CN116926051B - 一种查耳酮异构酶突变体及其制备方法和应用 - Google Patents
一种查耳酮异构酶突变体及其制备方法和应用 Download PDFInfo
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- CN116926051B CN116926051B CN202311207345.XA CN202311207345A CN116926051B CN 116926051 B CN116926051 B CN 116926051B CN 202311207345 A CN202311207345 A CN 202311207345A CN 116926051 B CN116926051 B CN 116926051B
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- chalcone
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- isomerase mutant
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公开了一种查耳酮异构酶突变体,该查耳酮异构酶突变体可以实现逆向催化二氢黄酮转化为查耳酮及其组合物,酶转化催化效率最高达99.36%;本发明公开的一种查耳酮异构酶突变体的制备方法,通过定向进化手段对查耳酮异构酶进行随机突变,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;本发明公开的一种查耳酮异构酶突变体在制备查耳酮组合物中的应用,利用查耳酮异构酶突变体催化转化二氢黄酮制备含查耳酮含量0.01%~99%的组合物。
Description
技术领域
本发明涉及酶工程领域,尤其涉及一种查耳酮异构酶突变体及其制备方法和应用。
背景技术
黄酮类化合物是一类多酚类次生代谢物质,在植物生长发育中有着重要的生理作用,参与紫外线防护、抗病原微生物、花色形成、植物育性、植物与微生物相互识别协作等。查耳酮属于黄酮类化合物中的一种,天然存在的查耳酮存在潜在的抗癌、抗炎、抗菌、抗氧化和抗寄生虫特性,并且具有独特的化学结构,因此促进了许多针对查尔酮衍生物的合成的研究。在植物体内,查尔酮是大部分类黄酮的重要前体,由苯丙烷酸途径产生的香豆酰辅酶A经丙二酰辅酶A延长碳链再环化生成。目前,工业上通过对在氢氧化钠的催化下由苯甲醛与苯乙酮的羟醛缩合反应来制备查耳酮。
然而,通过生物途径合成查耳酮的方法,目前仍未发现有关报道。植物中合成查耳酮的前体香豆酰辅酶A和丙二酰辅酶A原料价格昂贵,成本高,不适合大量制备查耳酮。查耳酮和与二氢黄酮之间属于异构化关系,因此考虑通过二氢黄酮的构象转换来制备查耳酮。查耳酮异构酶则是这两者转化的关键催化酶。查耳酮异构酶(chalcone isomerase,CHI,EC5.5.1.6)通过催化分子发生内环化反应,使双环的查耳酮转化为有生物学活性的三环(2S)-黄烷酮,而该步骤也可以在没有查耳酮异构酶的条件下在植物体内缓慢自发进行,但在查耳酮催化下可使该反应的反应速率提高107倍。因此,需要提供一种高效转化二氢黄酮为查耳酮的新型查耳酮异构酶。
发明内容
为了克服现有技术的不足,本发明的第一目的在于提供一种查耳酮异构酶突变体,其能解决目前查耳酮异构酶催化转化效果不佳的问题。
本发明的第二个目的在于提供一种查耳酮异构酶突变体的制备方法,其能解决目前查耳酮异构酶制备成本高和高转化的查耳酮异构酶制备困难的问题。
本发明的第三个目的在于提供一种查耳酮异构酶突变体的应用,其能解决目前二氢黄酮转化为查耳酮的问题。
本发明的第一个目的采用以下技术方案实现:
一种查耳酮异构酶突变体,所述查耳酮异构酶突变体为氨基酸序列如SEQ ID NO:1所示的查耳酮异构酶进行定向进化所获得的突变体。
进一步地,所述查耳酮异构酶突变体为氨基酸序列如SEQ ID NO:1所示的查耳酮异构酶的第120位的色氨酸突变成组氨酸。
进一步地,所述查耳酮异构酶突变体为氨基酸序列如SEQ ID NO:1所示的查耳酮异构酶的第183位的亮氨酸突变成酪氨酸。
进一步地,所述查耳酮异构酶突变体的氨基酸序列如SEQ ID NO:3所示。
进一步地,所述查耳酮异构酶突变体的核苷酸序列如SEQ ID NO:4所示。
本发明的第二个目的采用以下技术方案实现:
一种查耳酮异构酶突变体的制备方法,包括以下步骤:
S1、设计随机突变引物对查耳酮异构酶基因进行易错PCR,获得随机突变的查耳酮异构酶基因;
S2、根据步骤S1获得的随机突变的查耳酮异构酶基因,构建突变文库;
S3、对步骤S2获得的突变文库进行高通量筛选后,构建高表达工程菌株,得到查耳酮异构酶突变体。
进一步地,所述步骤S2中,将随机突变的查耳酮异构酶基因连接到质粒中,得到重组质粒后转化至宿主细胞中获得拟转化子,所述拟转化子组成突变文库。
进一步地,所述宿主细胞为大肠杆菌或酿酒酵母。
进一步地,步骤S1中,所述随机突变引物包括随机突变上游引物CmCHI-F和随机突变下游引物CmCHI-R;所述随机突变上游引物CmCHI-F的核苷酸序列如SEQ ID NO:5所示,所述随机突变下游引物CmCHI-R的核苷酸序列和SEQ ID NO:6所示。
本发明的第三个目的采用以下技术方案实现:
一种查耳酮异构酶突变体在制备查耳酮中的应用,所述查耳酮异构酶突变体将二氢黄酮催化转化为查耳酮含量在0.01%~99%的组合物。
相比现有技术,本发明的有益效果在于:
本发明的一种查耳酮异构酶突变体,可以实现逆向催化二氢黄酮转化为查耳酮及其组合物,该突变体的酶催化转化效率高,异构化处理仅需24h,在以黄酮类化合物(柚皮素、柚苷、橙皮苷、新橙皮苷或甲基橙皮苷)作为底物生成相应查耳酮时转化率高达71.30%-99.36%。
本发明的一种查耳酮异构酶突变体的制备方法,通过定向进化手段对查耳酮异构酶进行随机突变,构建突变库后以高通量的筛选手段快速鉴定转化效果提升的突变体,进行多轮的突变筛选,成本低廉且操作简便,无需使用昂贵的香豆酰辅酶A或者有毒的苯乙酮/苯甲醛进行反应。
本发明的一种查耳酮异构酶突变体在制备查耳酮组合物中的应用,利用查耳酮异构酶突变体催化转化二氢黄酮制备含查耳酮含量0.01%~99%的组合物。
附图说明
图1为突变库筛选96孔板反应强度图(M326为本发明的突变体CmCHIM)。
图2为大肠杆菌系统柚皮素转化为柚皮素查耳酮液相检测结果图。
图3为大肠杆菌系统柚苷转化为柚苷查耳酮液相检测结果图。
图4为大肠杆菌系统橙皮苷转化为橙皮苷查耳酮液相检测结果图。
图5为大肠杆菌系统新橙皮苷转化为新橙皮苷查耳酮液相检测结果图。
图6为大肠杆菌系统甲基橙皮苷转化为甲基橙皮苷查耳酮液相检测结果图。
图7为酿酒酵母系统柚苷转化为柚苷查耳酮液相检测结果图。
具体实施方式
下面将结合具体实施例对本发明的技术方案进行清楚、完整的描述。显然,所描述的实施例仅仅是本发明的部分实施例,而不是全部实施例。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的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。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
定向进化是蛋白质工程技术中的其中一个关键技术,是在不了解有关酶结构与功能之间的关系等信息的情况下通过对蛋白基因的随机突变或基因片段的重组来创造突变库, 然后通过高通量的筛选手段鉴定性能改善的突变体, 挑出的有益突变基因可以作为下一轮突变的模板, 进行多轮突变和筛选, 以进一步提高酶的性能。这项技术是对酶的催化功能进行反转设计的有力工具。本发明采用定向进化技术,对查耳酮异构酶进行定向进化,使其具备有二氢黄酮催化生成查耳酮的反转功能,并利用该突变体实现查耳酮的生物制备。
下面,结合附图和具体实施方式,对本发明做进一步描述,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查耳酮异构酶的突变克隆
取柑橘的叶子,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提取试剂盒进行柑橘叶子的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 Master Mix进行柑橘叶子RNA的反转录,获得柑橘叶子的cDNA。利用随机突变引物进行查耳酮异构酶的克隆,引物如下表1所示。
表1 本实验所用的引物
引物名称 | 序列(5’-3’)(下划线为酶切位点) | |
CmCHI-F | GAATTCATGAATCCCTCACCGTC | SEQ ID No. 5 |
CmCHI-R | GCGGCCGCTCATTTCATCTTATCACTAGTT | SEQ ID No. 6 |
引物对CmCHI -F和CmCHI-R用于克隆柑橘查耳酮异构酶CmCHI以及用于CmCHI的定向进化突变文库构建。
基因克隆的PCR体系如表2所示:
表2 PCR体系
2×PrimeSTAR® Max DNA Polymerase | 25 μL |
cDNA | 1 μL |
CmCHI-F | 1 μL |
CmCHI-R | 1 μL |
ddH2O | 补至50 μL |
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-CmCHI。
实施例2构建查耳酮异构酶突变文库
1、pMD18-T-CmCHI、pET-28a、pESC-HIS的质粒小量提取;
2、利用随机突变引物,以质粒pMD18-T-CmCHI为模板进行随机突变PCR扩增,100 μL的PCR体系如下:0.05U的Taq酶、0.2 mM的dATP、0.2 mM的dGTP、1 mM的dCTP、1 mM的dTTP、5mM的MgCl2、0.2 mM的MnCl2、0.4 mM的引物CmCHI-F和0.4 mM的引物CmCHI-R。
PCR扩增程序:预变性98℃ 3 min;循环设定:变性98℃ 10 s,退火55℃ 15 s,延伸72℃ 2 min,30个循环;最后延伸72℃ 10 min;反应结束后,利用试剂盒进行PCR产物回收。
3、酶切消化去除模板DNA,将PCR回收产物进行酶切,30 μL的酶切体系如下:PCR产物(≤1 μg)、QuickCut Dpn Ⅰ 1 μL、10×QuickCut Buffer 3 μL。酶切体系置于37℃金属浴中消化1 h,反应结束后利用试剂盒进行酶切产物CmCHI的回收。
4、酶切连接构建载体,酶切体系如下表3所示:
表3 酶切体系
10×QuickCut Buffer | 5 μL |
pET-28a/酶切产物CmCHI | ≤1 μg |
EcoR Ⅰ | 2 μL |
Not Ⅰ | 2 μL |
ddH2O | 补至50 μL |
酶切体系置于37℃金属浴中消化1 h,反应结束后利用试剂盒进行酶切产物回收,获得具有粘性末端的随机突变基因片段CmCHI和载体框pET-28a。
片段和载体框进行连接,连接体系如下表4所示:
表4 连接体系
10×T4 DNA Ligase Buffer | 2 μL |
T4 DNA Ligase | 1 μL |
CmCHI | 5 μL |
pET-28a | 7 μL |
ddH2O | 补至20 μL |
连接体系置于16℃金属浴中连接16 h,连接产物直接转化至大肠杆菌BL21(DE3)感受态细胞中,37℃倒置过夜培养,该平板上的拟转化子组成突变文库。
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。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100 μL 50 mM的Tris缓冲液(pH 7.5),含终浓度为100 mg/L的柚皮素,振荡混匀后置于30℃ 200 rpm反应12 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后在酶标仪上进行检测,波长为405 nm。如图1所示,吸光值越高表明查耳酮含量越高,突变酶的活性越好。通过上述实验进行多轮筛选,获得查耳酮异构酶突变体CmCHIM,其氨基酸序列如SEQ IDNO:3所示,核苷酸序列如SEQ ID NO:4所示。
实施例4突变体活性验证
重新活化该突变体,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1 mM的IPTG,在30℃下诱导6 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50 mM Tris缓冲液(pH 7.5)中含有50 mg/mL全细胞和100 mg/L柚皮素、柚苷、橙皮苷和新橙皮苷。30℃ 200 rpm反应24h,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=34:66,20 min,流速1 mg/min,检测波长360 nm,柱温30℃。图2-图6结果显示,该突变体能高效催化这5个底物生成相应的查耳酮,柚皮素转化为柚皮素查耳酮的转化率为94.41%,柚苷转化为柚苷查耳酮的转化率为97.45%,橙皮苷转化为橙皮苷查耳酮的转化率为87.76%,新橙皮苷转化为新橙皮苷查耳酮的转化率为99.36%,甲基橙皮苷转化为甲基橙皮苷查耳酮的转化率为71.30%。
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-CmCHIM以及酿酒酵母表达载体pESC-HIS的质粒小量提取;突变酶CmCHIM的编码基因的核苷酸序列如SEQ ID NO:4所示。
2、采用实施例2步骤4中的酶切体系,以质粒pET-28a-CmCHIM和pESC-HIS为模板进行酶切,酶切体系同表3,获得酶切片段CmCHIM和酶切表达框pESC-HIS,随后如下表5所示体系进行了连接:
表5
10×T4 DNA Ligase Buffer | 2 μL |
T4 DNA Ligase | 1 μL |
CmCHIM | 5 μL |
pESC-HIS | 7 μL |
ddH2O | 补至20 μL |
连接体系置于16℃金属浴中连接16 h,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-CmCHIM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-CmCHIM,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全细胞和100 mg/L柚苷。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,检测条件为:Diamonsil C18 (2) 5μm 250× 4.6mm,乙腈:0.5%乙酸水=34:66,20 min,流速1 mg/min,检测波长360 nm,柱温30℃。图7结果显示,该突变体在酿酒酵母中的表达和转化效果相比大肠杆菌较差,柚苷转化为柚苷查耳酮的转化率仅为34.07%,只有大肠杆菌转化效率的35%。
对本领域的技术人员来说,可根据以上描述的技术方案以及构思,做出其它各种相应的改变以及形变,而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
Claims (3)
1.一种查耳酮异构酶突变体,其特征在于,所述查耳酮异构酶突变体的氨基酸序列如SEQ ID NO:3所示。
2. 根据权利要求1所述的一种查耳酮异构酶突变体,其特征在于,所述查耳酮异构酶突变体的核苷酸序列如SEQ ID NO:4所示。
3.权利要求1-2任一项所述的一种查耳酮异构酶突变体在制备查耳酮组合物中的应用,其特征在于,所述查耳酮异构酶突变体将二氢黄酮催化转化为查耳酮含量在0.01%~99%的组合物。
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