CN108707590B - 一种Pictet-Spengler酶及其编码基因与应用 - Google Patents
一种Pictet-Spengler酶及其编码基因与应用 Download PDFInfo
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- CN108707590B CN108707590B CN201810510161.3A CN201810510161A CN108707590B CN 108707590 B CN108707590 B CN 108707590B CN 201810510161 A CN201810510161 A CN 201810510161A CN 108707590 B CN108707590 B CN 108707590B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种Pictet‑Spengler酶及其编码基因与应用。本发明提供的Pictet‑Spengler酶的编码基因nfcbB的核苷酸序列如SEQ ID NO.1所示,Pictet‑Spengler酶NfcbB的氨基酸序列如SEQ ID NO.2所示;构建含nfcbB基因的重组大肠杆菌BL21(DE3)可以生产β‑咔啉骨架1‑酰基‑3‑羧基‑β‑咔啉,纯化得到的NfcbB可催化L‑色氨酸和丙酮醛反应生产β‑咔啉骨架1‑酰基‑3‑羧基‑β‑咔啉。NfcbB的发现和功能鉴定对生物碱的大规模生产具有实际指导意义,可用于生物、医药、精细化工等领域。
Description
技术领域:
本发明属于生物工程技术领域,具体涉及一种Pictet-Spengler酶及其编码基因与应用。
背景技术:
Pictet-Spengler(P-S)反应是指具有苯乙胺结构的化合物与醛或酮反应形成席夫碱,接着通过Friedel-Crafts环化,形成异喹啉或β-咔啉生物碱类化合物的过程。该反应广泛应用于有机化学中天然产物和新型杂环物质的构建。它是1911年由Pictet和Spengler首次发现的,尽管它已有百年历史,至今仍是合成四氢异喹啉和β-咔啉衍生物的最为有效方法。自然界中,四氢异喹啉和β-咔啉是两类具有如抗病毒、抗肿瘤、抗菌、抗氧化等重要生物活性的生物碱。因此,作为多种生物碱合成过程中的基础反应,P-S反应近年来受到广泛关注。P-S反应在形成新的杂环体系中,原来的醛基碳原子成为一个新的手性中心,简单P-S反应得到的是外消旋混合物。然而,具有重要生物活性的物质往往是具有立体专一性的,因此,采用不同的不对称合成法,合成具有立体专一性的目标产物已成为P-S反应研究的热点,对于药物合成和天然产物全合成有十分重要的意义。由于自然界中存在具有立体专一性的天然产物,因此人们推测,自然界中存在能手性催化该类反应的物质,而生物体内的酶无疑是目前最完善的不对称合成技术中的关键,它作为一种具有高度立体专一性的生物催化剂引起人们的重视。
由酶催化的P-S反应在自然界中很少,迄今,从植物及动物脑内仅纯化和鉴定得到少数几种能催化P-S反应的酶,该类酶物质统称为“Pictet-Spengler酶”。该类酶中,有些为抗癌植物生物碱合成代谢途径中的关键酶,另一些酶的研究显示与帕金森氏病(PD)的致病机理相关。因此,将该类酶分离纯化并开展其生物学特性研究,具有重要的医学价值和社会意义,同时,对生物碱的大规模生产具有实际指导意义。
然而,迄今为止,从细菌中鉴定功能的P-S酶只有McbB一例,应用相应的酶催化形成β-咔啉及相应衍生物的报道更是罕见,制约着对该家族酶进行系统研究,并限制了其在生物、医药、精细化工等方面的应用。因此,未来应致力于新酶的发现,不断增添该家族的成员,并通过氨基酸或基因序列分析,以找出其序列同源性,为该类酶的定向进化及系统研究打下基础。
发明内容:
本发明的目的在于提供一种Pictet-Spengler酶NfcbB,编码该酶的基因,含有该编码基因的重组载体、重组菌、转基因细胞系和表达盒及其应用。
本发明采用的技术方案为如下:
本发明提供的酶NfcbB,选自下述a或b所示的蛋白质,其中:
a为由SEQ ID NO.2所示的氨基酸序列组成的蛋白质;
b为由SEQ ID NO.2所示的氨基酸序列经过一个或者几个氨基酸残基的取代和/或缺失和/或添加且具有Pictet-Spengler酶活性的由a衍生的蛋白质。
具体地,SEQ ID NO.2所示的氨基酸序列由314个氨基酸组成,分子量约为35.91kDa。
所述的蛋白质a和b可人工合成,也可先合成其编码基因,再进行生物表达得到。
本发明所提供的氨基酸序列可以用来分离所需要的蛋白并可用于抗体的制备。
包含本发明所提供的氨基酸序列或至少部分序列的多肽可能在去除或替代某些氨基酸之后仍有生物活性甚至有新的生物学活性,或者提高了产量或优化了蛋白动力学特征或其他致力于得到的性质。
进一步地,本发明还提供一种所述酶NfcbB的编码基因(nfcbB),所述的编码基因选自Ⅰ、Ⅱ或Ⅲ所示的DNA分子,其中:
Ⅰ为核苷酸序列如SEQ ID NO.1所示的DNA分子;
Ⅱ为与SEQ ID NO.1所示的核苷酸序列杂交且具有Pictet-Spengler酶活性的DNA分子;
Ⅲ为与SEQ ID NO.1所示的核苷酸序列有90%以上同源性且具有Pictet-Spengler酶活性的DNA分子。
如SEQ ID NO.1所示序列的第1-945位的碱基序列的互补序列可根据DNA碱基互补原则随时得到。且第1-945位的核苷酸序列或部分核苷酸序列可以通过聚合酶链式反应(PCR)或用合适的限制性内切酶对相应的DNA进行酶切或DNA体外合成技术或使用其他合适的技术得到。本发明提供了得到至少包含部分SEQ ID NO.1所示序列的第1-945位中DNA序列的重组DNA载体的途径。
本发明所提供的核苷酸列或部分核苷酸序列,可利用聚合酶链式反应(PCR)的方法或包含本发明SEQ ID NO.1所示序列的第1-945位的DNA作为探针以Southern杂交等方法从其他生物体中得到与nfcbB相似的基因。
包含本发明所提供的核苷酸序列或至少部分核苷酸序列可以被体内体外修饰或进行突变,包括插入、置换或缺失,聚合酶链式反应,错误介导聚合酶链式反应,位点特异性突变,不同序列的重新连接,序列的不同部分或与其他来源的同源序列进行定向进化,或通过紫外线或化学试剂诱变等。
包含本发明所提供的核苷酸序列或至少部分核苷酸序列的克隆可以通过合适的表达体系在外源宿主中表达以得到相应的酶或其他更高的生物活性物质或产量。这些外源宿主包括大肠杆菌、链霉菌、小单孢菌、假单孢菌、芽孢杆菌、酵母、植物和动物等。
包含本发明所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以在异源宿主中表达并了解它们在宿主代谢中的功能。
包含本发明所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以通过遗传重组来构建重组载体以获得新型生物合成途径,也可以通过插入、置换、缺失或失活进而获得其他新型生物合成途径或者产生新的化合物。
进一步地,含有所述NfcbB的编码基因nfcbB的重组载体、重组菌、转基因细胞系和表达盒也属于本发明的保护范围。
所述的重组载体是用常规方法将本发明编码基因nfcbB的核苷酸序列连接于各种载体上构建而成,该载体可以为本领域熟知的细菌质粒、噬菌体、酵母质粒、腺病毒、逆转录病毒或其它载体。
所述的重组菌是将包含本发明编码基因nfcbB核苷酸序列的表达载体转化到大肠杆菌BL21(DE3)即E.coli BL21(DE3)中得到的基因工程菌株。
进一步地,本发明还提供了酶NfcbB在催化L-色氨酸和丙酮醛形成如式Ⅰ所示的β-咔啉母核或其衍生物中的应用。
进一步地,本发明还提供了含有编码基因nfcbB的重组菌在制备如上式Ⅰ所示的β-咔啉母核或下式Ⅱ所示的β-咔啉衍生物中的应用。
所述的重组菌为重组大肠杆菌BL21(DE3)。
本发明的有益效果主要体现在:本发明提供的NfcbB的基因和蛋白信息,可用于生产β-咔啉骨架,增强人们对NfcbB的认识,为进一步遗传改造提供了材料和理论基础。本发明所提供的基因及其蛋白质也可以用来寻找和发现可用于医药、工业或农业的化合物、基因或蛋白。
本发明提供的Pictet-Spengler酶(NfcbB),来源于海洋放线菌Nocardiopsisflavescens(黄诺卡菌),该菌属于拟诺卡氏菌科(Nocardiopsaceae),是海洋放线菌新种,公开于非专利文献(Nocardiopsis flavescens sp.nov.,an actinomycete isolatedfrom marine sediment.Caiyuan Fang,Jianli Zhang,Huancheng Pang,Yuyi Li,YuhuaXin,Yabo Zhang.International Journal of Systematic and EvolutionaryMicrobiology.2011年,61卷,2640-2645页),申请人保证自本发明申请日起向公众提供。该菌现保存于普通微生物菌种保藏管理中心(China General Microbiological CultureCollection Center,CGMCC),保藏编号为:CGMCC 4.5723,该菌株对外销售,任何人都可以从该保藏中心购买到。
附图说明:
图1为β-咔啉生物碱类化合物1和2的化学结构式。
图2为酶NfcbB催化L-色氨酸和丙酮醛反应生成化合物1和2的化学反应式。
图3为酶NfcbB纯化的SDS-PAGE的电泳图。其中,泳道1为标准蛋白Marker;泳道2为菌体破碎离心得到的上清液;泳道3为纯化得到的NfcbB蛋白。
图4为NfcbB在不同温度下催化L-色氨酸和丙酮醛反应产物的HPLC分析结果。其中,i:对照实验,煮沸灭活的NfcbB;ii–x:分别为16℃、20℃、25℃、28℃、31℃、34℃、37℃、40℃和45℃;1和2代表图1所示的化合物1和2,横坐标为保留时间。
图5为nfcbB基因在E.coli BL21(DE3)中的表达得到的菌株在LB中发酵的发酵产物的HPLC分析图。其中,i:E.coli BL21(DE3)/pET28a(+)对照;ii-iv:E.coli BL21(DE3)/pET28a(+)/nfcbB的3个不同重复;v:化合物1的标准品;vi:化合物2的标准品;1和2代表图1所示的化合物1和2,横坐标为保留时间。
图6为L-色氨酸和丙酮醛(结构见图1)作为底物在酶NfcbB的催化作用下生成相应的β-咔啉母核化合物1及其类似物的HPLC分析图(反应温度为30℃)。其中,i:对照实验,煮沸灭活的NfcbB;ii:酶NfcbB;iii:化合物1的标准品;iv:化合物2的标准品;1和2代表图1所示的化合物1和2。
图7为化合物1的1H核磁共振谱的结果。
图8为化合物1的13C核磁共振谱的结果。
图9为化合物1的高分辨质谱结果。其中,化合物1:HR-ESI-MS(+)m/z[M+H]+=255.0777(cacld for C14H11N2O3,255.0764)。
图10为化合物2的高分辨质谱结果。其中,化合物2:HR-ESI-MS(+)m/z[M+H]+=211.0875(cacld for C13H11N2O,211.0866)。
具体实施方式:
本发明人通过对放线菌Marinactinospora thermotolerans SCSIO 00652中的咔啉生物碱(marincarbolines)生物合成基因簇中mcbB的编码蛋白McbB进行Blast分析,从中得到一个与McbB的一致性为67%的酶NfcbB,对应的编码基因为nfcbB。设计引物对nfcbB进行PCR扩增,得到的PCR产物依次经过胶回收、加“A”尾、TA克隆、测序验证,得到正确的重组载体pCR2.1/nfcbB,经酶切后克隆到pET28a(+)载体,得到重组表达载体pET28a(+)/nfcbB。然后转化E.coli BL21(DE3)从而得到重组表达菌株E.coli BL21(DE3)/pET28a(+)/nfcbB。
进一步地,本发明人通过在大肠杆菌中的异源表达实验和体外生化反应鉴定了NfcbB的功能,同时提供了一种通过大肠杆菌发酵或者酶反应制备β-咔啉母核(化合物1)的方法。
以下进一步提供实施例,这些实施实例有助于理解本发明,仅用作说明而不限制本发明的应用范围。
实施例1
放线菌Nocardiopsis flavescens CGMCC 4.5723基因组DNA的提取:
将放线菌Nocardiopsis flavescens CGMCC 4.5723的新鲜孢子按照体积分数5%的接种量接种于50mL的TSB培养基(胰蛋白胨17g,植物蛋白胨3g,氯化钠5g,磷酸氢二钾2.5g,葡萄糖2.5g,加水至1L,pH 7.2-7.4)中,28-30℃,振荡培养约2d,4000r/min离心10min收集菌丝体。菌丝体用STE溶液(NaCl 75mmol/L,EDTA 25mmol/L,Tris-HCl 20mmol/L,余量为水)洗涤两次,向洗涤后的菌丝体中加入30mL STE溶液和终浓度3mg/mL的溶菌酶,涡旋均匀,37℃温浴3h,加入至终浓度0.1-0.2mg/mL的蛋白酶K,混匀,37℃温浴10min,加入至终浓度1-2%的SDS,混匀,放入55℃水浴约1h,期间颠倒数次。加入等体积的酚-氯仿-异戊醇(V/V/V=25:24:1),混合均匀,置于冰上冷却30min。12000r/min,4℃离心10min,然后用剪过的大口径枪头小心吸取上清到新的离心管中,用同样的方法反复处理3次,然后用等体积的氯仿洗涤两次,12000r/min,4℃离心10min。用剪过的大口径枪头将水相吸出转移至新的离心管,加入1/10体积3mol/L NaAc(pH5.2),混匀后再加入等体积的异丙醇,混匀后冰上放置,沉淀DNA。小心地用玻璃棒将DNA纤维团转移至新的离心管中,用70%乙醇洗涤两次,将液体倾出,在37℃下略微烘干,加5mL TE溶解,并加入3-5U的RNA酶,由此得到放线菌Nocardiopsis flavescens CGMCC 4.5723的基因组DNA。
实施例2
NfcbB编码基因nfcbB在E.coli BL21(DE3)中的表达:
以Nocardiopsis flavescens CGMCC 4.5723的基因组DNA为模板,设计引物,上游引物F序列为:CATATGCGCCAGCTAGAGATCGAA,酶切位点为Nde I;下游引物R序列为:AAGCTTTCACCCCCGGGGGTAGGGGTA,酶切位点为Hind III。对基因nfcbB进行PCR扩增,PCR反应体系为:(50μL体系):5×Fast Pfu Buffer 10μL,dNTP Mix 5μL,上游引物F(10μM)1.5μL,下游引物R(10μM)1.5μL,DMSO 3μL,模板10ng,Fast Pfu DNA Polymerase 0.5μL,ddH2O27.5μL。反应条件和循环参数为:95℃预变性5min;95℃变性50sec,60℃50sec,72℃1.5min,30个循环;72℃延伸10min。得到的PCR产物依次经过胶回收、加“A”尾、TA克隆、测序验证(扩增出的基因nfcbB的核苷酸序列如SEQ ID NO.1所示),得到正确的重组载体pCR2.1/nfcbB(基因nfcbB插入到载体pCR2.1中)。经Nde I/Hind III酶切,克隆到与经同样的酶进行酶切的pET28a(+)载体,得到重组表达载体pET28a(+)/nfcbB。转化E.coli BL21(DE3)从而得到重组表达菌株E.coli BL21(DE3)/pET28a(+)/nfcbB。
实施例3
重组菌株E.coli BL21(DE3)/pET28a(+)/nfcbB的发酵和代谢产物的检测:
将得到的重组表达菌株E.coli BL21(DE3)/pET28a(+)/nfcbB划线培养至LB固体平板,37℃过夜培养后挑取单克隆过夜培养后按体积分数1%的接种量接入到250mL三角瓶的50mL LB培养液体,于28℃摇床180r/min培养至OD600约为0.6时,往培养液中加入终浓度为0.1mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)。于28℃继续培12h后,往发酵物加入适量1mol/L的HCl以调节其pH值至5,加入二倍体积的乙酸乙酯,超声5min破碎细胞,然后静置分层。将乙酸乙酯萃取液与水相分离,用旋转蒸发仪将乙酸乙酯蒸干,残留物溶解于甲醇形成样品,进行高效液相色谱(HPLC)检测,检测条件为Phenomenex(150×4.6mm,5μm)反相柱,流动相A相为体积分数15%乙腈水溶液,含0.1%乙酸,流动相B相为体积分数85%乙腈水溶液,含0.1%乙酸;流速为1mL/min,检测波长为215nm和285nm。HPLC程序:0-20min,0%-70%B相;20-20.1min,70%-100%B相;20.1-26min,100%B相;26-26.1min,100%-0%B相,26.1-30min为0%B相。以转有空载体pET28a(+)的E.coli BL21(DE3)重组菌E.coli BL21(DE3)/pET28a(+)作为对照。
结果如图5所示,从图5可以看出,对照重组菌E.coli BL21(DE3)/pET28a(+)的发酵产物没有检测到化合物1和2。而E.coli BL21(DE3)/pET28a(+)/nfcbB的发酵产物粗提物中可以检测到化合物1和2。由此可知,含nfcbB基因的重组基因工程菌株可表达β-咔啉生物碱类化合物。
实施例4
重组菌株E.coli BL21(DE3)/pET28a(+)/nfcbB的扩大发酵和部分产物的分离和鉴定:
Pictet-Spengler酶编码基因nfcbB在重组菌株E.coli BL21(DE3)的表达和扩大发酵见实施例2和实施例3。最后发酵物经乙酸乙酯萃取得到粗提物。粗提物首先通过正相硅胶柱层析方法,经氯仿-甲醇体系梯度洗脱(氯仿:甲醇=100:0,98:2,96:4,94:6,92:8,9:1,8:2v/v)后,顺序得到AFr.1-AFr.7共7个馏分,通过TLC结合HPLC追踪分析得到含有目标化合物的馏分。
AFr1主要含有化合物1,AFr2-AFr7再分别通过反相HPLC制备得BFr1、BFr2、BFr3三个馏分。反向制备用HPLC为KNAUER LC3000型高效液相色谱仪,制备柱为YMC C18250×20mm5μm,A流动相为100%ddH2O+0.1%乙酸,B流动相为100%乙腈+0.1%乙酸,制备条件:30%-80%流动相B,20min;80-100%流动相B,2min;100%流动相B,5min;0%流动相B,3min;流速8mL/min,由此分别得到BFr1(保留时间5-15min)、BFr2(保留时间18-22min)、BFr3(保留时间23-30min)三个馏分。
AFr1通过反向半制备HPLC制备得到化合物1。反向制备高效液相色谱仪为日立半制备型,制备柱为YMC C18 250×10mm 5μm,A流动相为100%ddH2O+0.1%乙酸,B流动相为100%乙腈+0.1%乙酸,制备条件:30%-80%流动相B,20min;80-100%流动相B,2min;100%流动相B,5min;0%流动相B,3min;流速2mL/min,由此得到化合物1馏分(保留时间约15min)。
将BFr1、BFr2、BFr3合并,通过反向半制备HPLC制备得到化合物2。反向制备高效液相色谱仪为日立半制备型,制备柱为YMC C18 250×10mm 5μm,A流动相为100%ddH2O+0.1%乙酸,B流动相为100%乙腈+0.1%乙酸,制备条件:30%-80%流动相B,20min;80-100%流动相B,2min;100%流动相B,5min;0%流动相B,3min;流速2mL/min,由此得到化合物2馏分(保留时间约18min)。
制备出来的馏分经旋转蒸发仪减压加热蒸馏后得到化合物1和2,用氯仿-甲醇转移至小的玻璃瓶中,干燥后称重并转移至核磁管中,备用。
将得到的化合物1和2进行鉴定,具体为:对化合物1进行核磁共振波谱法(1H–NMR和13C–NMR)鉴定,其NMR鉴定结果如图7、8和表1所示。对化合物1和2进行高分辨质谱法进行鉴定,其鉴定结果如图9、10所示。所得到的化合物1和2的化学结构式分别如图1中的式1和2所示。
表1:化合物1的NMR数据(500/125MHz,TMS为内标,ppm)
实施例5
酶NfcbB的表达、亲和层析纯化、SDS-PAGE电泳分析和Bradford法含量测定、体外生化反应及产物鉴定:
将重组菌株E.coli BL21/pET28a(+)/nfcbB过夜培养后,按体积分数1%的接种量接入到1L三角瓶(共计15个)的300mL LB培养液体,于28℃摇床200r/min培养至OD600约为0.6时,往培养物中加入终浓度为0.1mmol/L的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)。再于28℃150r/min继续培6-8h,4000r/min离心10min收集菌体,用50mmol/L Tris-HCl洗涤菌体两次,最后重悬于15-20mL的Binding Buffer中(20mmol/L Tris-HCl,500mmol/L NaCl,5mmol/L imidazole,pH 8.0),0℃超声裂解菌体。待菌体裂解后,4℃、10000r/min离心40min。然后按照以下过程纯化蛋白:(1)装柱:上清加入Ni-NTA亲和柱中,收集滤液;(2)洗涤:加入5-15mL Binding Buffer(大约5-15柱体积),收集滤液加入10-20mL WashingBuffer(大约15-20柱体积,20mmol/L Tris-HCl,pH 8.0,500mmol/L NaCl,50mmol/Limidazole)洗去与Ni-NTA柱相结合的杂蛋白;(3)洗脱:加入3.5mL的Elution Buffer 1(20mmol/L Tris-HCl,pH 8.0,500mmol/L NaCl,250mmol/L imidazole)把目的蛋白从Ni-NTA柱上洗脱下来,为防止目的蛋白与Ni-NTA柱过度结合,继续用1mL Elution Buffer 2(20mmol/L Tris-HCl,pH 8.0,500mmol/L NaCl,1mol/L imidazole)洗涤;(4)浓缩:将Elution Buffer I洗脱下来的3.5mL滤液转入15mL 10kD超滤管中,2500r/min、4℃离心20min,浓缩至小于2.5mL;(5)脱盐:将浓缩后的蛋白转入PD-10脱盐柱脱盐,用2.5mL的Storage Buffer洗脱(30%glycerol,100mmol/L磷酸钾,pH 7.0)把目的蛋白冲洗下来,再用10kD超滤管浓缩,浓缩后的目的蛋白于-80℃或-20℃保存备用,由此得到基因nfcbB编码的蛋白酶NfcbB(其氨基酸序列如SEQ ID NO.2所示)。
纯化后的蛋白酶NfcbB取出2-20μL,补水至20μL,加入5μL 5×SDS PAGE LoadingBuffer,充分混匀后沸水煮10-15min,取3-8μL上样SDS PAGE电泳(Tris-Glycin缓冲液系统),同时,纯化后的蛋白分别稀释1倍、2倍、10倍,补水至20μL,加入1mL 1×Bio-RadProtein Assay试剂,反应5-60min,测定595nm下的吸光度,通过和Bradford标准蛋白含量曲线比较,计算酰胺键合成酶McbA的浓度。
蛋白NfcbB的SDS-PAGE如图3所示,纯化后得到的NfcbB蛋白的浓度约为30μM。
实施例6
NfcbB的体外生化反应及产物鉴定:
1mmol/L L-色氨酸+2mmol/L丙酮醛(methylglyoxal)+2μmol/L酶NfcbB+50mmol/LpH7.5磷酸盐缓冲液,30℃反应10h,用1mL,用Agilent 1260 Infinity System HPLC分析产物,通过与标准品对照确定产物。HPLC检测条件为Phenomenex(150×4.6mm,5μm)反相柱,流动相A相为体积分数15%乙腈水溶液,含0.1%乙酸,流动相B相为体积分数85%乙腈水溶液,含0.1%乙酸;流速为1mL/min,检测波长为215nm和285nm。HPLC程序:0-20min,0%-70%B相;20-20.1min,70%-100%B相;20.1-26min,100%B相;26-26.1min,100%-0%B相,26.1-30min为0%B相。以煮沸灭活的酶NfcbB作为对照。
结果如图6所示,从图6可以看出,酶NfcbB负责L-色氨酸和丙酮醛的缩合反应,进而产生化合物1。
进一步地,本发明还考察了酶NfcbB分别在16℃、20℃、25℃、28℃、31℃、34℃、37℃、40℃或45℃下催化L-色氨酸和丙酮醛反应形成的产物,结果如图4所示,由图4可知,不同温度对酶催化L-色氨酸和丙酮醛的缩合形成化合物1具有重要的影响,温度偏低或偏高均不利于化合物1的生成。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,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。
序列表
<110> 安徽医科大学
<120> 一种Pictet-Spengler酶及其编码基因与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 945
<212> DNA
<213> 黄诺卡菌 CGMCC 4.5723(Nocardiopsis flavescens CGMCC 4.5723)
<400> 1
atgcgccagc tagagatcga atgggtccag ccgcggatca ccgtcaccgc cgacctcacc 60
gacgacaacc cggacctggt cgacatggtc tggaacaggc tggtccccta caacagcctg 120
cagaaccacg cgctggtctc cggcgaccac ctctaccacc tcctgcccga cccggaaccg 180
atccacaggg ccgccgagta caagatgcgg gaccgcaccg aggcgccgga cggcaccgtc 240
ttcctctccc agctgcagca catcggcatc aagtacggac cgctcaccga gtacctgccc 300
gccgcgccga tcggccacgt cgttcccgac gacgtcgacg ccctgcgcag cgccggccgc 360
tactgctggg acgcggccta ccggaccaag gaggtcgtcg aggtccgggt acggcgcaag 420
ggcgaccggg tggacgacta cccgctcccc cggcctcccc gcgtcggcga ccccgacgtg 480
cagcgcctca tcgacgagat gcacgacgcc acccggcgct cgtggacgga cccgcccgag 540
gagctcgtcg acatgcacgc gggccggatc ggcagccggg ccgggagcag ggaccagtac 600
ttctccacgc tggtcttcct caacggcgag gtgcggccgc tggggtacaa cgcgctcaac 660
ggactcgtcc gcctcgtgcg gaccaccgac ctcccgctgg agaccctccg ggagatcacg 720
cccggcttca tccgcacacc ggcggagttc ctgggctaca ccggactgca cgccctggag 780
gacttcaccg ggcgaagcgt caaggcactc ggcgcgctgg aggaccggga ggagtacttc 840
gcgctgatca gcaccctggc cctgtacacg aacatgctca acacctggaa cctccacctg 900
ttcccgtggg accacggaaa ggagtacccc tacccccggg ggtga 945
<210> 2
<211> 314
<212> PRT
<213> 黄诺卡菌 CGMCC 4.5723(Nocardiopsis flavescens CGMCC 4.5723)
<400> 2
Met Arg Gln Leu Glu Ile Glu Trp Val Gln Pro Arg Ile Thr Val Thr
1 5 10 15
Ala Asp Leu Thr Asp Asp Asn Pro Asp Leu Val Asp Met Val Trp Asn
20 25 30
Arg Leu Val Pro Tyr Asn Ser Leu Gln Asn His Ala Leu Val Ser Gly
35 40 45
Asp His Leu Tyr His Leu Leu Pro Asp Pro Glu Pro Ile His Arg Ala
50 55 60
Ala Glu Tyr Lys Met Arg Asp Arg Thr Glu Ala Pro Asp Gly Thr Val
65 70 75 80
Phe Leu Ser Gln Leu Gln His Ile Gly Ile Lys Tyr Gly Pro Leu Thr
85 90 95
Glu Tyr Leu Pro Ala Ala Pro Ile Gly His Val Val Pro Asp Asp Val
100 105 110
Asp Ala Leu Arg Ser Ala Gly Arg Tyr Cys Trp Asp Ala Ala Tyr Arg
115 120 125
Thr Lys Glu Val Val Glu Val Arg Val Arg Arg Lys Gly Asp Arg Val
130 135 140
Asp Asp Tyr Pro Leu Pro Arg Pro Pro Arg Val Gly Asp Pro Asp Val
145 150 155 160
Gln Arg Leu Ile Asp Glu Met His Asp Ala Thr Arg Arg Ser Trp Thr
165 170 175
Asp Pro Pro Glu Glu Leu Val Asp Met His Ala Gly Arg Ile Gly Ser
180 185 190
Arg Ala Gly Ser Arg Asp Gln Tyr Phe Ser Thr Leu Val Phe Leu Asn
195 200 205
Gly Glu Val Arg Pro Leu Gly Tyr Asn Ala Leu Asn Gly Leu Val Arg
210 215 220
Leu Val Arg Thr Thr Asp Leu Pro Leu Glu Thr Leu Arg Glu Ile Thr
225 230 235 240
Pro Gly Phe Ile Arg Thr Pro Ala Glu Phe Leu Gly Tyr Thr Gly Leu
245 250 255
His Ala Leu Glu Asp Phe Thr Gly Arg Ser Val Lys Ala Leu Gly Ala
260 265 270
Leu Glu Asp Arg Glu Glu Tyr Phe Ala Leu Ile Ser Thr Leu Ala Leu
275 280 285
Tyr Thr Asn Met Leu Asn Thr Trp Asn Leu His Leu Phe Pro Trp Asp
290 295 300
His Gly Lys Glu Tyr Pro Tyr Pro Arg Gly
305 310
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