CN114807193B - 一种碳酸酐酶基因及其用途 - Google Patents
一种碳酸酐酶基因及其用途 Download PDFInfo
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- CN114807193B CN114807193B CN202210519191.7A CN202210519191A CN114807193B CN 114807193 B CN114807193 B CN 114807193B CN 202210519191 A CN202210519191 A CN 202210519191A CN 114807193 B CN114807193 B CN 114807193B
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- carbonic anhydrase
- protein
- anhydrase gene
- gene
- glu
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
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- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
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- 102000003846 Carbonic anhydrases Human genes 0.000 description 18
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- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
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Abstract
本发明公开了一种碳酸酐酶基因及其用途。所述碳酸酐酶基因的核苷酸序列如SEQ IDNO:1所示或为与其互补的核苷酸序列；蛋白序列如SEQ ID NO:2所示或为与其互补的蛋白序列。还公开了重组载体和重组细胞。所述碳酸酐酶基因具有捕获CO2的作用。
Description
技术领域
本发明涉及基因重组领域，尤其涉及一种碳酸酐酶基因及其用途。
背景技术
碳酸酐酶能有效催化二氧化碳(CO2)的可逆水合反应：能实现CO2和碳酸氢根离子(HCO3 -)之间的快速转化。碳酸酐酶从真核生物领域到原核生物遍布系统发育树，不仅存在于几乎所有的哺乳动物组织中，而且在植物、藻类、细菌和古菌中也丰富存在，并且驱动着包括光合作用、呼吸作用、维持pH平衡、CO2的转运和离子转换以及钙化等在内的很多生理生化过程。碳酸酐酶资源已被应用于生物检测、天然活性物质的筛选、生物传感器、CO2捕集和生理诊断等方面，并取得了令人满意的结果。该碳酸酐酶有望应用于CO2捕集来调节碳封存，以改善由CO2排放引起的环境问题。
发明内容
本发明的目的在于提供一种碳酸酐酶基因。
为实现上述目的，本发明提供碳酸酐酶基因的核苷酸序列如SEQ ID NO:1所示或为与其互补的核苷酸序列；蛋白序列如SEQ ID NO:2所示或为与其互补的蛋白序列。
本发明还提供所述碳酸酐酶基因的重组载体。
本发明还提供所述碳酸酐酶基因的重组细胞。
本发明还提供所述碳酸酐酶基因具有捕获CO2的作用。本发明的实施例验证了碳酸酐酶基因增强了CaCO3的形成，蛋白碳酸酐酶基因具有捕获CO2的作用。
附图说明
图1为表达产物破碎离心后上清和沉淀的SDS-PAGE电泳图；
图2为表达产物纯化的SDS-PAGE电泳图；
图3为标准蛋白拟合曲线图；
图4为对硝基苯酚标准曲线图；
图5碳酸酐酶CA2催化的碳酸盐沉淀图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。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，均为可以通过市购获得的常规产品。
实施例1：碳酸酐酶基因的重组载体构建
人工合成SEQ ID NO:1,即为碳酸酐酶基因序列，该基因的蛋白序列为SEQ ID NO:2。
SEQ ID NO:1如下所示：
ATGGCAACAAAATTATTTCAGGGAATTAAAAAATTTAAGAAAAAGGATTTTGAAACACATAAAGAACTTTTTTCAAAACTAAAAAAAGGTCAAAATCCGTATACTTTTTTTATAGGCTGCAGTGACAGCAGGGTGGTGCCAAATATTATTACAAAAACACTTCCGGGTGAGCTTTTTGTAGTGAGAAATATTGCAAATGTTGTACCTCCTTATAAGGCAAGTGGAGAAGCAGGATATAAGTGTACGGCAAGTGCGCTTGAATATGCTGTTGAGTATTTGGAAGTGAGAAATATTGTTGTTTGCGGACACAGTAACTGCGGAGGATTAAAAGCACTTTTTTATGATGAAGAAAAATTAAATAAACTTCCAATTGTTAAAAAATGGCTAAATTTACTTGATGATGTTAAAAAGAGTGTAGAGCATATTGAAGATGAGGCTTTTAGGGAGTGGGAAATAGAACATCTTAACATTTTAAAGCAGATTGATAATCTAACAACTTACCCTTTTGTAGAAAAAAGATTTAAAGAAGGGAAACTTAATATTTTCGGATGGTATTATGTAATAGAAACGGAGGAAGTTTATAATTATAATTTTGAAACAAAAGAATTTGAGCTTATAAATTAA。
SEQ ID NO:2如下所示：
MATKLFQGIKKFKKKDFETHKELFSKLKKGQNPYTFFIGCSDSRVVPNIITKTLPGELFVVRNIANVVPPYKASGEAGYKCTASALEYAVEYLEVRNIVVCGHSNCGGLKALFYDEEKLNKLPIVKKWLNLLDDVKKSVEHIEDEAFREWEIEHLNILKQIDNLTTYPFVEKRFKEGKLNIFGWYYVIETEEVYNYNFETKEFELIN。
将人工合成的碳酸酐酶基因序列通过Ndel/Xhol酶切位点构建至pCold II载体，将重组质粒转入Escherichia coli BL21(DE3)感受态细胞内，由于pCold II带有氨苄青霉素抗性基因，因此需经带有氨苄青霉素(终浓度：100μg/mL)的抗性的LB平板进行筛选，筛选出的阳性经过测序验证，最终得到碳酸酐酶基因的重组菌株ABX14411-2 CA2。
实施例2：碳酸酐酶基因的重组表达
细菌扩大培养：从保种管中取200μL重组菌株ABX14411-2 CA2接种于带有氨苄青霉素(终浓度：100μg/mL)的20mL LB液体培养基中：37℃,200r/min培养至OD600 nm值为0.6～0.8。取出10mL活化液加入至1L带有氨苄青霉素(终浓度：100μg/mL)的LB培养基，继续同条件扩大培养至OD600 nm值为0.6～0.8。
诱导表达：加入终浓度为0.5mmol/L的IPTG(异丙基-β-D-硫代半乳糖)进行诱导表达，16℃、200r/min继续培养16～18h。发酵液经高速离心(8000r/min，20min)，收集菌体。使用20mL Tris-HCl缓冲液(10mM/L Tris-HCl，pH 8.0,150mM/L NaCl)重悬细菌。
细菌破碎离心：将重悬液吹打至无明显菌块后利用细胞破碎仪(SM-650D)进行高压破碎。循环破碎30min至菌液澄清，将破碎后的菌液在4℃、9000r/min条件下离心30min，收集上清。
SDS-PAGE蛋白胶检测：使用SDS-PAGE蛋白胶检测(浓缩胶电压，80V，40min；分离胶电压：200V，30min)收集到的上清液和沉淀，并使用考马斯亮蓝染色液进行过夜染色，最后使用沸水浴脱色直至蛋白胶变透明。结果见图1，可以看出，破碎后的上清中表达有较多的24KD大小的条带蛋白，与碳酸酐酶分子量相当。
实施例3：蛋白纯化
对实施例2中离心收集到的上清液蛋白进行纯化(AKTA蛋白纯化仪；镍柱型号：HisTrapTM HP 5mL)：缓冲液分A液(10mM/L Tris-HCl，pH 8.0,150mM/L NaCl)和B液(10mM/LTris-HCl，pH 8.0,150mM/L NaCl,500mM咪唑)。
依次使用超纯水，缓冲液A液冲洗管路(各5个柱体积，流速1.000mL/min)，随后安装柱子，使用缓冲液A液(10Mm/L Tris-HCl，pH 8.0,150mM/L NaCl)平衡柱子(10个柱体积)，最后将上清液经0.22μm膜过滤，使用注射器将上清液5mL/次加入到镍柱中，上清和镍基质结合后进行洗脱，先用缓冲液A液洗大约5个柱体积，然后再用含有20mmol/L咪唑的缓冲液(缓冲液A：100％；缓冲液B：4％配比)洗大约10个柱体积以除去少量杂蛋白，再用含有250mmol/L咪唑的缓冲液(缓冲液A：100％；缓冲液B：50％配比)进行洗脱，选择出峰位置进行收集，整个纯化过程在低温环境中进行。
使用超滤离心管(15mL，10KD)对收集的蛋白样品进行浓缩。
SDS-PAGE蛋白胶检测：用SDS-PAGE鉴定纯化后蛋白。加入1×甘氨酸电泳缓冲液，上样：蛋白marker(Pre-stained protein Marker)：5μL。目标蛋白(20μL)：10μL Loadingbuffer加30μL纯化后蛋白，混匀，热水浴95℃，5min，冰浴10min。取20μL混合样品上样，先80V电压，40min，然后切换至200V电压，30min。结果见图2，可以看出，纯化后的条带杂质少，主要条带都集中在24KD左右，与碳酸酐酶分子量相当，纯化的蛋白样品即为碳酸酐酶CA2。
实施例4碳酸酐酶活的测定
①BCA法测蛋白浓度：
1)蛋白浓度标准曲线。
配置不同梯度浓度蛋白标准液；
按照50:1配置A液和B液形成BCA工作液；
在不同浓度梯度蛋白标准液中加入200μL工作液，37℃培育30min；
根据OD562nm值数据绘制蛋白浓度标准曲线。
2)蛋白含量测定
加8μL体积碳酸酐酶CA2到96孔板的样品孔中，加入12μL缓冲液，并向孔中加入200μL BCA工作液，37℃放置30min。最终得到目标蛋白浓度为：1.964mg/mL。
②碳酸酐酶CA2活性测定
碳酸酐酶酶活单位：在室温下，每分钟水解1μmol乙酸对硝基苯酯所需的酶量为一个酶活单位。
绘制标准曲线：配制1mM的对硝基苯酚，称取对硝基苯酚(p-NP)0.0348g，加200mL60℃温水，搅拌溶解，定容至250mL容量瓶。
按照表1配制不同浓度的对硝基苯酚。
在OD348nm测定各浓度的吸光度，绘制标准曲线。
配制浓度为15mM乙酸对硝基苯酯(p-NPA),称取0.1359mg乙酸对硝基苯酯，加入6mL丙酮溶解，加水定容50mL容量瓶(母液)。
碳酸酐酶底物配制：
配制浓度为3mM乙酸对硝基苯酯(p-NPA)：1mL15 mM的乙酸对硝基苯酯加4mL水。
碳酸酐酶活的测定方法：
在酶标板上加入50μL缓冲液(10mM/L Tris-HCl，pH 8.0,150mM/L NaCl)，40μL酶液(1.964mg/mL)，50μL的p-NPA(3mM),反应进行5min时测定OD348 nm处的吸光度，以加入缓冲液代替酶的样品作为空白对照，每个样品三个平行。对照标准曲线，计算酶活性。
酶活的计算公式如下:
酶的比活力(U/mg)＝(np-NP)/mCA2t
其中，np-NP为反应生成乙酸对硝基苯酚的摩尔量，μmol；mCA2为所加入的碳酸酐酶CA2质量，mg；t为反应时间，min。
结果表明酶活约为457U/mg。
表1不同浓度的对硝基苯酚的配置表
实施例5碳酸酐酶基因的功能验证实验
配置15mL含有0.9g CaCl2·2H2O的Tris(2.52g)缓冲液备用。
将200μL含有CaCl2的Tris缓冲液以及300μL碳酸酐酶CA2(0.6mg)加入到透明小试管中，向试管中加入1mL CO2饱和溶液(将CO2(标准级)引入试管中的100mL MilliQ级水中，pH≈5.6,在4℃下放置1小时)，并充分混合，反应在30℃下进行10分钟，缓冲液在对照中代替碳酸酐酶CA2做对照实验。结果见图5，可以看出，对照管里清澈，实验管浑浊，有白色沉淀生产；说明与对照相比，碳酸酐酶CA2增强了CaCO3的形成，蛋白碳酸酐酶CA2具有捕获CO2的作用。
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不脱离本发明的原理和宗旨的情况下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。
SEQUENCE LISTING
<110> 厦门大学
<120> 一种碳酸酐酶基因及其用途
<130> XMDXB-22001-CNI
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 624
<212> DNA
<213> 人工序列
<400> 1
atggcaacaa aattatttca gggaattaaa aaatttaaga aaaaggattt tgaaacacat 60
aaagaacttt tttcaaaact aaaaaaaggt caaaatccgt atactttttt tataggctgc 120
agtgacagca gggtggtgcc aaatattatt acaaaaacac ttccgggtga gctttttgta 180
gtgagaaata ttgcaaatgt tgtacctcct tataaggcaa gtggagaagc aggatataag 240
tgtacggcaa gtgcgcttga atatgctgtt gagtatttgg aagtgagaaa tattgttgtt 300
tgcggacaca gtaactgcgg aggattaaaa gcactttttt atgatgaaga aaaattaaat 360
aaacttccaa ttgttaaaaa atggctaaat ttacttgatg atgttaaaaa gagtgtagag 420
catattgaag atgaggcttt tagggagtgg gaaatagaac atcttaacat tttaaagcag 480
attgataatc taacaactta cccttttgta gaaaaaagat ttaaagaagg gaaacttaat 540
attttcggat ggtattatgt aatagaaacg gaggaagttt ataattataa ttttgaaaca 600
aaagaatttg agcttataaa ttaa 624
<210> 2
<211> 207
<212> PRT
<213> 人工序列
<400> 2
Met Ala Thr Lys Leu Phe Gln Gly Ile Lys Lys Phe Lys Lys Lys Asp
Phe Glu Thr His Lys Glu Leu Phe Ser Lys Leu Lys Lys Gly Gln Asn
Pro Tyr Thr Phe Phe Ile Gly Cys Ser Asp Ser Arg Val Val Pro Asn
Ile Ile Thr Lys Thr Leu Pro Gly Glu Leu Phe Val Val Arg Asn Ile
Ala Asn Val Val Pro Pro Tyr Lys Ala Ser Gly Glu Ala Gly Tyr Lys
Cys Thr Ala Ser Ala Leu Glu Tyr Ala Val Glu Tyr Leu Glu Val Arg
Asn Ile Val Val Cys Gly His Ser Asn Cys Gly Gly Leu Lys Ala Leu
Phe Tyr Asp Glu Glu Lys Leu Asn Lys Leu Pro Ile Val Lys Lys Trp
Leu Asn Leu Leu Asp Asp Val Lys Lys Ser Val Glu His Ile Glu Asp
Glu Ala Phe Arg Glu Trp Glu Ile Glu His Leu Asn Ile Leu Lys Gln
Ile Asp Asn Leu Thr Thr Tyr Pro Phe Val Glu Lys Arg Phe Lys Glu
Gly Lys Leu Asn Ile Phe Gly Trp Tyr Tyr Val Ile Glu Thr Glu Glu
Val Tyr Asn Tyr Asn Phe Glu Thr Lys Glu Phe Glu Leu Ile Asn
Claims (4)
1.一种碳酸酐酶基因，其特征在于，所述碳酸酐酶基因的核苷酸序列如SEQ ID NO:1所示或为与其互补的核苷酸序列；所述碳酸酐酶基因编码的蛋白序列如SEQ ID NO:2所示。
2.含有权利要求1所述碳酸酐酶基因的重组载体。
3.含有权利要求1所述碳酸酐酶基因的重组细胞。
4.权利要求1所述碳酸酐酶基因在捕获CO2中的用途。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002306172A (ja) * | 2001-04-09 | 2002-10-22 | Kanegafuchi Chem Ind Co Ltd | 炭酸脱水酵素遺伝子 |
| WO2013028512A1 (en) * | 2011-08-19 | 2013-02-28 | Novozymes, Inc. | Recombinant microorganisms for production c4-dicarboxylic acids |
| CN104328104A (zh) * | 2014-10-10 | 2015-02-04 | 上海立足生物科技有限公司 | 一种热稳定性碳酸酐酶、制备方法及应用 |
| CN110218715A (zh) * | 2019-06-14 | 2019-09-10 | 中国石油大学(华东) | 碳酸酐酶XiCA及其编码基因和应用 |
| KR20210120388A (ko) * | 2020-03-26 | 2021-10-07 | 경상국립대학교산학협력단 | 발현량이 증가된 써모설퍼리모나스 디스뮤탄스 유래 탄산무수화효소 단백질 변이체 및 이의 용도 |
| CN113549609A (zh) * | 2021-06-07 | 2021-10-26 | 中国石油大学(华东) | 碳酸酐酶SaCA及其编码基因和应用 |
-
2022
- 2022-05-13 CN CN202210519191.7A patent/CN114807193B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002306172A (ja) * | 2001-04-09 | 2002-10-22 | Kanegafuchi Chem Ind Co Ltd | 炭酸脱水酵素遺伝子 |
| WO2013028512A1 (en) * | 2011-08-19 | 2013-02-28 | Novozymes, Inc. | Recombinant microorganisms for production c4-dicarboxylic acids |
| CN104328104A (zh) * | 2014-10-10 | 2015-02-04 | 上海立足生物科技有限公司 | 一种热稳定性碳酸酐酶、制备方法及应用 |
| CN110218715A (zh) * | 2019-06-14 | 2019-09-10 | 中国石油大学(华东) | 碳酸酐酶XiCA及其编码基因和应用 |
| KR20210120388A (ko) * | 2020-03-26 | 2021-10-07 | 경상국립대학교산학협력단 | 발현량이 증가된 써모설퍼리모나스 디스뮤탄스 유래 탄산무수화효소 단백질 변이체 및 이의 용도 |
| CN113549609A (zh) * | 2021-06-07 | 2021-10-26 | 中国石油大学(华东) | 碳酸酐酶SaCA及其编码基因和应用 |
Non-Patent Citations (2)
| Title |
|---|
| Structure and mechanism of carbonic anhydrase;S Lindskog;Pharmacol Ther .;第74卷(第1期);全文 * |
| 碳酸酐酶Ⅱ基因克隆及在毕赤酵母中的异源表达;卫玲;赵莹;徐晓晶;;上海医药(第17期);全文 * |
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