CN112725323B - 一种重组耐盐腺苷酸环化酶及其编码基因和应用 - Google Patents
一种重组耐盐腺苷酸环化酶及其编码基因和应用 Download PDFInfo
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- CN112725323B CN112725323B CN202110163676.2A CN202110163676A CN112725323B CN 112725323 B CN112725323 B CN 112725323B CN 202110163676 A CN202110163676 A CN 202110163676A CN 112725323 B CN112725323 B CN 112725323B
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Abstract
本发明公开了一种重组耐盐腺苷酸环化酶及其应用,其核苷酸序列如SEQ ID NO.1所示,长度为1008 bp,其氨基酸序列如SEQ ID NO.2所示,共335个氨基酸,理论分子量为36.09 kDa。该耐盐腺苷酸环化酶最适pH为10.0,最适温度为45℃,该重组耐盐腺苷酸环化酶活性随着氯化钠浓度的提高而增强,在氯化钠浓度为2.5 M时酶活性达到最大(约22倍),具有良好的耐盐性,与现有报道的腺苷酸环化酶相比,其在高渗条件下能更好地提高生产效率,具有良好的工业应用前景。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种重组耐盐腺苷酸环化酶及其应用。
背景技术
环磷腺苷(cAMP)是生物体内广泛存在的一种生理活性物质,对糖代谢、脂肪代谢以及核酸和蛋白质合成等具有重要的调节作用。在生物体内,各类激素通过调节细胞内cAMP的生成与分解而发挥作用。激素将改变细胞活动的信息传递给靶细胞,再由cAMP传递到靶细胞内相应酶系而发挥作用。因此,通常将激素称之为“第一信使”,将cAMP称之为“第二信使”。cAMP作为激素的第二信使,参与生物体内多种代谢反应过程,对维持生物体正常生命活动起到重要作用,除此以外,cAMP还可以应用在植物生长调节、临床治疗和畜牧业生产等方面。
国内外工业化生产几乎全部采用化学合成法制备cAMP,除此之外,cAMP的制备方法还有发酵法以及酶法,酶法反应条件温和、周期短、副产物少、收率高、环境友好,所以具有一定的优势。利用腺苷酸环化酶生产cAMP,是一条可行的途径。腺苷酸环化酶的来源,可以是破碎的菌体细胞,或是从细胞中提取纯化的酶或固定化酶。现有的腺苷酸环化酶耐盐稳定性较差,其在较高的盐浓度以及逆境下活性降低的性质限制了它在食品、医药行业以及工业生产中的应用。通常高稳定性被认为是一个经济优势可以降低酶的消耗和补偿速率,此外,更稳定的酶可能对反应速率有着积极的影响,对于提高产品品质、获得更好地工业应用潜力、实现该酶的诸多应用具有重要的意义。目前,关于高渗条件下腺苷酸环化酶的应用研究鲜有报道。
发明内容
发明目的:针对现有技术的不足,本发明的目的在于提供了一种重组耐盐腺苷酸环化酶。
本发明另一个目的在于提出了该酶编码基因。
本发明进一步的目的在于提出包含上述编码基因的重组载体和重组菌。
本发明还有一个目的在于提供上述重组酶的制备方法及其应用。
为实现上述目的,本发明提供了一种重组耐盐腺苷酸环化酶,其氨基酸序列如SEQID NO.2所示,共335个氨基酸,理论分子量为36.09kDa。
本发明进一步提出了上述重组耐盐腺苷酸环化酶的编码基因,其核苷酸序列如SEQ ID NO.1所示,基因长度为1008bp,所述编码基因来自于Thermobifida,halotolerans其序列由NCBI数据库获得。
进一步地,本发明提出了一种包含上述耐盐腺苷酸环化酶编码基因的重组载体。优选地,所述重组载体为融合型表达载体,优选出发载体为pET28a,所述耐盐腺苷酸环化酶编码基因插入pET28a的EcoR I和Nde I酶切位点之间。
同时本发明提供了包含所述的耐盐腺苷酸环化酶编码基因的重组菌株,优选地,所述宿主菌为Escherichia coli BL21(DE3)。
本发明同时提出了上述重组耐盐腺苷酸环化酶的获得方法,包括以下步骤:
(1)构建重组菌E.coli BL21(DE3)(pET28a-Thcya)并活化,将活化的重组菌接种到LB培养基中,培养至OD600nm为0.6-0.8时,加入诱导剂IPTG诱导表达,离心收集菌泥;
(2)将步骤(1)得到的菌泥进行超声破碎,离心,上清液即为含耐盐腺苷酸环化酶的粗酶液。
其中,重组菌的构建方法如下:
1)耐盐腺苷酸环化酶编码基因片段插入出发载体pET28a的EcoR I和Nde I酶切位点之间得到重组载体pET28a-Thcya;
2)将重组载体pET28a-Thcya通过热击法转化至E.coli BL21(DE3)中,即得重组菌E.coli BL21(DE3)(pET28a-Thcya)。
优选地,步骤(1)中,所述LB培养基中包含50mg/L卡那霉素,IPTG的终浓度为0.1-1.2mM,诱导表达4-15h。更优选地IPTG的终浓度为0.8mM,诱导表达8h。
优选地,将步骤(1)收集的菌泥用100mM Tris-HCl(pH8.0)清洗两遍,然后加入20mL的100mM Tris-HCl(pH8.0)重悬,超声破碎,离心得到的上清即为粗酶液。
本发明还提供重组耐盐腺苷酸环化酶在制备cAMP中的应用。
具体地,向Tris-HCl缓冲液中添加重组耐盐腺苷酸环化酶的粗酶液,底物ATP、金属离子和氯化钠,建立酶催化反应体系,进行酶催化反应,生成cAMP;或者向Tris-HCl缓冲液中添加含重组耐盐腺苷酸环化酶的菌泥,底物ATP、金属离子、氯化钠、表面活性剂,建立全细胞催化反应体系,进行全细胞催化反应,生成cAMP。
其中,所述金属离子为30-80mM Mg2+,所述酶用量为0.1-20U/mL反应液,ATP浓度为10-100mM,氯化钠浓度为1-5M,,所述表面活性剂为非离子型表面活性剂、阳离子型表面活性剂或阴离子型表面活性剂,所述催化条件为pH7.0-11.0,25-60℃反应4-8h。
优选地,所述金属离子的浓度为50mM,所述ATP的浓度为30mM,所述表面活性剂用量为0.5%(v/v)的Triton X-100,所述催化条件为pH 10.0,45℃反应6h。
有益效果:本发明首次构建了来源于T.halotolerans的重组耐盐腺苷酸环化酶,并实现了该酶在E.coli BL21(DE3)中的高效表达。该重组耐盐腺苷酸环化酶最适pH为10.0,最适温度为45℃,该重组酶酶活性随着氯化钠浓度的提高而增强,在氯化钠浓度为2.5M时酶活性达到最大(约22倍)。将该重组酶应用于cAMP的制备,在最优底物浓度条件下,cAMP产率达91.8%。本发明提供的重组腺苷酸环化酶具有良好的耐盐性,与现有报道的腺苷酸环化酶相比,其在高渗条件下能更好地提高生产效率,具有良好的工业应用前景。
附图说明
图1为实施例1提供的pET28a-Thcya质粒图谱;
图2为实施例2提供的重组菌提取质粒的电泳结果,其中,泳道M为10,000bpMarker,泳道1为重组菌提取质粒结果;
图3为实施例3提供的诱导表达耐盐腺苷酸环化酶粗酶液蛋白电泳结果,其中泳道M为标准蛋白Marker,泳道1为耐盐腺苷酸环化酶粗酶液,泳道2为基因Arcya表达的腺苷酸环化酶粗酶液,泳道3为E.coli BL21(DE3)对照菌破碎上清液;
图4为实施例5提供的耐盐腺苷酸环化酶最适氯化钠浓度结果图;
图5为实施例6提供的耐盐腺苷酸环化酶的最适pH结果图;
图6为实施例7提供的耐盐腺苷酸环化酶的最适温度结果图;
图7为实施例8提供的耐盐腺苷酸环化酶粗酶液催化生成cAMP的过程图。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。给出了详细的实施方式和具体的操作过程,实施例将有助于理解本发明,但是本发明的保护范围不限于下述的实施例。
实施例1耐盐腺苷酸环化酶基因的克隆及重组载体的构建。
以目的基因Thcya(SEQ ID NO.1)为模板,使用引物1(SEQ ID NO.3)和引物2(SEQID NO.4),在下述表1反应体系中进行PCR,扩增目的基因Thcya。
其中,引物1含有EcoR I酶切位点,引物2含有Nde I酶切位点。
PCR反应体系及反应条件见表1。
表1 Thcya基因的PCR反应体系及反应条件
将载体pET28a进行线性酶切,酶切体系及反应条件见表2。
表2载体pET28a的酶切体系及反应条件
利用ClonExpressTMⅡ重组反应体系(ClonExpress II One Step Cloning Kit;Cat:C112-01/02)进行一步克隆得到重组载体pET28a-Thcya,反应体系及反应条件见表3。
表3 ClonExpressTMⅡ重组反应体系及反应条件
待一步克隆反应完成后,立即将反应管置于冰水浴中冷却5min,然后将该连接产物转化E.coli DH5a感受态细胞(购自生工生物工程(上海)股份有限公司),涂布于带有卡那霉素抗性的LB琼脂平板上,37℃培养过夜,挑取单菌落,在LB液体培养基中过夜培养,质粒DNA小量提取试剂盒(购自AxyPrep公司,Cat:AP-MN-P-250G)提取质粒,测序结果正确后可获得阳性重组质粒。构建的重组质粒pET28a-Thcya的图谱如图1所示。
其中,含卡那霉素抗性的LB琼脂平板的配方如下:蛋白胨10g/L,酵母膏5g/L,NaCl10g/L,琼脂15-20g/L,卡那霉素50mg/L。
LB液体培养基的配方如下:蛋白胨10g/L,酵母膏5g/L,NaCl 10g/L。
实施例2重组E.coli BL21(DE3)(pET28a-Thcya)的构建。
将构建好的重组质粒pET28a-Thcya通过热击法转化至E.coli BL21(DE3)(购自生工生物工程(上海)股份有限公司),具体操作如下:取9μL的重组质粒(实施例1得到),加入到100μL的E.coli BL21(DE3)感受态细胞液中,冰上放置30min,42℃热击90s,立即取出放冰上2min。加入900μL的LB液体培养基,37℃,200rpm,培养40min后,取200μL培养液涂布含有卡那霉素的LB固体培养基中,37℃培养12h后所得的单菌落即为重组菌。挑取单菌落,在LB液体培养基中过夜培养,用质粒DNA小量提取试剂盒(购自AxyPrep公司,Cat:AP-MN-P-250G)提取转化子质粒,用EcoRⅠ和NdeⅠ双酶切转化子质粒,凝胶电泳验证获得阳性重组菌,电泳结果如图2所示。
实施例3重组耐盐腺苷酸环化酶的诱导表达。
将构建好的重组菌接种至含有50mg/L卡那霉素的LB液体培养基中,37℃,200rpm条件下培养过夜,然后按1%(v/v)的接种量接种到含有50mg/L卡那霉素的100mL LB培养基的500mL三角瓶中,待OD600nm长到0.6-0.8时,加入终浓度为0.8mM的IPTG,30℃,200rpm诱导8h。诱导结束后,4℃,8000rpm离心10min收集菌泥。
将上述收集的菌泥用100mM Tris-HCl(pH8.0)清洗两遍,然后加入20mL的100mMTris-HCl(pH8.0)重悬,超声破碎(200w,超3s,停7s,共10min),离心得到的上清即为粗酶液,其蛋白电泳结果如图3所示。
实施例4重组耐盐腺苷酸环化酶的粗酶酶活测定。
酶活测定反应体系(1mL)及反应条件如下:实施例3的得到的粗酶液,终浓度为100mM的Tris-HCl,30mM的ATP,50mM的MgCl2,2.5M的氯化钠,pH为9.0,35℃,反应30min,95℃加热10min灭酶,离心,上清液过膜。采用HPLC检测产物cAMP的含量,具体为C18HPLC色谱柱,250mm×4.6mm;流动相:磷酸三乙胺水溶液(其中磷酸含量0.6%(v/v),用三乙胺调节pH至6.6):甲醇=75:25;紫外检测器,波长254nm;柱温25℃,流速0.8mL/min,进样量20μL,标品浓度0.1g/L。
酶活定义如下:在35℃下每分钟生成1微摩尔cAMP所需的酶量定义为一个酶活单位U。经检测,粗酶酶活为45.9U/mL。
对比例1
作为本发明申请的重组耐盐腺苷酸环化酶的对照,将发明专利CN102212538中公布的来源于Arthrobacter的腺苷酸环化酶基因Arcya按实施例1的方法克隆到pET28a载体上,按实施例2的方法将重组质粒导入E.coli BL21(DE3)感受态,按实施例3的方法诱导表达获得粗酶液,其蛋白电泳结果如图3所示。通过实施例4的酶活测定方法检测粗酶液酶活,经检测,该腺苷酸环化酶粗酶液酶活为3.05U/mL。
实施例5重组菌全细胞催化生成cAMP。
全细胞反应体系(1mL)及反应条件如下:实施例3获得的菌泥(5g/L),100mM Tris-HCl(pH9.0),50mM MgCl2,2.5M的氯化钠,30mM的底物ATP,和0.5%(v/v)的Triton X-100。35℃,反应2h,95℃加热10min灭酶,离心,上清液过膜。采用HPLC检测cAMP含量,最终cAMP产率为82.5%。
实施例6重组耐盐腺苷酸环化酶最适氯化钠浓度的测定。
利用实施例3得到的粗酶液,按照实施例4中的酶活测定反应体系及反应条件,其中氯化钠浓度分别为0、1、1.5、2、2.5、3、3.5、4.0、5.0M,测定不同浓度的氯化钠对重组酶酶活的影响。测得结果如图4所示,该酶活性随着氯化钠浓度的提高而增强,在氯化钠浓度为2.5M时酶活性达到最大,约为初始酶活的22倍。
实施例7重组耐盐腺苷酸环化酶的最适pH的测定。
利用实施例3得到的粗酶液,按照实施例4中的酶活测定反应体系及反应条件,其中pH分别为7.0、7.5、8.0、8.5、9.0、9.5、10.0、10.5、11.0,测定不同pH对重组酶酶活的影响。测得结果如图5所示,该酶的最适pH为10。
实施例8重组耐盐腺苷酸环化酶的最适温度的测定。
利用实施例3得到的粗酶液,按照实施例4中的酶活测定反应体系及反应条件,其中温度分别为25、30、35、40、45、50、55、60,测定不同温度对重组酶酶活的影响。测得结果如图6所示,该酶的最适温度为45℃。
实施例9利用重组耐盐腺苷酸环化酶的粗酶液催化生成cAMP。
取50mL烧杯配制总体积为20mL的如下反应液:100mM Tris-HCl(pH 10),50mMMgCl2,2.5M的氯化钠,30mM的底物ATP和实施例3获得的粗酶液0.4mL(18.36U)。于45℃,200rpm反应7h,反应过程中采用NaOH调节控制pH至10.0。反应过程中与结束后取催化液1mL,95℃加热10min灭酶,离心,上清液过膜。采用HPLC对ATP和cAMP进行定量分析,经检测,最终产生27.5mM的cAMP(图7),cAMP产率为91.8%。
对比例2
将对比例1中获得的粗酶液0.4mL(1.22U),按实施例8的酶反应体系与反应条件,催化生成cAMP,经检测,最终生成8.73mM的cAMP,cAMP产率为29.1%(图7)。
本申请提出的重组腺苷酸环化酶具备耐盐性,与现有报道的腺苷酸环化酶相比,其在高渗条件下能更好地提高生产效率,具有良好的工业应用前景。
序列表
<110> 南京工业大学
<120> 一种重组耐盐腺苷酸环化酶及其编码基因和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1008
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtcgtctc gtcccgaccc caaggagatc gaagccgtcc tgttgggagg cgaggcgcgg 60
tatacccgtg aagaggtggt gcggctcgcg gggacctcgc ccgagctcgc ctcccgcatc 120
tggcgggccc tggggttcgc cacacgcggg gaggacacgc ccgcctacac cgacagcgac 180
gtcgaggcca tgcgggtcac cgccgaactg ctgcgcagcg gcacactcac cgaggaggcc 240
gcggtccgcc tggcccgggc catggggcag accatgacca ggctcgccga atggcagacc 300
agcatcctga ccaccctgtc ctacgagccc gaggtcgacc tcgacggaca cctgggaccg 360
ctggtggggc tggccaagga gctgcttccc gacgtcgaga cactgctgct gcacatctgg 420
cggcgccagc tcgccgcgtc ggtctcgcgc agcctggcgg tccaggagca gcgggaggac 480
gcctcccccc actacttccc gctggtcgtc ggcttcgcgg acctggtgtc cttcaccgcg 540
ctcagccggg agctgaacga ggtcggcctg gccgacgtgg tggaggggtt cgaggcgacc 600
tcggccgaca tcgtcgcctc ctgcgggggc cggatcgtca agaccctggg cgacgagatc 660
ctctacgtcg cggacaacgc ccggcaggcc gccgacatcg ccctgcagct cgccgccgga 720
gtcaagaccc acgtcgaggt tcccgacgtg cgggtcggcc tggcctacgg cccggtgctg 780
gcgctgctcg gcgacgtgtt cggcatcacc gtcaaccggg ccagccgcct gacctccttc 840
gcgcggccgg ggaccgtcct catcgacgag gccctggcgg accagctcaa gggcgaggac 900
ggcttccagg tggtggccgt gcgcgcccgc cacgcccacg gtctcggaca gctccagccc 960
tacgcgctgc gccggagttt cggcgcgaac gtgtcgctgc gcgggtga 1008
<210> 2
<211> 335
<212> PRT
<213> 耐盐腺苷酸环化酶(Artificial Sequence)
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Gly Glu Ala Arg Tyr Thr Arg Glu Glu Val Val Arg Leu Ala Gly Thr
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Ser Pro Glu Leu Ala Ser Arg Ile Trp Arg Ala Leu Gly Phe Ala Thr
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Arg Gly Glu Asp Thr Pro Ala Tyr Thr Asp Ser Asp Val Glu Ala Met
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Arg Val Thr Ala Glu Leu Leu Arg Ser Gly Thr Leu Thr Glu Glu Ala
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Ala Val Arg Leu Ala Arg Ala Met Gly Gln Thr Met Thr Arg Leu Ala
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Glu Trp Gln Thr Ser Ile Leu Thr Thr Leu Ser Tyr Glu Pro Glu Val
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Asp Leu Asp Gly His Leu Gly Pro Leu Val Gly Leu Ala Lys Glu Leu
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Leu Pro Asp Val Glu Thr Leu Leu Leu His Ile Trp Arg Arg Gln Leu
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Ala Ala Ser Val Ser Arg Ser Leu Ala Val Gln Glu Gln Arg Glu Asp
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Ala Ser Pro His Tyr Phe Pro Leu Val Val Gly Phe Ala Asp Leu Val
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Ser Phe Thr Ala Leu Ser Arg Glu Leu Asn Glu Val Gly Leu Ala Asp
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Val Val Glu Gly Phe Glu Ala Thr Ser Ala Asp Ile Val Ala Ser Cys
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Gly Gly Arg Ile Val Lys Thr Leu Gly Asp Glu Ile Leu Tyr Val Ala
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Asp Asn Ala Arg Gln Ala Ala Asp Ile Ala Leu Gln Leu Ala Ala Gly
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Val Lys Thr His Val Glu Val Pro Asp Val Arg Val Gly Leu Ala Tyr
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Gly Pro Val Leu Ala Leu Leu Gly Asp Val Phe Gly Ile Thr Val Asn
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Arg Ala Ser Arg Leu Thr Ser Phe Ala Arg Pro Gly Thr Val Leu Ile
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Asp Glu Ala Leu Ala Asp Gln Leu Lys Gly Glu Asp Gly Phe Gln Val
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Val Ala Val Arg Ala Arg His Ala His Gly Leu Gly Gln Leu Gln Pro
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Tyr Ala Leu Arg Arg Ser Phe Gly Ala Asn Val Ser Leu Arg Gly
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<210> 3
<211> 38
<212> DNA
<213> 引物1(Artificial Sequence)
<400> 3
gcttgtcgac ggagctcgaa ttcatgtcgt ctcgtccc 38
<210> 4
<211> 36
<212> DNA
<213> 引物2(Artificial Sequence)
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gtgccgcgcg gcagccatat gtcacccgcg cagcga 36
Claims (2)
1.一种在高渗条件下制备环磷腺苷的方法,其特征在于,所述,包括以下步骤:向Tris-HCl缓冲液中添加重组耐盐腺苷酸环化酶的酶液、ATP、金属离子和氯化钠,建立酶催化反应体系,进行酶催化反应,生成cAMP;或者向Tris-HCl缓冲液中添加ATP、金属离子、氯化钠、表面活性剂和生产所述重组耐盐腺苷酸环化酶的重组菌的菌泥,建立全细胞催化反应体系,进行全细胞催化反应,生成cAMP,
其中,所述金属离子的浓度为50 mM,所述ATP的浓度为30 mM,所述表面活性剂用量为0.5 % (v/v)的Triton X-100,所述催化条件为pH 10.0,2.5M的氯化钠,反应温度为45℃;
所述重组耐盐腺苷酸环化酶的氨基酸序列如SEQ ID NO.1所示,编码所述耐盐腺苷酸环化酶的基因的核苷酸序列如SEQ ID NO.2所示;所述重组菌的宿主菌为Escherichia coli BL21(DE3),重组载体为pET28a,所述耐盐腺苷酸环化酶编码基因插入pET28a的EcoRI和Nde I酶切位点之间;
重组耐盐腺苷酸环化酶的酶液和生产所述重组耐盐腺苷酸环化酶的重组菌的菌泥的获得方法,包括以下步骤:
(1)构建重组菌并活化,将活化的重组菌接种到LB培养基中,培养至OD600nm为0.6-0.8时,加入诱导剂IPTG诱导表达,离心收集,生产所述重组耐盐腺苷酸环化酶的重组菌的菌泥;
(2)将步骤(1)得到的菌泥进行超声破碎,离心,上清液即为含耐盐腺苷酸环化酶的酶液。
2. 根据权利要求1所述的方法,其特征在于,所述菌泥在超声破碎前用pH8.0、100 mMTris-HCl,清洗两遍,然后加入20 mL的pH8.0、100 mM Tris-HCl重悬。
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