CN107384886B - 一种过氧化氢酶及其应用 - Google Patents
一种过氧化氢酶及其应用 Download PDFInfo
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- CN107384886B CN107384886B CN201710750633.8A CN201710750633A CN107384886B CN 107384886 B CN107384886 B CN 107384886B CN 201710750633 A CN201710750633 A CN 201710750633A CN 107384886 B CN107384886 B CN 107384886B
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- hydrogen peroxide
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Abstract
本发明属于基因工程技术领域，具体涉及一种新型过氧化氢酶及其应用。所述过氧化氢酶的最适作用pH值为8.5，最适作用温度为50℃，且耐热效果显著。本发明提供的新型过氧化氢酶能有效催化过氧化氢分解生成水和氧气，10min时即将过氧化氢全部分解；而对照组所述市售过氧化氢酶，在20min时才将过氧化氢全部分解，取得了意料不到的技术效果，可广泛运用于食品、纺织、医药、造纸等领域中去除生产过程中的过氧化氢。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种新型过氧化氢酶及其应用。
背景技术
过氧化氢酶（Hydrogen Peroxidase）又称触酶（Catalase，简称 CAT），是一类催化底物氧化还原反应的酶，其主要功能是催化过氧化氢分解成氧气和水。
过氧化氢酶的研究可追溯到19世纪初，迄今它已成为农业，以及与之相关的食品与乳制品业、纸浆和造纸业、以及农业环保产业中有应用价值的酶之一。过氧化氢酶存在于红细胞及某些组织内的过氧化体中，它的主要作用是催化过氧化氢分解为水和氧气，使得过氧化氢不至于与氧分子在铁螯合作用下反应生成非常厉害的-OH，过氧化氢酶的作用是使过氧化氢还原成水和氧分子。
过氧化氢酶可促进漂白的进行，可显著提高羊毛的白度和亲水性。这是由于过氧化氢酶促进羊毛纤维初始受到快速的浸蚀，致使羊毛漂白较易进行。因此，工业中可先利用过氧化氢酶对羊毛进行预处理，是纤维表面裸露，再进行漂白，效果会更好，且纤维损伤也易控制。
过氧化氢酶广泛分布于动物、植物组织中和绝大多数的好氧菌和少数的厌氧微生物中。动物肝脏、红细胞、植物叶绿体以及放线菌、细菌、真菌也含有过氧化氢酶，其中哺乳动物组织中 CAT 含量差异较大，肝脏和结缔组织含量分别为最高和最低，过氧化氢酶在上述细胞组织中主要是与细胞器如线粒体和过氧化物体结合的形式存在，红细胞中则以可溶的状态存在。不同来源的过氧化氢酶的性质差距较大，从动植物和人体内获得的过氧化氢酶的热稳定性较差；而微生物来源的过氧化氢酶的稳定性相对表现较好，此外，微生物还具有易培养、来源广、生产周期短且成本低等优点。因此，微生物过氧化氢酶必将成为过氧化氢酶工业化发展的重要方向，利用基因工程技术手段开发出新型活性高的过氧化氢酶菌株具有重要的应用价值。
发明内容
本发明的目的是提供一种新型过氧化氢酶及其应用。本发明通过构建含有过氧化氢酶基因的表达载体，并将其转化入黑曲霉（Aspergillus niger）中，构建得到黑曲霉工程菌株。该菌株能高效分泌表达过氧化氢酶，所产过氧化氢酶可广泛运用于食品、纺织、医药、造纸等领域中去除生产过程中的过氧化氢。
本发明一方面提供了一种过氧化氢酶，所述过氧化氢酶为：
（a）氨基酸序列为SEQ ID NO:1的酶；
（b）在（a）中的氨基酸上发生取代、缺失或添加一个或数个氨基酸得到的，具有过氧化氢酶活性的酶。
本发明，另一方面提供了编码所述过氧化氢酶的基因，其一种编码核苷酸序列为SEQ ID NO: 2。
本发明还涉及一种表达载体，其携带有所述编码过氧化氢酶的基因。
本发明还涉及一种宿主细胞，其携带有所述表达载体。
本发明还涉及上述过氧化氢酶在降解过氧化氢中的应用。
有益效果
本发明提供了一种新型过氧化氢酶，并构建了高效重组表达该酶的黑曲霉工程菌株，摇瓶发酵酶活高达3150U/mL。所述过氧化氢酶的最适作用pH值为8.5，最适作用温度为50℃，其在4-50℃水浴中静置1个小时，酶活比较稳定，几乎没有变化，在50-60℃水浴中静置1小时后仍能保留超过83%的酶活，70℃水浴中静置1小时后仍然能保留超过50%的活力，耐热效果显著。本发明提供的新型过氧化氢酶能有效催化过氧化氢分解生成水和氧气，10min时即将过氧化氢全部分解；而对照组所述市售过氧化氢酶，在20min时才将过氧化氢全部分解，取得了意料不到的技术效果，可广泛运用于食品、纺织、医药、造纸等领域中去除生产过程中的过氧化氢。
具体实施方式
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001)和CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，这并不意味着将本发明限定于所述的任何具体方法、实验方案和试剂，因为它们可以改变。
除非在本文中另作限定，本文所用的全部技术术语和科学术语具有本发明所属领域的普通计数人员通常所理解的相同含义。DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY, 3nd Ed. (Singleton et al., 2006)和COLLINS DICTIONARY BIOLOGY (Haleet al., 2003)为技术人员提供了本发明中所使用的许多术语的一般性解释。
下面结合具体实施例对本发明进行详细的描述。
实施例1 基因克隆
申请人首先提取曲霉（Aspergillus sp.）WLP（该菌株由发明人王艺璇于2017年1月筛选自山东省青岛市崂山区林场的落叶表面）的基因组总DNA。然后以基因组总DNA为模板，利用上下游引物进行扩增。
PCR扩增条件为95℃ 4min；94℃ 30S；55℃ 40S，72℃ 1min 30个循环；72℃7min。
凝胶回收试剂盒回收PCR扩增产物，送至北京华大基因研究中心进行测序分析。结果显示，扩增产物的核苷酸序列为SEQ ID NO:2，其编码的氨基酸序列为SEQ ID NO:1。经NCBI Blast比对发现，该序列与烟曲霉(Aspergillus fumigatus)的过氧化氢酶序列的相似性仅为47%，为一新的等位基因。
实施例2 表达载体构建
对实施例1中得到的PCR产物和黑曲霉表达质粒pGAMD分别进行Xba I单酶切；回收酶切产物；用T4连接酶把酶切产物和表达载体在22℃条件下连接过夜。将连接产物导入大肠杆菌DH5α；然后通过PCR验证连接正确与否；将相应的阳性克隆表达质粒命名为pVL-Cs。
实施例3 黑曲霉工程菌的构建
3.1 原生质体制备
接种黑曲霉菌丝于PDA平板上生长4 天；切取直径约3cm的菌落置于约100mL CMA（2%麦芽提取物、2%葡萄糖，0.1%细菌蛋白胨）的液体培养基中，30℃，200 rpm振荡培养过夜；多层纱布过滤收集菌丝；将菌丝置于盛有20 mL裂解酶液（Sigma L1412）酶解2-3小时；取出酶解液，加入0.8M MgSO4溶液，轻轻摇晃，倒于三层灭菌擦镜纸过滤，收集滤液，3000rpm，离心10 min；弃上清，加10 mL 1.2M山梨醇悬浮，然后3000 rpm，离心10 min；加适量山梨醇悬浮分装（200 µL/管，108个/mL）。
转化与验证
取10µg pVL-Cs DNA加入到200µL原生质体中，接着加入50µL 25%PEG轻轻混匀，室温静置20 min；然后加2mL 25%PEG，轻轻混匀，室温静置5min，把原生质体加到50 mL左右熔化后冷却至45-55℃的上层半固体培养基（0.059%乙酰胺，0.152%KH2PO4，0.34%CsCl，0.052%KCl，1%葡萄糖, 21.85%山梨醇, 0.35%琼脂糖），轻轻混匀后倒入下层基础培养基平板（0.059%乙酰胺, 0.152%KH2PO4，0.34%CsCl，0.052%KCl，1%葡萄糖，1%琼脂粉），30℃黑暗培养数天至转化子长出；验证阳性转化子。将获得的一株工程菌命名为黑曲霉Cs-1（Aspergillus niger Cs-1）。
实施例4 发酵与酶活测定
将黑曲霉Cs-1接种于50mL 黑曲霉发酵培养基 (1.2％NaNO3，0.05％KCl，0.15%KH2PO4，0.205% MgSO4·7H2O，0.35％NaH2PO4·H2O，7%柠檬酸钠，4.5%胰蛋白大豆肉汤，微量元素1mL，4.1%葡萄糖），30℃培养4-5天，离心取上清，进行SDS-PAGE检测分析。结果可知，在33kDa处有一明显的蛋白条带，即为重组表达的过氧化氢酶，重组蛋白大小与预测的一致。
将上清液进行酶活检测，结果显示，发酵液上清酶活为3150U/mL。
酶活测定方法：紫外分光光度法
（1）测定的原理：过氧化氢在240nm波长下有强吸收，过氧化氢酶能分解过氧化氢，使溶液的吸光度（A240）随反应时间而降低。根据测量吸光度的变化速度可测出过氧化氢酶的活性。
（2）测定过程：将紫外分光光度计通电后预热30min，在吸收波长240nm处机械调零后用磷酸盐缓冲液调零。用5ml的移液管准确移取2.9ml底物溶液至1cm石英比色皿中，用50-250ul的微量移液器移取100ul待测样品，加入上述石英比色皿中，摇匀后立即在吸收波长240nm处测A值，每隔5s记录一个A值，测定时间为30s.在Excel中将吸光度对时间做一次方程曲线，选取曲线方程斜率K的绝对值计算酶活。控制K值在0.0031-0.0020之间，若K不在此区域内，需将样品重新稀释到合适倍数测定。
（3）酶活力计算：
酶活力（IU/ml或IU/g）=N×K×4.13×104
式中：
N-稀释倍数，
K-吸光度变化的曲线的斜率的绝对值，
实施例5 过氧化氢酶酶学性质分析
1、最适作用pH分析
用pH值分别为2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0的缓冲液对上述发酵上清液进行稀释，在温度35℃条件下测定上述发酵上清液的酶活，以最高酶活为100%，计算相对酶活，做pH-相对酶活曲线。结果显示：本发明提供的新型过氧化氢酶的最适作用pH值为8.5。
、最适作用温度分析
分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃，pH5.5的条件下测定酶活，以最高酶活为100%，计算相对酶活，做温度-相对酶活曲线。结果显示：本发明提供的新型过氧化氢酶的最适作用温度为50℃。
、温度稳定性
将上述发酵上清液取500ul分装成小份，分别放置于4℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃和70℃的水浴锅中保温1h后，检测酶活，以4℃水浴下样品的酶活为100%，其它温度点的酶活除以该酶活力，从而得到其相对酶活值。结果显示，本发明提供的过氧化氢酶在4-50℃水浴中静置1个小时，酶活比较稳定，几乎没有变化，在50-60℃水浴中静置1小时后仍能保留超过83%的酶活，70℃水浴中静置1小时后仍然能保留超过50%的活力，耐热效果显著。
实施例6 本发明提供的过氧化氢酶的应用
1、试剂：
1）用NaH2PO4-Na2HPO4为缓冲溶液（pH7.0）配制浓度为200 mg/L 过氧化氢 1L；
3）本发明提供的过氧化氢酶1000U/mL。
2、仪器：
容量瓶等玻璃仪器；BL-310A精密电子天平；DragonMed标准移液器100~1000ul；5~50ul； DMI-010钻石牌数字式石英电子秒表。
3、测定方法：
1）测定原理：
过氧化氢酶能够分解过氧化氢生成氧气和水，将酶与过氧化氢作用一段时间后，用过氧化氢试纸测试残余的过氧化氢量，残余量越小说明过氧化氢酶的效果越好。
2）具体测定：
量取100mL 过氧化氢溶液（200 mg/L），40℃水浴，预热5min；然后加入0.5mL本发明所述酶（1000U/mL），立即计时（加液时开始计算时间）；加完酶液后，搅拌均匀，即得待测液；分别于5min、10min、15min、20min时，将过氧化氢试纸（默克）浸于待测液中，1sec后取出，15sec后读数。同时以市售过氧化氢酶产品作为对照组，稀释至1000U/mL，在相同添加量的情况下，与本发明所述酶进行效果比较。具体结果如表1所示：
表1 过氧化氢酶对过氧化氢浓度的影响
从表1的结果可以看出，利用本发明所述酶处理过氧化氢，10min时即将过氧化氢全部分解；而对照组所述市售过氧化氢酶，在20min时才将过氧化氢全部分解，且其在5min，10min，15min时对过氧化氢的分解效果均低于本发明所述酶。从而说明本发明所述酶能有效分解过氧化氢，且效果要优于市售产品，应用前景广泛。
本发明所述酶具有过氧化氢酶活性，能有效催化过氧化氢分解生成水和氧气，因此可广泛运用于食品、纺织、医药、造纸等领域中去除生产过程中的过氧化氢，比传统的方法更高效，节能，环保。
序列表
<110> 王艺璇
<120> 一种新型过氧化氢酶及其应用
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<210> 1
<211> 305
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<213> 曲霉属(Aspergillus sparsus)
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Lys Gly Cys Arg Lys Lys Ser Lys Glu Ile Val Gly Gly Arg Leu Cys
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Ala Thr Pro Arg Ser Ser Thr Ile Gly Arg Leu Ile Cys Ala Val Ser
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Ser Lys Ser Glu Asp Val Trp Ser Gln Leu Arg Val Phe Phe Lys Ser
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Asn Thr Asn Val Lys Leu Pro Glu Val Arg Ser Thr Ala Ser Ser Gln
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Leu Ile Arg Ser Glu Ser Glu Arg Gly Arg Arg Ala Thr Gly Glu Val
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Ile Met Val Gly Val Leu Gly Ser Val Glu His Ser Gly Leu Phe Lys
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Gly Gly Val Asn Phe Ser Asp Thr Leu Glu Gly Lys Gly Cys Gly Arg
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atgttcattc ccgtcaaccc tcacgcgtac ttgctaaaga cctacttaaa agggtgccga 60
aagaaatcca aggaaattgt gggtggtaga ttatgtgcaa cgcctagaag tagcacgatt 120
ggcaggctta tctgtgcagt aagctctaag tcggaggatg tgtggtctca gctacgggtc 180
ttcttcaagt ctttggtgcc tggcgaaaag aagttagtta tagacgccgt ccactctcaa 240
aatacgaacg taaagttacc cgaggtaagg agcaccgcca gcagtcaatt gatccgtagc 300
gagagtgagc gtggacgtcg tgctacgggc gaagtgggta tccccgggcc agagccagaa 360
cctaggttcg atcagaacaa caaaactgct gatatcggaa cttttgaagc taagcagaag 420
aagcgtgaag gcatcatggt aggcgtcttg ggaagtgtag agcattcggg cttgtttaag 480
ggaggtgtca acttcagcga cacgttggaa gggaaagggt gtggacgttg ttgttgtcgc 540
agagcgtctg gccgacggtg ttgcccagac ctattctact tcagatgcaa tccaattaaa 600
cgctgtggtc gttgccgccg gtgcagagag tctcttcgcg gcatcctcgt ttaccggtgg 660
gtccgccaac tcagcttttg gtgcaaggtt tctttacccc accggtcggc tccttcagat 720
tctcatggac ggattccggt tcggcaagac tgttggggcg ctgggaagcg gcacagctgc 780
tctccgcaac gcggggattg caacgtctcg ggacggcgtg tatgtggctc agtcagtcac 840
ggaggacttt gccaattttc taaagggggg tctaaggacc ttaaaattgt tgggaggatt 900
tcccgtggaa cttta 915
Claims (5)
1.一种过氧化氢酶，其特征在于，所述过氧化氢酶的氨基酸序列为SEQ ID NO:1。
2.用于编码权利要求1所述过氧化氢酶的基因，其核苷酸序列为SEQ ID NO:2。
3.一种表达载体，其携带有权利要求2所述的基因。
4.一种宿主细胞，其携带有权利要求3所述的表达载体。
5.权利要求1所述过氧化氢酶在食品、纺织、制药、造纸领域中的应用。
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