液;
(3)培养所述的重组表达转化体,分离含有所述羰基还原酶的转化体细胞,对含有所述羰基还原酶的转化体细胞进行破碎,获得的细胞破碎液,将所述羰基还原酶的细胞破碎液经冷冻干燥而得到的冻干酶粉;
(4)所述羰基还原酶OdCR2或羰基还原酶OdCR2的突变体,所述羰基还原酶OdCR2的突变体是指SEQ ID No.2所示氨基酸序列中经过取代、缺失或添加几个氨基酸且可以催化γ-/δ-酮酸不对称还原制备光学活性γ-/δ-羟基酸,同时催化活性提高的蛋白质。
本发明还提供所述羰基还原酶催化剂的制备方法。
本发明所述重组羰基还原酶OdCR2的制备方法较佳地为:培养如上所述的重组表达转化体,分离获得重组表达的羰基还原酶OdCR2。其中所述重组表达转化体培养所用的培养基为本领域任何可使转化体生长并产生本发明的重组羰基还原酶的培养基。所述培养基优选LB培养基,其配方为:蛋白胨10g/L,酵母膏5g/L,NaCl 10g/L,pH 7.0。培养方法和培养条件没有特殊的限制,可以根据宿主细胞类型和培养方法等因素的不同,按本领域常规知识进行适当的选择,只要使转化体能够生长并生产所述羰基还原酶OdCR2即可。重组表达转化体培养的具体操作可按本领域常规操作进行。优选的,将本发明所述的重组大肠杆菌,例如E.coli BL21(DE3)/pET28a-OdCR2,接种至含卡那霉素的LB培养基中,37℃培养,当培养液的光密度OD600达到0.5~1.0(优选0.6)时,加入终浓度为0.1~1.0mmol/L(优选0.5mmol/L)的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行产酶诱导,在16℃继续培养24h,即可高效表达本发明所述的羰基还原酶OdCR2。培养结束后,离心收集沉淀的菌体细胞,即为重组表达转化体的静息细胞;将收获的细胞悬浮于PBS缓冲液(100mM,pH 6.0)中,超声破碎,破碎液离心,收集上清液,即可获得所述重组羰基还原酶OdCR2的粗酶液;将离心收获的细胞沉淀冷冻干燥,可以获得冻干细胞,有利于长期存放,方便以后使用。
羰基还原酶OdCR2的活性测定:将含2mmol/Lγ-羰基癸酸和0.1mmol/L NADPH的1ml反应体系(100mmol/L磷酸钠缓冲液,pH 6.0)预热至30℃,然后加入适量的羰基还原酶OdCR2粗酶液,混合均匀,30℃保温反应,在分光光度计上检测340nm处NADPH的吸光度变化,记录一定时间内吸光度的变化值。
根据下式计算得到酶活力:
酶活力(U)=EW×V×103/(6220×l)
式中,EW为1分钟内340nm处吸光度的变化;V为反应液的体积,单位为ml;6220为NADPH的摩尔消光系数,单位为L/(mol·cm);l为光程距离,单位为cm。1个酶活力单位(U)对应于上述条件下每分钟氧化1μmol NADPH所需的酶量。
本发明的技术方案之六:提供所述羰基还原酶催化剂在不对称还原长链酮酸中的应用。
其中所述长链酮酸化学结构如下所示:
进一步地,提供所述羰基还原酶催化剂催化长链γ-/δ-酮酸不对称还原,进而制备光学活性长链烷基内酯中的应用。
在本发明的一个实施方式中,所述长链酮酸的不对称还原,可按下述示例性方法进行:在pH 6-7的磷酸盐缓冲液中,在葡萄糖脱氢酶、葡萄糖和NADP+的存在下,在所述羰基还原酶催化剂的作用下,催化所述长链酮酸的不对称还原反应。
不对称还原反应结束后,通过化学法对长链羟基酸中间产物进行内酯化,获得相应的光学活性长链γ-或δ-内酯。
所述应用中,底物在反应液中的浓度可以为0.1~50mmol/L。根据所采用的反应体系,所述羰基还原酶的用量可以为1~500U/L。酶促长链酮酸不对称还原时,辅酶NADPH氧化生成NADP+,为了进行辅酶NADPH的循环再生,向反应体系中额外添加葡萄糖和来自巨大芽孢杆菌的葡萄糖脱氢酶(J Ind Microb Biotechnol,2011,38:633–641)。取决于不同的反应体系,葡萄糖脱氢酶的活力单位上载可以与所述羰基还原酶相等。葡萄糖与底物的摩尔比可以为1.0~1.5,额外添加的NADP+的用量可以为0~1.0mmol/L。所述缓冲液为磷酸钠缓冲液,优选pH范围为6.0~8.0,更优选pH 6.0。磷酸盐缓冲液的浓度可以为0.05~0.2mol/L。所述的酶促不对称还原反应的温度可以是25~40℃,优选30℃。反应过程中,间歇取样测定反应转化率,反应时间以底物完全转化或反应转化率停止增长的时间为准,一般为1~24小时。
反应转化率和产物对映体过量值(ee)可采用气相色谱法进行分析,优选的,使用MS毛细管色谱柱(30m×0.25mm×0.25μm)进行转化率分析,载气为氮气,检测器为氢火焰离子化检测器(FID)。进样口温度为280℃,检测器温度为280℃,柱温恒定为160℃。使用GC/CP-Chirasil-Dex CB毛细管色谱柱(25m×0.25mm×0.25μm)进行还原产物的ee值分析,载气为氮气,检测器为氢火焰离子化检测器(FID)。进样口温度280℃,检测器温度280℃,柱温采用程序升温:初始温度为110℃,按2℃/min的速率升温至140℃,保持10min。
酶促不对称还原反应结束后,对反应生成的光学活性长链γ-/δ-羟基酸产物进行内酯化。对于长链羟基酸产物,酶促反应结束后,分离除去催化剂,将反应液酸化至pH 1~2,80~90℃加热1~2h,促使羟基酸环化生成相应的内酯。反应液冷却至室温,用等量本领域常规的水不溶性有机溶剂,如乙酸乙酯、乙酸丁酯、甲苯、二氯甲烷、三氯甲烷、异丙醚、甲基叔丁基醚等进行萃取,重复萃取两次,合并萃取液,用饱和食盐水洗涤,加入无水硫酸钠干燥过夜。旋转蒸发除去溶剂,即可获得相应光学活性长链烷基内酯粗产品。
与现有技术相比,本发明的积极进步效果在于:本发明提供的羰基还原酶OdCR2,可立体选择性催化γ-/δ-长链酮酸的不对称还原,生成相应的光学活性长链γ-/δ-羟基酸,进而通过化学法环化生成γ-/δ-长链烷基内酯,反应条件温和,转化率高,产品光学纯度好,ee值可高于99%,具有很好的工业应用前景。
具体实施方式
发明内容中所述的各反应或检测条件,可根据本领域常识进行组合或更改,并可通过实验得到验证。下面通过实施例的方式进一步说明本发明,应当理解,所列实施例虽然列举了本发明优选的实施方式,但所列具体实施例仅是为了更好地阐明本发明而给出,并不因此将本发明限制在所述的实施例范围之内。
下列实施例中的材料来源为:
酿酒酵母(Saccharomyces cerevisiae),记为S288C,该酿酒酵母(Saccharomycescerevisiae)S288C保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.22135,保藏时间为2021年04月06日,保藏地点为:北京市朝阳区北辰西路1号院3号。
载体pUC118和限制性内切酶Sau3AI购于宝生物工程(大连)有限公司。
表达质粒pET28a购自Novagen公司。
E.coli DH5α和E.coli BL21(DE3)感受态细胞、2×Taq PCR MasterMix、琼脂糖凝胶DNA回收试剂盒均购自北京天根生化科技有限公司。
限制性内切酶EcoR I和Xho I均为New England Biolabs(NEB)公司的市售产品。
除非另有说明,下列实施例中的具体实验按照本领域常规方法和条件进行,或遵照试剂盒的商品说明书。
实施例1羰基还原酶OdCR2的基因克隆
采用高盐法获取完整的酿酒酵母(Saccharomyces cerevisiae)S288C的基因组。将酿酒酵母(Saccharomyces cerevisiae)S288C接种到马铃薯培养基中,30℃培养24h,取100ml菌液,8,000rpm离心10min,收集菌体,用20ml生理盐水洗涤,重复两次,然后用20ml生理盐水重悬菌体。悬浮菌液中加入1ml溶菌酶液(50mg/ml),37℃水浴恒温1h;加入1.6ml的十二烷基硫酸钠溶液(SDS,10%,w/v),160μl蛋白酶K(20mg/ml),55℃水浴保温,直到悬浮液变清。然后加入三分之一体积的饱和NaCl溶液,振荡混匀至溶液浑浊,然后高速离心10min,弃去细胞碎片。用等体积的酚/氯仿/异戊醇(25:24:1)反复萃取,直到两相界面上看不到蛋白质时停止。吸取萃取之后的水相清液,加入0.6倍体积的异丙醇,混匀后置于-20℃,低温析出DNA,离心弃去上清,用75%的乙醇洗涤沉淀DNA,室温干燥,最后加入20μL的TE缓冲液(100mM Tris-HCl,10mM EDTA,pH 8.0)将基因组DNA溶解。
使用限制性内切酶Sau3AI对基因组DNA进行酶切,对酶切片段进行电泳分离,收集2~6kb的DNA片段。将空载质粒pUC 118用限制性内切酶Bam HI酶切,碱性磷酸酶去磷酸化。使用T4连接酶将回收的DNA片段与酶切的质粒pUC118片段进行连接,两者的浓度比为3:1,16℃过夜连接。将连接产物全部转化到大肠杆菌DH5α中,涂布含有100μg/ml氨苄青霉素的LB固体培养基平板,37℃培养12h。用无菌牙签将所有的单克隆分别挑至96孔深孔板的孔洞中,每个孔洞中装有300μL含有100μg/ml氨苄青霉素的LB培养基。37℃振荡培养12h,取50μL培养液转接至600μL含有100μg/ml氨苄青霉素的LB培养基,37℃振荡培养3h后加入终浓度为0.2mmol/L的异丙醇硫代半乳糖苷(IPTG),16℃诱导24h。3500×g离心10min,弃去培养基,放入-80℃冰箱冷冻2h。从冰箱中拿出深孔板,待菌液融化后,每个孔洞中加入200μL溶菌酶液(750mg溶菌酶和10mg DNA酶溶解于1L去离子水中),振荡混匀,37℃静置1h。4℃、3500×g离心10min,分别取50μL破碎离心上清液转移到新的96孔板中,加入150μL反应液(100mM KPB,pH 7.0,含有1mM 4-羰基癸酸以及0.2mM NADPH),随后加入50μL酶液,30℃震荡混匀,在酶标仪上读取340nm处吸光度值的减少。对吸光度值显著下降的克隆子进行活性复筛,将其接种到4ml含有100μg/ml氨苄青霉素的LB培养基中,37℃培养2h,加入终浓度为0.2mmol/L的IPTG,16℃培养24h,8000×g离心10min,弃上清,加入500μl的PBS缓冲液(100mM,pH 7.0)重悬菌液,然后加入底物4-羰基癸酸至终浓度为10mM,葡萄糖脱氢酶1U,葡萄糖25mg,NADP+终浓度1mM,30℃,1,100rpm的条件下反应24h。反应结束后离心去除细胞,反应液酸化后用等体积的乙酸乙酯进行萃取。萃取液用无水硫酸钠干燥12h,GC检测有没有产物生成。有产物的克隆为阳性克隆子,委托上海桑尼生物技术有限公司进行序列测定,根据开放阅读框,获得如SEQ ID No.1所示的核酸序列,根据该核酸序列所推测的氨基酸序列如SEQ ID No.2所示,将该序列表达的羰基还原酶命名为OdCR2。
实施例2羰基还原酶OdCR2的基因克隆
根据羰基还原酶OdCR2的开放阅读框,设计上、下游引物,以酿酒酵母(Saccharomyces cerevisiae)S288C的基因组DNA为模板,进行PCR扩增。
设计的上、下游引物如下:
上游引物SEQ ID No.3:
5’-CCG GAATTC ATGTCGGAGTTACAGTCACAACC-3’;
下游引物SEQ ID No.4:
5’-CCG CTCGAG CTAATCGTCCTTATTCTTCTGTTTCG-3’;
其中,上游引物下划线部分为限制性内切酶EcoR I的酶切位点,下游引物下划线部分为限制性内切酶Xho I的酶切位点。
PCR体系为:2×Taq PCR MasterMix 25μl,上游引物和下游引物(10ng/μl)各2.5μl,1μl酿酒酵母(Saccharomyces cerevisiae)S288C的基因组DNA(100ng/μl),以及19μl的ddH2O。PCR扩增程序为:95℃预变性5分钟后进行32次如下循环:94℃变性30秒,50℃退火30秒,72℃延伸50分钟;循环结束后,最后再72℃延伸10分钟。PCR扩增产物进行凝胶电泳纯化后,用DNA回收试剂盒回收目的片段。经过DNA测序,该序列编码的开放阅读框全长894bp,其碱基序列如SEQ ID No.1所示。
实施例3羰基还原酶OdCR2重组表达质粒和重组表达转化体的制备
将实施例2中PCR扩增所得的羰基还原酶目的DNA片段以及pET28a空质粒同时用限制性内切酶EcoR I和Xho I双酶切过夜,然后经琼脂糖凝胶电泳纯化、DNA试剂盒回收。将回收的酶切目的片段和空载体在T4 DNA连接酶的作用下,于4℃连接12小时,得到重组质粒pET28a-OdCR2。
将所得重组质粒转化至E.coli DH5α,涂布到含有50μg/ml卡那霉素的LB培养基平板上,37℃培养8小时,对长出来的菌落进行菌落PCR验证,挑取成功扩增出长度约894bp的目的条带的阳性克隆。经测序验证后,提取相应的质粒,进一步转化至E.coli BL21(DE3),挑取阳性克隆,即获得重组表达转化体E.coli BL21(DE3)/pET28a-OdCR2。
实施例4羰基还原酶OdCR2的诱导表达
将实施例2中所得的重组表达转化体E.coli BL21(DE3)/pET28a-OdCR2,接种至含50μg/ml卡那霉素的LB培养基中,37℃摇床振荡培养12小时,之后按1%(v/v)的接种量接种至装有100ml的LB培养基(含50μg/ml卡那霉素)的500ml三角烧瓶中,放入摇床中,37℃、180rpm振荡培养,当培养液的OD600达到0.6时,加入IPTG至终浓度0.2mmol/L进行诱导,16℃诱导24小时后,将培养液以8000rpm转速离心,收集细胞沉淀,并用生理盐水洗涤,得到静息细胞,将其冷冻干燥,制得冻干细胞,其比活力为34.8U/gDCW。
将5g如上方法所得的静息细胞悬浮于100ml的磷酸钠缓冲液(100mM,pH 6.0)中,冰水浴中进行超声破碎,离心收集上清液,即为重组羰基还原酶OdCR2的粗酶液。所得粗酶液经聚丙烯酰胺凝胶电泳分析,重组羰基还原酶OdCR2以可溶的形式存在。将获得的重组羰基还原酶OdCR2的粗酶液进行冷冻干燥,制得重组羰基还原酶OdCR2的粗酶粉。
实施例5 pH对羰基还原酶OdCR2催化活性的影响
在pH 5~11的范围内按标准方法测定pH对重组羰基还原酶OdCR2活性的影响。缓冲液分别为柠檬酸-柠檬酸钠缓冲液(5.0~6.0),磷酸钠缓冲液(6.0~8.0),Tris-HCl缓冲液(7.0~9.0),甘氨酸-NaOH缓冲液(8.0~11)。
在1ml上述缓冲液体系中,加入γ-羰基癸酸和NADPH至终浓度分别为2mmol/L和0.1mmol/L,预热至40℃,然后加入适量的羰基还原酶,混合均匀,40℃保温反应,在分光光度计上检测340nm处NADPH的吸光度变化,测定不同pH的缓冲溶液中,羰基还原酶OdCR2的活性差异,结果如表1所示。优选酶促反应的pH范围为6.0~7.0,更优选pH 6.5。
表1 pH对OdCR2催化γ-羰基癸酸不对称还原活性的影响
实施例6温度对羰基还原酶OdCR2催化活性的影响
在1ml磷酸钠缓冲液(100mM,pH 6.5)体系中,加入γ-羰基癸酸和NADPH至终浓度分别为2mmol/L和0.1mmol/L,在20~60℃环境中预热2min,然后加入适量的羰基还原酶,混合均匀,在与预热温度相同的温度环境中保温反应,在分光光度计上检测340nm处NADPH的吸光度变化,测定不同温度条件下,羰基还原酶OdCR2的活性差异,结果如表2所示。优选酶促反应的温度范围为30~40℃。
表2温度对OdCR2不对称催化还原的影响
实施例7重组羰基还原酶OdCR2催化4-羰基癸酸合成γ-癸内酯
将2.5g如实施例4所述的OdCR2冻干细胞和3U的葡萄糖脱氢酶冻干酶粉加入100ml磷酸钠缓冲液(100mmol/L,pH 6.0)中,加入4-羰基癸酸、葡萄糖和NADP+至终浓度分别为50mmol/L、100mmol/L和0.2mmol/L。30℃,磁力搅拌反应。转化6小时,使用气相色谱测得底物转化率为>99%。
反应结束后,使用20%硫酸将反应液的pH调节至2左右,80℃加热1h进行环化反应。反应液冷却,使用2倍体积的乙酸乙酯进行萃取,萃取有机相使用无水硫酸钠干燥过夜,过滤,旋转蒸发去除溶剂。使用石油醚:乙酸乙酯=8:1的流动相,对粗产品进行硅胶柱层析,精制后获得361mgγ-癸内酯,ee值高于99%,分离得率为85%。
实施例8重组还原酶OdCR2催化5-羰基癸酸合成δ-癸内酯
在100ml磷酸钠缓冲液(100mmol/L,pH 6.0)中加入3g如实施例4所述的冻干细胞,以及3U的葡萄糖脱氢酶粗酶液,加入5-羰基癸酸、葡萄糖和NADP+至终浓度分别为50mmol/L、100mmol/L和0.2mmol/L。30℃,磁力搅拌反应。转化4小时,使用气相色谱测得底物转化率为>99%。
反应结束后,使用硫酸调节pH至2左右,90℃加热2h将5-羟基癸酸进行内酯化,最后使用2倍体积的乙酸乙酯进行萃取,萃取有机相使用无水硫酸钠干燥过夜后过滤,选择蒸发去除溶剂。硅胶柱层析将粗产品进行纯化,使用石油醚:乙酸乙酯=8:1的为流动相,精制后获得391mgδ-癸内酯,ee值为98%,分离得率为92%。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 华东理工大学
<120> 酿酒酵母羰基还原酶及其在制备光学活性烷基内酯中的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 894
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 1
atgtcggagt tacagtcaca acctaaaaag attgccgttg ttacaggcgc ctcaggtggt 60
attggatatg aggtaacgaa ggaattagcc agaaatggct atttggttta tgcatgtgca 120
agaagactag aaccaatggc acaattggcc atccaatttg gtaacgatag tatcaaacca 180
tataagttgg atatttccaa gccagaagaa attgtaacat tttctggttt tttgcgagct 240
aatttacctg atggtaaatt agatttattg tacaacaatg ccggtcagag ttgtacattc 300
cctgcattgg atgcaacaga tgctgcagta gaacaatgct ttaaggtcaa tgtgtttggt 360
catatcaaca tgtgccgtga attgtcggag ttccttatta aagcgaaagg aaccattgtc 420
ttcactggtt ccttggcagg agttgtgtca ttcccctttg gtagcattta ttccgcttct 480
aaagctgcta tccaccaata tgctcgtggg ttacatctag agatgaagcc cttcaacgtg 540
agagtgatta acgctattac tggcggtgtg gctactgata tcgctgataa aaggcccttg 600
cctgaaacct caatatacaa cttccctgaa ggtagggaag cttttaattc aagaaaaacc 660
atggctaagg acaataagcc catgccagct gatgcgtacg caaagcaact ggttaaagat 720
atcttgagca ctagtgaccc tgtagatgtt tatagaggaa cgttcgccaa catcatgcgt 780
tttgtcatga tatttgttcc ttactggcta ttggaaaaag gactttccaa aaaatttaaa 840
ttggacaaag ttaataatgc tttgaagtcg aaacagaaga ataaggacga ttag 894
<210> 2
<211> 297
<212> PRT
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 2
Met Ser Glu Leu Gln Ser Gln Pro Lys Lys Ile Ala Val Val Thr Gly
1 5 10 15
Ala Ser Gly Gly Ile Gly Tyr Glu Val Thr Lys Glu Leu Ala Arg Asn
20 25 30
Gly Tyr Leu Val Tyr Ala Cys Ala Arg Arg Leu Glu Pro Met Ala Gln
35 40 45
Leu Ala Ile Gln Phe Gly Asn Asp Ser Ile Lys Pro Tyr Lys Leu Asp
50 55 60
Ile Ser Lys Pro Glu Glu Ile Val Thr Phe Ser Gly Phe Leu Arg Ala
65 70 75 80
Asn Leu Pro Asp Gly Lys Leu Asp Leu Leu Tyr Asn Asn Ala Gly Gln
85 90 95
Ser Cys Thr Phe Pro Ala Leu Asp Ala Thr Asp Ala Ala Val Glu Gln
100 105 110
Cys Phe Lys Val Asn Val Phe Gly His Ile Asn Met Cys Arg Glu Leu
115 120 125
Ser Glu Phe Leu Ile Lys Ala Lys Gly Thr Ile Val Phe Thr Gly Ser
130 135 140
Leu Ala Gly Val Val Ser Phe Pro Phe Gly Ser Ile Tyr Ser Ala Ser
145 150 155 160
Lys Ala Ala Ile His Gln Tyr Ala Arg Gly Leu His Leu Glu Met Lys
165 170 175
Pro Phe Asn Val Arg Val Ile Asn Ala Ile Thr Gly Gly Val Ala Thr
180 185 190
Asp Ile Ala Asp Lys Arg Pro Leu Pro Glu Thr Ser Ile Tyr Asn Phe
195 200 205
Pro Glu Gly Arg Glu Ala Phe Asn Ser Arg Lys Thr Met Ala Lys Asp
210 215 220
Asn Lys Pro Met Pro Ala Asp Ala Tyr Ala Lys Gln Leu Val Lys Asp
225 230 235 240
Ile Leu Ser Thr Ser Asp Pro Val Asp Val Tyr Arg Gly Thr Phe Ala
245 250 255
Asn Ile Met Arg Phe Val Met Ile Phe Val Pro Tyr Trp Leu Leu Glu
260 265 270
Lys Gly Leu Ser Lys Lys Phe Lys Leu Asp Lys Val Asn Asn Ala Leu
275 280 285
Lys Ser Lys Gln Lys Asn Lys Asp Asp
290 295
<210> 3
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccggaattca tgtcggagtt acagtcacaa cc 32
<210> 4
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccgctcgagc taatcgtcct tattcttctg tttcg 35
Claims (10)
1.一种羰基还原酶,其特征在于,其是如下(a)或(b)的蛋白质:
蛋白质(a):由SEQ ID No.2所示氨基酸序列组成的蛋白质;
蛋白质(b):SEQ ID No.2所示氨基酸序列中经过取代、缺失或添加几个氨基酸且可以催化γ-/δ-酮酸不对称还原制备光学活性γ-/δ-羟基酸的由(a)衍生,同时催化活性高于(a)的蛋白质。
2.一种分离的核酸,其特征在于,所述核酸编码如权利要求1所述的羰基还原酶。
3.根据权利要求2所述的一种分离的核酸,其特征在于,羰基还原酶基因的核苷酸序列如SEQ ID No.1所示。
4.一种重组表达载体,其特征在于,其包含如权利要求2所述的核酸序列。
5.一种重组表达转化体,其特征在于,其包含如权利要求4所述的重组表达载体。
6.一种羰基还原酶催化剂,其特征在于,选自以下形式中的任意一种:
(1)培养如权利要求5所述的重组表达转化体,分离含有如权利要求1所述羰基还原酶的转化体细胞;
(2)培养如权利要求5所述的重组表达转化体,分离含有如权利要求1所述羰基还原酶的转化体细胞,对含有所述羰基还原酶的转化体细胞进行破碎,获得的细胞破碎液;
(3)培养如权利要求5所述的重组表达转化体,分离含有如权利要求1所述羰基还原酶的转化体细胞,对含有所述羰基还原酶的转化体细胞进行破碎,获得的细胞破碎液,将所述羰基还原酶的细胞破碎液经冷冻干燥而得到的冻干酶粉;
(4)如权利要求1所述羰基还原酶。
7.如权利要求6所述羰基还原酶催化剂的应用,其特征在于,所述羰基还原酶催化剂在催化γ-/δ-酮酸不对称还原制备光学活性γ-/δ-羟基酸中的应用。
8.如权利要求7所述羰基还原酶催化剂的应用,其特征在于,在葡萄糖脱氢酶、葡萄糖和NADP+的存在下,在所述羰基还原酶催化剂的作用下,催化γ-/δ-酮酸不对称还原制备光学活性γ-/δ-羟基酸。
9.如权利要求8所述羰基还原酶催化剂的应用,其特征在于,不对称还原反应结束后,通过化学法对长链γ-/δ-羟基酸进行内酯化,获得相应的光学活性长链γ-或δ-内酯。
10.如权利要求8所述羰基还原酶催化剂的应用,其特征在于,底物在反应液中的浓度为0.1~50mmol/L,所述羰基还原酶催化剂中羰基还原酶的用量为1~500U/L,葡萄糖与底物的摩尔比为1.0~1.5,额外添加的NADP+的用量为0~1.0mmol/L;
反应体系pH范围为6.0~8.0,不对称还原反应的温度是25~40℃。
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