CN117467627B - 一种烯醛还原酶突变体及其编码基因和应用 - Google Patents
一种烯醛还原酶突变体及其编码基因和应用 Download PDFInfo
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Abstract
本发明公开了一种烯醛还原酶突变体及其编码基因和应用,其中,烯醛还原酶突变体可以实现高效催化姜黄素转化为四氢姜黄素及其组合物,烯醛突变体在酿酒酵母中的表达和转化效果相比大肠杆菌好,酶转化催化效率最高达95.8%;本发明的烯醛还原酶突变体是通过定点突变技术对烯醛还原酶进行饱和组合突变,构建突变库后以高通量的筛选手段快速鉴定,成本低廉且操作简便;本发明的烯醛还原酶突变体在制备四氢姜黄素组合物中的应用,利用烯醛还原酶突变体催化转化姜黄素制备含四氢姜黄素含量0.01%~99%的组合物。
Description
技术领域
本发明涉及烯醛还原酶技术领域,具体涉及一种烯醛还原酶突变体及其编码基因和应用。
背景技术
姜黄素是从姜科植物姜黄、莪术、芥末、咖哩、郁金等根茎中提取的一种天然的酚类抗氧化剂,是常用的调料及食用色素,同时具有良好的降血脂、抗肿瘤、抗炎、利胆、抗氧化等作用。
据研究表明,姜黄素在体内迅速代谢为葡萄糖醛酸结合物、硫酸结合物、二氢姜黄素、四氢姜黄素和六氢姜黄素,而二氢和六氢后续又会转化为四氢姜黄素。四氢姜黄素作为姜黄素在体内代谢过程中产生的最为活跃和主要的代谢产物,可以从姜黄素对人或小鼠给药后的小肠和肝脏的细胞质中分离得到,能有效抑制酪氨酸酶、抑制氧自由基的生成并消除已经形成的自由基,具有明显的抗氧化作用,目前已作为天然功能性美白原料用于化妆品的研究开发。
目前,四氢姜黄素主要以化学合成和植物提取为主,化学合成法有通过已姜黄素和乙醇为原料,已铂-铁镍氢氧化物复合纳米颗粒为催化剂在室温条件下反应,经浓缩和冷藏静置析晶制备;也有以姜黄素和丙酮为原料通过催化氢化制备。而生物合成法均处于实验室研发阶段,郑彬彬等人挖掘了一株库德里阿兹威毕赤酵母ZJPH0802,利用其进行生物转化姜黄素制备得到四氢姜黄素,不过底物浓度仅50mg/L,转化率77.43%,仅处于实验室研究水平;天津大学刘彬的毕业课题合成姜黄素和四氢姜黄素的工程菌株研究中,工程菌株在添加0.1mM姜黄素前体下,四氢姜黄素的产量为25mg/L,该产量也是实验室水平;有科研工作者从姜黄根茎中分离得到内生真菌,利用该内生真菌转化得到四氢姜黄素,但是同样转化效率不高。
半理性设计是蛋白质工程技术中的其中一个关键技术,是在对有关酶结构与功能之间的关系等信息有部分了解的情况下通过对蛋白基因的多位点饱和组合突变来创造突变库, 然后通过高通量的筛选手段鉴定性能改善的突变体, 以获得高性能的酶。这项技术是对酶的催化功能进行定向设计的有力工具。本发明采用半理性设计技术,对烯醛还原酶进行定向改造,使其具备高效催化姜黄素生成四氢姜黄素的功能,并利用该突变体实现四氢姜黄素的生物制备。
发明内容
本发明首先要解决的技术问题是提供一种烯醛还原酶突变体,其具备高效催化姜黄素生成四氢姜黄素的功能,因此,本发明可利用该烯醛还原酶突变体实现四氢姜黄素的生物制备。
为解决上述技术问题,本发明的技术方案如下:
一种烯醛还原酶突变体,其是将氨基酸序列如SEQ ID NO.1所示的烯醛还原酶进行序列定点饱和组合突变得到的,烯醛还原酶突变体的氨基酸序列如SEQ ID NO.3所示。
SEQ ID NO.1:
MTATNKQVILKDYVSGFPTESDFDFTTTTVELRVPEGTNSVLVKNLYLSCDPYMRIRMGKPDPSTAALAQAYTPGQPIQGYGVSRIIESGHPDYKKGDLLWGIVAWEEYSVITPMTHAHFKIQHTDVPLSYYTGLLGMPGMTAYAGFYEVCSPKEGETVYVSAASGAVGQLVGQLAKMMGCYVVGSAGSKEKVDLLKTKFGFDDAFNYKEESDLTAALKRCFPNGIDIYFENVGGKMLDAVLVNMNMHGRIAVCGMISQYNLENQEGVHNLSNIIYKRIRIQGFVVSDFYDKYSKFLEFVLPHIREGKITYVEDVADGLEKAPEALVGLFHGKNVGKQVVVVARE*
烯醛还原酶基因核苷酸序列如SEQ ID NO.2所示:
ATGACGGCGACGAACAAGCAAGTCATATTGAAAGACTACGTGAGTGGTTTCCCTACGGAATCCGATTTCGATTTCACTACCACCACCGTCGAACTTAGGGTTCCGGAAGGTACTAACTCTGTTCTAGTGAAGAATCTCTACTTGTCATGCGATCCTTACATGAGAATTCGCATGGGGAAACCTGATCCTTCCACTGCTGCTCTTGCTCAAGCTTACACTCCCGGCCAGCCAATCCAAGGGTATGGAGTGTCTAGAATAATAGAATCTGGACATCCAGATTACAAGAAAGGAGACTTACTCTGGGGTATAGTTGCATGGGAGGAGTACAGTGTTATCACTCCAATGACTCACGCGCATTTCAAGATCCAACATACTGATGTTCCATTATCTTATTACACTGGACTTTTAGGTATGCCTGGTATGACTGCCTATGCTGGGTTTTATGAAGTTTGTTCTCCAAAGGAAGGAGAGACAGTTTATGTGTCAGCTGCATCTGGTGCTGTTGGTCAGCTTGTGGGACAACTTGCTAAGATGATGGGCTGTTATGTTGTTGGAAGCGCTGGAAGTAAAGAGAAGGTTGATCTTCTGAAGACCAAGTTTGGGTTTGATGATGCATTTAACTACAAGGAAGAATCTGACCTTACTGCTGCCCTAAAAAGGTGTTTCCCTAATGGCATTGACATATACTTTGAGAATGTAGGAGGCAAAATGCTAGATGCAGTGCTTGTGAACATGAACATGCACGGGCGTATCGCTGTCTGTGGAATGATCTCACAGTACAATCTTGAGAACCAGGAAGGTGTACACAACCTATCCAACATAATCTACAAAAGAATCCGCATTCAAGGCTTTGTAGTGTCTGATTTCTACGACAAATACTCAAAGTTCTTGGAGTTTGTGCTTCCCCACATTAGAGAAGGGAAGATAACGTACGTGGAAGATGTAGCTGATGGGCTTGAGAAAGCTCCCGAAGCTCTTGTGGGACTCTTCCATGGTAAGAATGTTGGGAAACAAGTTGTTGTTGTTGCTCGTGAGTGA
SEQ ID NO.3:
MTATNKQVILKDYVSGFPTESDFDFTTTTVELRVPEGTNSVLVKNLYLSCDPYMRIRMGKPDPSTAALAQAYTPGQPIQGSGVSRIIESGHPDYKKGDLLWGIVAWEEYSVITPMTHAHFKIQHTDVPLSYYTGLLGMPGMTAYAGFYEVCSPKEGETVYVSAASGAVGQLVGQLAKMMGCYVVGSAGSKEKVDLLKTKFGFDDAFNYKEESDLTAALKRCFPNGIDIYFENVGGKMLDAVLVNMNMHGRIAVCGMISQYNLENQEGVHNLSNIIYKRIRIQGFYVSDFYDKYSKFLEFVLPHIREGKITYVEDVADGLEKAPEALVGLFHGKNVGKQVVVVARE*
由于氨基酸序列的特殊性,任何含有SEQIDNO.2所示氨基酸序列的多肽的片段或其变体,如其保守性变体、生物活性片段或衍生物,只要该多肽的片段或多肽变体与前述氨基酸序列同源性在90%以上、且具有相同的酶活性,均可。具体的,所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换;其中,对于变体的保守性改变,所替换的氨基酸具有与原氨基酸相似的结构或化学性质,如用亮氨酸替换异亮氨酸,变体也可具有非保守性改变,如用色氨酸替换甘氨酸。
本发明还公开了上述烯醛还原酶突变体的编码基因,具体地,所述烯醛还原酶突变体的编码基因的核苷酸序列如SEQIDNO.4所示。
SEQIDNO.4:
ATGACGGCGACGAACAAGCAAGTCATATTGAAAGACTACGTGAGTGGTTTCCCTACGGAATCCGATTTCGATTTCACTACCACCACCGTCGAACTTAGGGTTCCGGAAGGTACTAACTCTGTTCTAGTGAAGAATCTCTACTTGTCATGCGATCCTTACATGAGAATTCGCATGGGGAAACCTGATCCTTCCACTGCTGCTCTTGCTCAAGCTTACACTCCCGGCCAGCCAATCCAAGGGAGCGGAGTGTCTAGAATAATAGAATCTGGACATCCAGATTACAAGAAAGGAGACTTACTCTGGGGTATAGTTGCATGGGAGGAGTACAGTGTTATCACTCCAATGACTCACGCGCATTTCAAGATCCAACATACTGATGTTCCATTATCTTATTACACTGGACTTTTAGGTATGCCTGGTATGACTGCCTATGCTGGGTTTTATGAAGTTTGTTCTCCAAAGGAAGGAGAGACAGTTTATGTGTCAGCTGCATCTGGTGCTGTTGGTCAGCTTGTGGGACAACTTGCTAAGATGATGGGCTGTTATGTTGTTGGAAGCGCTGGAAGTAAAGAGAAGGTTGATCTTCTGAAGACCAAGTTTGGGTTTGATGATGCATTTAACTACAAGGAAGAATCTGACCTTACTGCTGCCCTAAAAAGGTGTTTCCCTAATGGCATTGACATATACTTTGAGAATGTAGGAGGCAAAATGCTAGATGCAGTGCTTGTGAACATGAACATGCACGGGCGTATCGCTGTCTGTGGAATGATCTCACAGTACAATCTTGAGAACCAGGAAGGTGTACACAACCTATCCAACATAATCTACAAAAGAATCCGCATTCAAGGCTTTTATGTGTCTGATTTCTACGACAAATACTCAAAGTTCTTGGAGTTTGTGCTTCCCCACATTAGAGAAGGGAAGATAACGTACGTGGAAGATGTAGCTGATGGGCTTGAGAAAGCTCCCGAAGCTCTTGTGGGACTCTTCCATGGTAAGAATGTTGGGAAACAAGTTGTTGTTGTTGCTCGTGAGTGA
本发明的烯醛还原酶突变体的制备方法,包括以下步骤:
S1,设计饱和突变引物对利用克隆引物克隆的烯醛还原酶基因进行PCR;
S2,将PCR扩增片段基因进行连接,得到重组质粒;
S3,将重组载体转化至宿主细胞中,获得拟突变体;
S4,在96孔板中进行底物反应后,利用姜黄素褪色的特征进行突变体筛选,得到高效转化的突变体表达菌株,最后表达得到烯醛还原酶突变体。
进一步地,所述的克隆引物序列如SEQ ID NO.5和SEQ ID NO.6所示;突变引物序列如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10所示。
进一步地,所述宿主细胞为酿酒酵母菌或大肠杆菌。
本发明的烯醛还原酶突变体在制备四氢姜黄素及其组合物中的应用。
相比现有技术,本发明的有益效果在于:
本发明的烯醛还原酶突变体可实现催化姜黄素转化制备四氢姜黄素及其组合物,酶解时间短,转化率高,酶转化催化效率最高达95.8%。
本发明的烯醛还原酶突变体的制备是通过定点包和组合突变与高通量筛选方法,转化至宿主细胞后,诱导表达,得到高效的烯醛还原酶突变体。
本发明的烯醛还原酶突变体在制备四氢姜黄素组合物时,利用烯醛还原酶突变体催化转化姜黄素制备含四氢姜黄素含量0.01%~99%的组合物。
附图说明
图1为突变库筛选96孔板实景图;
图2为突变库筛选96孔板的反应强度图;
图3为姜黄素对照品液相检测结果图;
图4为四氢姜黄素对照品液相检测结果图;
图5为野生型大肠杆菌系统姜黄素转化为四氢姜黄素液相检测结果图;
图6为AtAERM大肠杆菌系统姜黄素转化为四氢姜黄素液相检测结果图;
图7为AtAERM酿酒酵母系统姜黄素转化为四氢姜黄素液相检测结果图。
具体实施方式
下面,结合附图和具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。以下是本发明具体的实施例,在下述实施例中所采用的原材料、设备等除特殊限定外均可以通过购买方式获得。
实施例1
烯醛还原酶的获得:
取拟南芥的叶子,利用购买自天根生化科技(北京)有限公司的RNAprep Pure多糖多酚植物总RNA提取试剂盒进行拟南芥叶子的RNA提取,提取步骤参考说明书。随后利用购买自宝日医生物技术(北京)有限公司(TAKARA)的反转录试剂盒PrimeScript™RT MasterMix进行拟南芥叶子RNA的反转录,获得拟南芥叶子的cDNA。利用克隆引物进行烯醛还原酶的克隆,引物如下表1所示。
表1 本实验所用的克隆引物
引物对AtAER -F和AtAER-R用于克隆拟南芥烯醛还原酶AtAER。
基因克隆的PCR体系如表2所示:
表2PCR体系
PCR扩增程序:预变性98℃ 3 min;循环设定:变性98℃ 10 s,退火58℃ 15 s,延伸72℃ 1 min,30个循环;最后延伸72℃ 10 min;反应结束后,加入适量购买自北京聚合美生物科技有限公司的2×M5 HiPer plus Taq HiFi PCR mix(with blue dye),放入72℃静置30 min,随后利用试剂盒进行产物回收。
回收的产物连接到TAKARA公司的pMD18-T载体上,获得后续实验需要用的模板载体pMD18-T-AtAER。
实施例2
烯醛还原酶突变文库的获得:
1、pMD18-T-AtAER和pET-28a的质粒小量提取;
2、利用酶切连接方法构建表达载体pET-28a-AtAER:
对质粒pMD18-T-AtAER和pET-28a进行EcoR Ⅰ和NotⅠ的双酶切处理并利用试剂盒回收AtAER基因片段和pET-28a载体片段,利用T4 DNA连接酶进行基因片段和载体片段的连接,获得表达载体pET-28a-AtAER;
3、突变片段的获得:
(1)项目组前期研究结果发现AtAER基因中的两个氨基酸位点对姜黄素的转化起着重要作用,因此围绕这两个位点进行饱和组合突变,设计突变引物如下表3所示。
表3 本实验所用的突变引物
(2)以pET-28a-AtAER作为模板,利用高保真酶PrimeSTAR® Max DNA Polymerase进行基因片段的扩增,分别以引物对Y81-F/V285-R和引物对V285-F/Y81-R进行片段扩增,得到一个627 bp的突变片段1和一个5761 bp的突变片段2,通过试剂盒凝胶电泳切胶回收获得。
4、突变文库的建立:
突变片段1和突变片段2利用金沙生物的一步快速克隆试剂盒Uniclone One StepSeamless Cloning Kit (SC612)进行片段连接,连接产物转化至大肠杆菌感受态细胞BL21(DE3)中,平板长出的菌落即为突变文库。
实施例3
突变文库的高通量筛选:
在96孔板中分别加入200 μL含Kan(100 μg/mL)的LB液体培养基,将突变平板上的所有菌落分别挑入96孔板中,如图1所示。经37℃ 200 rpm振荡培养过夜后,将96孔板中的菌液按照10%接种量接种到装有新鲜LB液体培养基的96孔板中,37℃ 200 rpm震荡培养3 h后加入终浓度1 mM的IPTG,在30℃下诱导6 h,随后经4000 rpm离心15 min弃上清,加入100μL 50 mM的Tris缓冲液(pH 7.5),含终浓度为750 μM的姜黄素和1.5 mM的NADH,振荡混匀后置于30℃ 200 rpm反应1 h后加入等体积的DMSO进行反应终止,反应液经4000 rpm离心15 min后肉眼观测,同时在酶标仪上进行检测,波长为405 nm,肉眼观测越黄(如图1、2所示),吸光值越高,表明姜黄素剩余含量越高,突变酶的活性越差。
通过上述实验进行多轮筛选,获得烯醛还原酶突变体AtAERM。
实施例4
突变体活性进一步验证:
重新活化该突变体(同时做野生型作对比),将其接入5 mL含Kan(100 μg/mL)的LB液体培养基,经37℃ 200 rpm振荡培养过夜后,按照1%接种量接种到100 mL新鲜LB液体培养基中,37℃ 200 rpm震荡培养3 h后加入终浓度0.4 mM的IPTG,在20℃下诱导16 h,随后经8000 rpm离心15 min弃上清,收集细胞,按照下列体系进行全细胞催化反应:5 mL的50mM Tris缓冲液(pH 7.5)中含有50 mg/mL全细胞和1 g/L姜黄素、3.3 g/L NADH。30℃ 200rpm反应1 h,取样加入等体积的DMSO后,经0.22 μm的微孔滤膜处理,样品进行HPLC检测,检测条件为:Diamonsil C18 (2) 5μm 250 × 4.6mm,乙腈:0.5%乙酸水=48:52,16-20 min,流速1 mL/min,检测波长280 nm,柱温30℃。结果显示,该突变体能高效催化姜黄素,转化率达73.9%,比野生型(8.3%)提高8.9倍,底物姜黄素对照品的液相检测结果如图3所示,产物四氢姜黄素对照品的液相检测结果如图4所示,野生型大肠杆菌系统姜黄素转化为四氢姜黄素液相检测结果如图5所示,AtAERM大肠杆菌系统姜黄素转化为四氢姜黄素液相检测结果如图6所示。
实施例5
突变酶重组酿酒酵母工程菌的构建:
1、重组质粒pET-28a-AtAERM以及酿酒酵母表达载体pESC-HIS的质粒小量提取;突变酶AtAERM的编码基因的核苷酸序列如SEQ ID NO.3所示。
2、采用酶切连接的方法,以质粒pET-28a-AtAERM和pESC-HIS为模板进行酶切得到AtAERM基因片段和pESC-HIS载体片段,再利用T4 DNA 连接酶进行连接,连接产物直接转化至大肠杆菌DH5α感受态细胞中,37℃倒置过夜培养,挑取转化子提质粒,随后转化至酿酒酵母BY4742中,30℃倒置培养2 d,获得酿酒酵母转化子。
3、转化子经菌落PCR鉴定,酿酒酵母工程菌株BY4742/pESC-HIS-AtAERM构建成功。
实施例6
酿酒酵母工程菌株反应验证
活化重组酿酒酵母工程菌BY4742/pESC-HIS-AtAERM,将其接种至15 mL的SD-HIS液体培养基中,30℃,200 r/min振荡培养48 h。震荡培养后,4℃,6000 rpm离心5 min收集酿酒酵母细胞,并转移至50 mL的SD-HIS诱导培养基(将SD-HIS原配方的葡萄糖换为半乳糖,添加量不变)中,30℃,200 r/min发酵培养4 d。发酵结束后经8000 rpm离心15 min弃上清,收集细胞,按照下列体系进行全细胞催化反应:5 mL的50 mM Tris缓冲液(pH 7.5)中含有50 mg/mL全细胞、1 g/L姜黄素和3.3 g/L NADH。30℃ 200 rpm反应24 h,取样加入等体积的DMSO后,经0.22 μm的微孔滤膜处理,样品进行HPLC检测,检测条件同实施例4。液相检测结果如图7所示,结果显示,该烯醛突变体在酿酒酵母中的表达和转化效果相比大肠杆菌好,转化率为95.8%。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种烯醛还原酶突变体,其特征在于,其氨基酸序列如SEQ ID No.3所示。
2.权利要求1所述的烯醛还原酶突变体的编码基因。
3.如权利要求2所述的编码基因,其特征在于,所述编码基因的核苷酸序列如SEQ IDNo.4所示。
4.含有权利要求2或3所述的编码基因的重组质粒。
5.一种用权利要求4所述的重组质粒转化得到的重组基因工程菌。
6.根据权利要求5所述的重组基因工程菌,其特征在于,重组基因工程菌为酿酒酵母工程菌。
7.权利要求2或3所述的编码基因、权利要求4所述的重组质粒、权利要求5或6所述的重组基因工程菌在制备烯醛还原酶突变体中的应用。
8.如权利要求1所述的烯醛还原酶突变体在制备四氢姜黄素或含有四氢姜黄素的组合物中的应用。
9.如权利要求7所述的应用,其特征在于,含有四氢姜黄素的组合物中四氢姜黄素含量在0.01%~99%。
10.一种烯醛还原酶突变体的制备方法,其特征在于,包括如下步骤:培养权利要求5或6所述的基因工程菌,获得烯醛还原酶突变体。
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