CN117264936A - 一种催化活性提高的苯丙氨酸解氨酶突变体及其用途 - Google Patents
一种催化活性提高的苯丙氨酸解氨酶突变体及其用途 Download PDFInfo
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Abstract
本发明属于基因工程和酶工程技术领域,具体公开了一种高催化活性的苯丙氨酸解氨酶突变体,可用于制备非天然手性氨基酸。本发明的突变体催化挑战性底物如4‑甲氧基‑肉桂酸、3‑(2‑萘基)‑丙烯酸等活性大幅度提升且具有优异的立体选择性,展现出了良好的催化效果与潜力。
Description
技术领域
本发明属于基因工程和酶工程技术领域,具体涉及一种苯丙氨酸解氨酶突变体及其应用。
背景技术
生物催化过程具有高效、高选择性、条件温和、环境友好等优点,已经成为当前绿色生物制造的一大潮流。尤其是随着新酶的发现和改造技术的飞速发展,生物催化剂的研发速度和成功率都得到了显著提升,应用范围也更加广泛。苯丙氨酸解氨酶能够催化L-苯丙氨酸及其类似物与相应反式肉桂酸发生可逆反应,在生产非天然手性氨基酸中具有明显优势,如优异的立体选择性、反应体系中无需添加辅因子或额外的再生系统等等。
目前,苯丙氨酸解氨酶仅用于极少数非天然手性氨基酸的生产,对多数大位阻底物没有催化活性或者催化活性很低。然而,这些产物却是重要的医药中间体,需求量很大。例如,L-4-甲氧基-苯丙氨酸可用于合成降血压药坦索罗辛。3-(2-萘基)-丙氨酸常作为苯丙氨酸类似物用于多肽药物的开发。4,4’-联苯基-丙氨酸类化合物则是多种生物活性肽抑制剂的组成部分。其中L-4,4’-联苯基-丙氨酸已被纳入二肽酶(DPPIV)抑制剂、α4β7整合素抑制剂和内皮素转换酶抑制剂中;D-对映体则在肉毒杆菌毒素抑制剂、淀粉样β肽聚集抑制剂、驱动蛋白KIFC1抑制剂中作为重要组成砌块。
利用蛋白质工程技术对天然酶进行分子改造是获得高活性苯丙氨酸解氨酶的有力工具。目前蛋白质工程已被广泛应用于提高酶的催化活性、区域选择性和立体选择性,以及酶蛋白的热稳定性和耐溶剂性等。
发明内容
本发明对来源于莴苣(Lactuca sativa L.)的苯丙氨酸解氨酶LsPAL3进行分子改造,将单个或者多个氨基酸进行替换,从而获得催化活性大幅度提高的突变体。
具体而言,所述苯丙氨酸解氨酶突变体是以LsPAL3(核苷酸序列为SEQ ID NO.1;氨基酸序列为SEQ ID NO.2)为出发序列,将以下氨基酸位点进行突变得到的突变体。
单点突变体LsPAL3-I454V:将第454位的异亮氨酸突变为缬氨酸。
三位点突变体LsPAL3-F129C/L130V/I454V:在LsPAL3-I454V的基础上,将第129位的苯丙氨酸突变为半胱氨酸且第130位的亮氨酸突变为缬氨酸。
根据本领域公共知识,构建的能表达上述突变体的载体、基因工程菌等也属于本发明的保护范围。
为了达到以上目的,本发明将此前获得的苯丙氨酸解氨酶LsPAL3进行酶分子改造(参考文献Journal of Agricultural and Food Chemistry,2023,71,6,2935-2942)。首先通过定点突变技术构建了单点突变体LsPAL3-I454V,然后以此为母本,构建了包含F129和L130两个位点的突变文库。以L-4-甲氧基-苯丙氨酸为底物,经过96孔板初筛及高效液相色谱HPLC复筛确认,获得了三位点突变体LsPAL3-F129C/L130V/I454V。双位点文库构建中,采用了简并引物NDT以控制突变氨基酸种类,减少筛选的工作量。具体方法见实施例1和实施例2。
本发明的优点:
本发明获得的突变体与野生型相比,其催化挑战性底物的能力显著增强,对L-4-甲氧基-苯丙氨酸的催化活性提高了3倍多;对L-3-(2-萘基)-丙氨酸的催化活性提高了4-5倍。在加氨反应中,两个突变体对4-甲氧基-肉桂酸的催化活性提高了6-7倍;对3-(2-萘基)-丙烯酸的催化活性提高了5-8倍。且具有优异的L-立体选择性。特别是对于L-4,4’-联苯基-丙氨酸和4,4’-联苯基-丙烯酸底物,母本不能催化,LsPAL3-F129C/L130V/I454V突变体对它们的催化能力大幅度提升。因此,本发明的突变体具有进一步开发应用的潜力。
具体实施方式
实施例1:构建LsPAL3的I454V单点突变体
首先,我们构建了苯丙氨酸解氨酶LsPAL3的I454V单点突变体LsPAL3-I454V(LsPAL3核苷酸序列为SEQ ID NO.1所示,氨基酸序列为SEQ ID NO.2所示)。以野生型酶构建的质粒pET-28a(+)-LsPAL3为模板,所用的正向引物为:5′-GTTTCAAAGGTGGCGAAGTGGCGATGGCGAGCTACTG-3′;反向引物为:5′-CAGTAGCTCGCCATCGCCACTTCGCCACCTTTGAAAC-3′。采用常规的PCR定点突变技术。PCR体系如下:质粒模板(50ng/μL)1μL;正反向引物(10μM)各2μL;dNTP(10mmol/L)1μL;pfu酶(1U)1μL;buffer(2×)25μL;补充ddH2O至50μL。扩增条件:95℃预变性3min,95℃变性30s,60℃退火30s和72℃延伸7min 30s,共16个循环,再72℃延伸10min。PCR产物通过DpnI酶在37℃处理2h,以去除甲基化的质粒模板。取10μLPCR产物按化学法转入E.coli DH5α感受态细胞,挑取单克隆并提取质粒后送生物公司测序验证,获得pET-28a(+)-LsPAL3-I454V质粒。
实施例2:构建组合突变文库和高通量筛选
为了识别LsPAL3的有益突变位点,对该酶利用SWISS-MODEL进行同源建模,根据活性口袋残基分布和分子对接结果,选择22个位点残基进行丙氨酸扫描。根据扫描结果,以突变体LsPAL3-I454V为起始母本,将其中的潜在位点(F129和L130)进行组合突变,构建包含这两个随机化位点的组合突变文库。
文库构建中,采用简并引物NDT以减少筛选的工作量,正向引物为:5′-CAGAAAGAACTGATTCGCNDTNDTAACGCAGGTATTTTCGG-3′;反向引物为:5′-CCGAAAATACCTGCGTTAHNAHNGCGAATCAGTTCTTTCTG-3′。PCR扩增的模板是实施例1构建的pET-28a(+)-LsPAL3-I454V质粒,PCR反应体系和条件同实施例1。以L-4-甲氧基-苯丙氨酸为底物对文库进行筛选。
高通量筛选方法如下:取2μL文库质粒库转入E.coli BL21(DE3)感受态细胞,在LB平板(卡那霉素100μg/mL)上37℃过夜培养;然后从平板上挑取单个菌落,接种至含有200μLLB液体培养基(卡那霉素100μg/mL)的无菌96孔板中,37℃培养过夜,制备种子液。以多通道移液器转接30μL种子液至另一个96孔板中,其中每孔已含有170μL TB液体培养基(包括0.5mmol/LIPTG和100μg/mL卡那霉素)。将96孔板在30℃,180r/min空气摇床中培养22h,促进菌体生长和蛋白表达。然后在4℃离心(5000r/min、10min)收集细胞微球,每孔用200μLNaCl(0.85%)缓冲液洗涤菌体2次。将细胞微球重悬于160μL的50mmol/L Tri-HCl缓冲液(pH 9.0)中,在每孔中加入40μL底物溶液(L-4-甲氧基-苯丙氨酸,终浓度1mmol/L)。在摇床(45℃,200r/min)中孵育2h后,加入6mol/L HCl终止反应。4℃、5000r/min离心10min,每孔小心吸取60μL上清液至新的96孔板中,多功能酶标仪测定290nm处的吸光度变化,从而识别催化活性提升的突变子。筛选过程中,我们同时以野生型LsPAL3和单点突变体LsPAL3-I454V作为参照。将初筛获得的有益突变子在500mL摇瓶中重新挑菌培养,收集菌体后在1mL体系中进行反应,并利用HPLC进行复筛检测,确认有益突变子催化活性。
通过筛选,得到的三位点突变体LsPAL3-F129C/L130V/I454V对L-4-甲氧基-苯丙氨酸的催化活性是野生型的3倍,与单点突变体LsPAL3-I454V相当。
实施例3:酶的表达与纯化
将野生型、单点突变体LsPAL3-I454V和三位点突变体LsPAL3-F129C/L130V/I454V突变体质粒分别转化至E.coli BL21(DE3)感受态细胞进行蛋白表达。具体过程为:挑取单克隆至5mL LB培养基中(含卡那霉素100μg/mL),在37℃、180r/min培养过夜,制备种子液。接种1mL种子液至200mL TB培养基中进行扩大培养;待OD600≈0.8时,加入IPTG(终浓度为0.5mmol/L),在20℃、180r/min条件下继续培养18h,诱导目的蛋白表达。然后收集菌体,既可以作为催化剂直接使用,也可以进一步破碎后进行蛋白纯化。蛋白纯化操作按照已发表文献方法进行(Journal of Agricultural and Food Chemistry,2023,71,6,2935-2942)。所有酶均以纯酶催化确认了无副产物产生。
实施例4:突变体的生物催化
脱氨反应:将离心后的湿菌体重悬于50mmol/L Tris-HCl缓冲液(pH 9.5)中进行脱氨反应。1mL反应体系中包含Tris-HCl缓冲液(50mmol/L,pH 9.5)、底物和45mg湿菌体细胞。底物在Tris-HCl缓冲液(50mmol/L,pH 9.5)和MeOH(1:1,V/V)中预溶解配制成贮存液,再加入到反应体系中。底物分别为L-4-甲氧基-苯丙氨酸(催化体系的终浓度为25mmol/L)、L-3-(2-萘基)-丙氨酸(催化体系的终浓度为25mmol/L)以及L-4,4’-联苯基-丙氨酸(催化体系的终浓度为10mmol/L)。反应条件为:50℃的水浴震荡(200r/min)反应0.5h或者2h。每个底物的具体催化反应条件见表1。反应结束后加入100μL HCl(6mol/L)终止反应。取200μL反应混合液加入1mLMeOH,漩涡混匀后离心(13000r/min)3min,将上清液过滤(0.22μm尼龙滤膜)后,直接用于HPLC分析。
加氨反应:将底物(分别为4-甲氧基-肉桂酸、3-(2-萘基)-丙烯酸、4,4’-联苯基-丙烯酸)溶解于4M氨基甲酸铵(NH2CO2NH4)缓冲液和MeOH(1:1,V/V)的混合溶液中,配制成浓度50mmol/L的贮存液。取100μL底物贮存液加入到含有全细胞(45mg湿菌体重)的900μL 4M氨基甲酸铵溶液(pH~10.0,未调节)中,使底物终浓度为5mmol/L。反应在37℃、200rpm水浴摇床中反应12h,反应后处理同脱氨反应的操作。
转化率测定:采用反相C18(Zorbax Extend)色谱柱(3.5μm,4.6×100mm,Agilent),使用岛津LC-20AD液相系统与PDA检测器,测定脱氨反应和加氨反应的转化率。柱温设置为40℃,流动相选择NH4OH缓冲液(pH为10.0)和纯MeOH,流速通常设置为1.0mL/min,根据检测目标检测物不同适当调整流速及比例。
对映体选择性(ee值)测定:采用反相CROWNPAK CR(+)色谱柱(5μm,4.0×150mm,Daicel)测定对映体过量值(ee)。柱温设为25℃,流动相选择三氟乙酸水溶液(pH为2.0),流速通常设置为1.0mL/min,根据检测目标检测物不同适当调整流速及比例。
野生型、单点突变体LsPAL3-I454V和三位点突变体LsPAL3-F129C/L130V/I454V突变体的脱氨和加氨反应结果如表1和表2所示。在野生型酶的基础上,两个突变体催化3种挑战性底物的能力显著增强,对L-4-甲氧基-苯丙氨酸的催化活性提高了3倍以上;对L-3-(2-萘基)-丙氨酸的催化活性提高了4-5倍。LsPAL3-F129C/L130V/I454V突变体对L-4,4’-联苯基-丙氨酸催化能力提升最大,从母本不能转化,提升到2h可以转化10mmol/L底物,转化率达到97%。已有报道的野生型PcPAL在16h对rac-4,4’-联苯基-丙氨酸(1mmol/L,其中L-4,4’-联苯基-丙氨酸的含量为0.5mmol/L)的转化率为<1%,其最佳突变体PcPAL-F137A在16h内的转化率为35%。因此,本发明的突变体的催化活性远远高于已有报道。
加氨反应具有类似的结果,两个突变体对4-甲氧基-肉桂酸的催化活性提高了6-7倍;对3-(2-萘基)-丙氨酸的催化活性提高了5-8倍。LsPAL3-F129C/L130V/I454V突变体可以催化5mmol/L 4,4’-联苯基-丙烯酸,转化率20%,而母本不能转化。加氨反应具有优异的立体选择性,L-4-甲氧基-苯丙氨酸等产物的ee值大于99%。
表1野生型和有益突变子的脱氨反应
a底物浓度25mmol/L,反应时间0.5h,温度50℃;b底物浓度10mmol/L,反应时间2h,温度50℃。
表2野生型和有益突变子的加氨反应
Claims (2)
1.一种苯丙氨酸解氨酶突变体,其特征在于以苯丙氨酸解氨酶LsPAL3的氨基酸序列SEQ ID NO.2为出发序列,经突变后特征如下:将第454位的异亮氨酸突变为缬氨酸。
2.一种苯丙氨酸解氨酶突变体,其特征在于:将权利要求1所述的突变体的第129位的苯丙氨酸突变为半胱氨酸且第130位的亮氨酸突变为缬氨酸。
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