CN118147178A - 甲硫氨酸裂解酶、编码基因、重组载体、宿主细胞及应用 - Google Patents
甲硫氨酸裂解酶、编码基因、重组载体、宿主细胞及应用 Download PDFInfo
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Abstract
本发明涉及甲硫氨酸裂解酶、编码基因、重组载体、宿主细胞及应用,涉及基因工程技术领域。所述的甲硫氨酸裂解酶的cDNA序列为(a)、(b)和(c)中的任意一项所示:(a)如SEQ ID NO:1所示的核苷酸;(b)与SEQ ID NO:1互补的序列,在严谨杂交条件能够进行杂交的核苷酸;(c)与如SEQ ID NO:1所示的核苷酸至少有80%以上同源性的核苷酸序列,且表达所述的甲硫氨酸裂解酶。本发明的甲硫氨酸裂解酶的编码基因,能够在宿主细胞中高效表达甲硫氨酸裂解酶,该甲硫氨酸裂解酶能够在20℃低温条件下达到较高的甲硫氨酸裂解酶效率。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及甲硫氨酸裂解酶、编码基因、重组载体、宿主细胞及应用。
背景技术
甲硫氨酸裂解酶具有广泛的工业和医药用途。在工业领域,甲硫氨酸裂解酶以甲硫氨酸为底物,直接催化合成甲基硫醇及其衍生物二甲基硫、二甲基二硫和二甲基三硫,这些含硫化合物是重要的食品香料添加剂,主要用于配制玉米、番茄、土豆、奶制品、菠萝和橘子类果香及青香型香精【Martinez-Cuesta Mdel,C.,Pelaez,C.,Requena,T.,2013.Methionine metabolism:major pathways and enzymes involved and strategiesfor control and diversification of volatile sulfur compounds in cheese.CritRev Food Sci Nutr.53,366-385.】;功能酶在常温条件下(25-40℃)处理食品需要消耗更多的能源、易引发常温微生物的污染,且酶促反应产物VOSCs在常温条件下易挥发,不易保持食品香味和品质【Siddiqui KS.Some like it hot,some like it cold:Temperaturedependent biotechnological applications and improvements in extremophilicenzymes.Biotechnology Advances 2015,33:1912-1922;Duarte AWF,Santos JA,ViannaMV,Vieira JMF,Mallagutti VH,Inforsato FJ,et al.Cold-adapted enzymes producedby fungi from terrestrial and marine antarctic environments.Critical Reviewsin Biotechnology 2018,38:1-19;Mhetras N,Mapare V,Gokhale D.Cold activelipases:Biocatalytic tools for greener technology.Applied Biochemistry andBiotechnology 2021,193:2245-2266】。另外,主产物甲基硫醇也是一种重要的燃料添加剂,同时可以作为生产甲硫氨酸和杀虫剂的前体化合物【Gutierrez,O.Y.,Zhong,L.S.,Zhu,Y.Z.,Lercher,J.A.,2013.Synthesis of methanethiol from CS2 on Ni-,Co-,andK-Doped MoS2/SiO2 catalysts.Chemcatchem.5,3249-3259;Zhang,Y.H.,Chen,S.P.,Wu,M.,Fang,W.P.,Yang,Y.Q.,2012.Promoting effect of SiO2 on the K2WO4/Al2O3catalysts for methanethiol synthesis from methanol and H2S.Catal Commun.22,48-51】。甲硫氨酸裂解酶在医药领域也有广泛的应用范围,通过食品添加甲硫氨酸裂解酶或者体内注射以降低哺乳动物体内甲硫氨酸浓度成为一种治疗依赖性甲硫氨酸癌症的重要技术【Sun,X.,Yang,Z.,Li,S.,Tan,Y.,Zhang,N.,Wang,X.,Yagi,S.,Yoshioka,T.,Takimoto,A.,Mitsushima,K.,Suginaka,A.,Frenkel,E.P.,Hoffman,R.M.,2003.In vivoefficacy of recombinant methioninase is enhanced by the combination ofpolyethylene glycol conjugation and pyridoxal 5'-phosphatesupplementation.Cancer Res.63,8377-8383;Lu WC,Saha A,Yan W,Garrison K,Lamb C,Pandey R,Irani S,Lodi A,Lu X.,Tiziani S.,Zhang YJ.,Georgiou G.,DiGiovanni J.,Stone E.,2020.Enzyme-mediated depletion of serum l-Met abrogates prostatecancer growth via multiple mechanisms without evidence of systemictoxicity.Proc Natl Acad Sci U S A.117(23),13000-13011;Hu,Y.,Liu,Y.,Zhang,J.,Zhou,Z.,Wang,J.,Chen,H.,Huang,M.,Hu,H.,Dai,Z.,&Jia,K.,2023Depletion of L-Methionine in Foods with an Engineered Thermophilic Methionineγ-lyaseEfficiently Inhibits Tumor Growth.J.Agric.Food Chem.71,17141-17152】;同时,甲硫氨酸裂解酶亦可通过感染的方式进入癌症细胞,引发癌症细胞的死亡和严重聚集,是基因治疗癌症的一种前沿技术【Venkatachalam,KV,2015.Novel cancer therapy:Targetingmethionine metabolism.29(1):897】。鉴于此,有必要研发一种耐低温甲硫氨酸裂解酶,以解决现有技术的不足。
发明内容
本发明所要解决的技术问题是提供甲硫氨酸裂解酶、编码基因、重组载体、宿主细胞及应用。目的在于在低温下采用甲硫氨酸裂解酶使食品中的甲硫氨酸裂解。
解决上述技术问题的技术方案如下:
第一方面,一种甲硫氨酸裂解酶的编码基因,所述的甲硫氨酸裂解酶的cDNA序列为(a)、(b)和(c)中的任意一项所示:
(a)如SEQ ID NO:1所示的核苷酸;
(b)与SEQ ID NO:1互补的序列,在严谨杂交条件能够进行杂交的核苷酸;
(c)与如SEQ ID NO:1所示的核苷酸至少有80%以上同源性的核苷酸序列,且表达所述的甲硫氨酸裂解酶。
本发明的有益效果是:本发明的甲硫氨酸裂解酶的编码基因,能够在宿主细胞中高效表达甲硫氨酸裂解酶,该甲硫氨酸裂解酶能够在20℃低温条件下达到较高的甲硫氨酸裂解酶效率。
上述(b)中的“严谨杂交条件”,意指在所属领域中已知的低离子强度和高温条件。通常,在严谨条件下,探针与其靶序列杂交的可检测程度比其他序列杂交的可检测程度更高(例如超过本底至少2倍)。严谨序列杂交是序列依赖性的,在不同的环境条件下将会不同,较长的序列在较高温度下特异杂交。通过控制杂交的严谨性或洗涤条件可鉴定与探针100%互补的靶序列。对于核酸杂交的详细指导,可参考文献3。更具体的,所述严谨条件通常被选择为低于特异序列在规定离子强度及pH下的热熔点(Tm)5-10℃。Tm为在平衡状态下50%与目标互补的探针杂交到目标序列时所处的温度(在指定离子强度、pH和核酸浓度下)。因为目标序列过量存在,在Tm下在平衡状态下50%的探针被占据。严谨条件可为以下条件:其中pH值在7.0-8.3下,盐浓度低于约1.0M钠离子浓度,通常为0.01M-1.0M钠离子浓度(或其它盐),并且温度对于短探针(包括(但不限于)10到50个核苷酸)而言为至少约30℃,而对于长探针(包括(但不限于)大于50个核苷酸)而言为至少约60℃。严谨条件也可通过加入诸如甲酰胺的去稳定剂来实现。对于选择性或特异性杂交而言,正信号可为至少两倍的背景杂交,视情况为10倍背景杂交。例示性严谨杂交条件可如下:50%甲酰胺,5×SSC和1%SDS,在42℃下培养;或5×SSC,1%SDS,在65℃下培养,在0.2×SSC中洗涤和在65℃下于0.1%SDS中洗涤。所述洗涤可进行5min、15min、30min、60min、120min或更长时间。
第二方面,一种甲硫氨酸裂解酶,所述的甲硫氨酸裂解酶由所述的编码基因表达得到。
采用上述方案的有益效果是:本发明的甲硫氨酸裂解酶能够在20℃低温条件下达到较高的甲硫氨酸裂解酶效率。
进一步,所述的甲硫氨酸裂解酶具有SEQ ID NO:2所示氨基酸序列。
其中,与如SEQ ID NO:2所示的核苷酸至少有90%以上同源性的核苷酸序列。或与如SEQ ID NO.1所示的核苷酸至少有95%以上同源性的核苷酸序列。或与如SEQ ID NO.1所示的核苷酸至少有97%以上同源性的核苷酸序列。均属于本发明的保护的范围。
第三方面,一种重组载体,所述的重组载体含有所述的甲硫氨酸降解酶的编码基因。
采用上述方案的有益效果是:该重组载体含有上述编码基因,并能够在宿主细胞中高效表达编码基因。
进一步,所述的重组载体为pET28a-yMGL(1-252)。
第四方面,一种宿主细胞,所述的宿主细胞含有所述的甲硫氨酸降解酶的编码基因或如所述的重组载体。
采用上述方案的有益效果是:该宿主细胞含有上述编码基因,该基因基于宿主细胞密码子偏好性进行密码子优化,能够在宿主细胞中高效启动表达。
进一步,所述的宿主细胞为大肠杆菌。
进一步,所述的宿主细胞为大肠杆菌Escherichia coli BL21。
第五方面,一种甲硫氨酸裂解酶的制备方法,包括如下步骤:
(1)合成含有SEQ ID NO:1表达甲硫氨酸裂解酶的重组序列;
(2)将所述重组序列组装为重组载体;
(3)表达所述的重组载体,得到甲硫氨酸裂解酶。
采用上述方案的有益效果是:通过该方法得到的该甲硫氨酸裂解酶能够在20℃低温条件下达到较高的甲硫氨酸裂解酶效率,且该方法操作简易,市场前景广阔,适合规模化推广应用。
优选的,得到甲硫氨酸裂解酶的过程为:收集诱导表达后的菌体,采用液氮中速冻、高压匀质进行破碎菌体,后纯化得到。
其中,上述的纯化的方法包括如下步骤:清洗并再生的His-标记蛋白纯化柱,用Lysis Buffer缓冲液进行柱平衡;平衡完毕后,将收集诱导表达后的菌体用Lysis Buffer缓冲液重悬,高压匀质破碎细胞壁,离心取上清进行上样结合;上样完毕后,用含有10,20,50,100mM含有咪唑的裂解缓冲液将柱上的非特异性杂质洗去;用含有200mM咪唑的洗脱缓冲液收集目的蛋白;将收集的目的蛋白过脱盐柱进行纯化并收集,即可得到纯化的甲硫氨酸裂解酶。
第六方面,一种甲硫氨酸裂解酶的应用,将所述的甲硫氨酸裂解酶用于食品中甲硫氨酸的降解。
采用上述方案的有益效果是:本发明的甲硫氨酸裂解酶能够在20℃低温条件下,较高的甲硫氨酸裂解酶效率降解食品中存在的甲硫氨酸,具备广泛的应用前景。
术语解释:
术语“宿主细胞”意指包含本发明多核苷酸的细胞,而不管使用何种方法进行插入以产生重组宿主细胞,例如直接摄取、转导、f配对或所属领域中已知的其它方法。宿主细胞可为原核细胞或真核细胞,宿主细胞还可为单子叶或双子叶植物细胞。
术语“核苷酸”意指单股或双股形式的脱氧核糖核苷酸、脱氧核糖核苷、核糖核苷或核糖核苷酸及其聚合物。除非特定限制,否则所述术语涵盖含有天然核苷酸的已知类似物的核酸,所述类似物具有类似于参考核酸的结合特性并以类似于天然产生的核苷酸的方式进行代谢。除非另外特定限制,否则所述术语也意指寡核苷酸类似物,其包括PNA(肽核酸)、在反义技术中所用的DNA类似物(硫代磷酸酯、磷酰胺酸酯等等)。除非另外指定,否则特定核酸序列也隐含地涵盖其保守修饰的变异体(包括(但不限于)简并密码子取代)和互补序列以及明确指定的序列。特定而言,可通过产生其中一个或一个以上所选(或所有)密码子的第3位经混合碱基和/或脱氧肌苷残基取代的序列来实现简并密码子取代。
术语“多肽”、“酶”和“蛋白质”在本文中互换使用以意指氨基酸残基的聚合物。即,针对多肽的描述同样适用于描述肽和描述蛋白,且反之亦然。所述术语适用于天然产生氨基酸聚合物以及其中一个或一个以上氨基酸残基为非天然编码氨基酸的氨基酸聚合物。如本发明中所使用,所述术语涵盖任何长度的氨基酸链,其包括全长蛋白(即抗原),其中氨基酸残基经由共价肽键连接。
本发明中所述的“替换”是指分别用不同的氨基酸残基取代一个或多个氨基酸残基;所述的“缺失”是指氨基酸残基数量的减少,也即是分别缺少其中的一个或多个氨基酸残基;所述的“插入”是指氨基酸残基序列的改变,相对天然分子而言,所述改变导致添加一个或多个氨基酸残基。
附图说明
图1为本发明筛选菌株的菌落PCR鉴定图;其中,1至10分别为挑选的菌株编号;
图2为本发明纯化的甲硫氨酸降解酶yMGL(1-252)的SDS-PAGE图;
图3为本发明不同温度下的降解效果图;
图4为甲硫氨酸降解酶yMGL与yMGL(1-252)降解效果对照图。
具体实施方式
以下对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购得的常规产品。
实施例:
1、实验材料
大肠杆菌Escherichia coli BL21,购自武汉淼灵生物科技有限公司;大肠杆菌感受态细胞Escherichia coli BL21(DE3),购自Trans Gen Biot ech公司。
2、实验仪器
CX41倒置相差显微镜和BX51荧光显微镜,均购自日本Olympus公司;DTX880酶联免疫检测仪,购自美国Beckman公司;751GD紫外分光光度计,购自杭州汇尔仪器有限公司;CytoFLEX流式细胞仪,购自贝克曼库尔特国际贸易(上海)有限公司。
3、实验方法
LB培养基配方:胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L和卡纳霉素50mg/L。
(1)重组载体为pET28a-yMGL(1-252)的构建
选择SEQ ID NO:1所示的碱基序列,5端位点为NdeI,3端位点为Xh oI,目标载体为pET28a,委托北京擎科生物科技股份有限公司进行基因合成,将合成后的4μg质粒加入100μL的ddH2O,取出3μL的质粒加入到50μL的Escherichia coli BL21(DE3)感受态细胞中,将EP管重置于冰中30mi n,然后放入42℃水浴热激45s,再放入冰中静置2min。每管加入900μLLB培养基,在37℃、180rpm摇床培养60min;然后2000g离心5min,取850μL上清,将沉淀轻轻混匀,涂布在含有卡纳霉素100mg/L的LB平板上,在37℃恒温箱中培养10-12h。
(2)阳性产物的筛选
将平板上长出的单菌落接种于含有50mg/L卡纳霉素的LB平板上,37℃培养5h,挑取单菌落测序,测序序列结果显示载入的yMGL(1-252)-His片段长度为756bp,表明yMGL(1-252)合成成功(图1)。
(3)重组载体为pET28a-yMGL(1-252)的表达和纯化
取少量活化后菌液加入含有卡那霉素的10mL LB培养基中,生长10小时。收集试管培养基中的菌液,接种于1L的含卡纳霉素LB培养基中,生长5小时,OD600达0.6~0.8时加终浓度为0.5mM的IPTG进行诱导培养,18℃、180rpm诱导18h。用高速冷冻离心机(J-26XP,Beckma n),2000g,4℃离心30min,收集诱导后的菌体。
将收集的菌体在液氮中速冻,解冻后加入35mL的Lysis buffer重悬,进行高压匀质离心(1100MPa,5min)至溶液变为透明状态为止,匀质结束后,15000rpm,4℃离心30min,收集破碎细胞后离心得到的上清。
准备His-标记蛋白纯化柱,超滤水,超纯水洗3遍,Lysis buffer缓冲液洗3次,将上清过His-标记蛋白纯化柱,流穿1次;用裂解缓冲液清洗His-标记蛋白纯化柱上的杂蛋白,每次40mL,洗1次;上样完毕后,用含有10,20,50,100mM含有咪唑的裂解缓冲液将柱上的非特异性杂质洗去;用含有200mM咪唑的洗脱缓冲液收集目的蛋白;将收集的目的蛋白过脱盐柱进行纯化并收集,即可得到纯化的甲硫氨酸裂解酶(图2)。
所述的Lysis Buffer柱平衡缓冲液由以下部分组成:pH值为8.0的20mM Tris-HCl,300mM KCl,10%体积甘油。
所述裂解缓冲液由如下组分组成:pH值为8.0的10mM Tris-HCl,300mM KCl,10mM咪唑,10%体积甘油和1mM苯甲基磺酰氟;
所述裂解缓冲液由如下组分组成:pH值为8.0的10mM Tris-HCl,300mM KCl,20mM咪唑,10%体积甘油和1mM苯甲基磺酰氟;
所述裂解缓冲液由如下组分组成:pH值为8.0的10mM Tris-HCl,300mM KCl,50mM咪唑,10%体积甘油和1mM苯甲基磺酰氟;
所述裂解缓冲液由如下组分组成:pH值为8.0的10mM Tris-HCl,300mM KCl,100mM咪唑,10%体积甘油和1mM苯甲基磺酰氟;
所述洗脱缓冲液由如下组分组成:pH值为8.0的20mMTris-HCl,300mMKCl,200mM咪唑,10%体积甘油和1mM苯甲基磺酰氟。
浓缩后使用分子筛去除杂质后,采用BCA(Bicinchoninic Acid)法对通过镍柱纯化的yMGL(1-252)洗脱液浓度进行测定,浓度为6.2mg/mL,共收集了2mL蛋白洗脱液,蛋白最终质量为12.4mg。出发菌液为2L,计算得到的甲硫氨酸裂解酶的得率大约为6.2mg/L。
(4)甲硫氨酸裂解酶yMGL(1-252)的降解应用
(4-1)最适温度测定
为测定yMGL(1-252)最适温度,故构建酶促反应体系,在pH=8.0(在此pH值条件下,MGL的催化活性最高),50mM Tris-HCl缓冲溶液中加入20mM甲硫氨酸、5μM PLP和0.6mg已纯化的MGL酶,总体积为1mL。反应温度分别为0℃,4℃,10℃,15℃,20℃,30℃,37℃,45℃,65℃,反应时间为2h。
高效液相色谱法检测甲硫氨酸浓度的降低(或α-酮丁酸产物的积累):
取200μL的酶促反应液与乙腈(LC-MS级)1:1等比例混合,充分振荡混匀。再取200μL混合液加入含有内衬管的液相瓶中,拧紧瓶盖,按照样品顺序,放置于液相进样盘中。甲硫氨酸和α-酮丁酸的保留时间分别为5.08min和5.81min。yMGL(1-252)催化甲硫氨酸反应产物的高效液相检测条件为:
在装有Reprosil Pur Basic C18柱(4.6mm×150mm×5μm)色谱柱的Ultimate3000HPLC仪器中进行检测。流动相为乙腈:磷酸二氢钾=13:87(v:v),并用磷酸调节pH至3.0。流速调节至0.5mL/min,柱温保持在40℃,检测波长为230nm。在20℃酶活达到做高,表明yMGL(1-252)的最适温度为20℃,20℃属于在低温的范围之内,表明yMGL(1-252)为耐低温酶。(图3)
(4-2)酶活比对实验
为了比较yMGL和yMGL(1-252)的酶活。其中yMGL降解甲硫氨酸的活力为248.22±2.03nmol·min-1·mg recombinant protein-1,详见文献记载:Zhao,Q.L.,Wang,Z.L.,Yang,L.,Zhang,S.,&Jia,K.Z.(2021).YALI0C22088g from Yarrowia lipolyticacatalyses the conversion ofl-methionine into volatile organic sulfur-containing compounds.Microbi al biotechnol.14(4),1462–1471.
本发明构建了酶促反应体系如上,反应温度在20℃,得到yMGL(1-252)的酶活是yMGL的7.2倍。(图4)
综上可知,本发明的甲硫氨酸裂解酶的编码基因,能够在宿主细胞中高效表达甲硫氨酸裂解酶,该甲硫氨酸裂解酶能够在20℃低温条件下达到较高的甲硫氨酸裂解酶效率。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
1.一种甲硫氨酸裂解酶的编码基因,其特征在于,所述的甲硫氨酸裂解酶的cDNA序列为(a)、(b)和(c)中的任意一项所示:
(a)如SEQ ID NO:1所示的核苷酸;
(b)与SEQ ID NO:1互补的序列,在严谨杂交条件能够进行杂交的核苷酸;
(c)与如SEQ ID NO:1所示的核苷酸至少有80%以上同源性的核苷酸序列,且表达所述的甲硫氨酸裂解酶。
2.一种甲硫氨酸裂解酶,其特征在于,所述的甲硫氨酸裂解酶由权利要求1所述的编码基因表达得到。
3.根据权利要求2所述一种甲硫氨酸裂解酶,其特征在于,所述的甲硫氨酸裂解酶具有SEQ ID NO:2所示氨基酸序列。
4.一种重组载体,其特征在于,所述的重组载体含有如权利要求1所述的甲硫氨酸降解酶的编码基因。
5.根据权利要求4所述一种重组载体,其特征在于,所述的重组载体为pET28a-yMGL(1-252)。
6.一种宿主细胞,其特征在于,所述的宿主细胞含有如权利要求1所述的甲硫氨酸降解酶的编码基因或如权利要求4-5任一项所述的重组载体。
7.根据权利要求6所述一种宿主细胞,其特征在于,所述的宿主细胞为大肠杆菌。
8.根据权利要求6所述一种宿主细胞,其特征在于,所述的宿主细胞为大肠杆菌Escherichia coli BL21。
9.基于权利要求2或3所述一种甲硫氨酸裂解酶的制备方法,其特征在于,包括如下步骤:
(1)合成含有SEQ ID NO:1表达甲硫氨酸裂解酶的重组序列;
(2)将所述重组序列组装为重组载体;
(3)表达所述的重组载体,得到甲硫氨酸裂解酶。
10.一种甲硫氨酸裂解酶的应用,其特征在于,将所述的甲硫氨酸裂解酶用于食品中甲硫氨酸的降解。
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