CN106010981B - 一株亚硝酸盐降解菌 - Google Patents
一株亚硝酸盐降解菌 Download PDFInfo
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Abstract
本发明公开了一株亚硝酸盐降解菌，该亚硝酸盐降解菌保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，保藏名称Daldinia sp.HYTC‑TB2（以下简称HYTC‑TB2），保藏编号：CCTCC M 2016067，保藏日期2016年2月22日。本发明的亚硝酸盐降解菌是从土壤中筛选出的，具有较高亚硝酸盐耐受性及较高降解活性，高效安全，发挥其在环境修复方面的优势，解决水体修复等问题。
Description
技术领域
本发明属于生物技术领域，具体涉及一种亚硝酸盐降解菌Daldinia sp. HYTC-TB2及其在生产亚硝酸盐还原酶和亚硝酸盐降解中的应用。
背景技术
亚硝酸盐，是氮循环中无机结合氮的一种分子形态，其氮处于中间价态，酸性条件下具有较强的氧化性，在血液中可将血红蛋白氧化为高铁血红蛋白，从而对高等动物体内氧的运输有抑制作用。在体外，用亚硝酸钠处理人红细胞后，会引发红细胞中酶和非酶介导的氧化应激，导致血红蛋白失活聚集，脂质和蛋白质被氧化，且红细胞内的主要代谢途径亦发生改变。
自然界可利用生物类群对被污染的环境，如亚硝酸盐污染，进行自净化作用。生物体可利用体内的亚硝酸盐还原酶（Nitrite reductase，NiR）把亚硝酸盐还原为一氧化氮或氨。NiR广泛存在于植物和微生物中，国内外的研究者们已经从植物、红藻、细菌中分离并提纯到亚硝酸盐还原酶。早在1960s，Alcaligenes sp.，Neurospora crassa，Pseudomonas sp.等亚硝酸盐降解菌就从污水中被分离出来了。但关于能降解亚硝酸盐的真菌的研究起步较晚，1995年，Kobayashi, M等首次从真菌Fusarium oxysporum中分离出NiR，后有多种真菌的Cu-NiRs被报道。
目前关于亚硝酸盐还原酶及亚硝酸盐降解菌的应用主要在生物传感器、水体修复、食品保鲜等方面。其中水体养殖中亚硝酸盐还原酶及亚硝酸盐降解菌的应用比较广泛，但已报道的该类酶或菌株的适用条件为弱酸性、温度大于30℃，限制了其应用。
发明内容
本发明的目的是：提供一株亚硝酸盐降解菌，从土壤中筛选出具有较高亚硝酸盐耐受性及较高降解活性的真菌新种，高效安全，发挥在环境修复方面的独特优势，解决水体修复等问题。
本发明的技术解决方案是：该亚硝酸盐降解菌保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，保藏名称Daldinia sp. HYTC-TB2，保藏编号：CCTCC M 2016067，保藏日期2016年2月22日。
本发明的亚硝酸盐降解菌种：Daldinia sp.HYTC-TB2是从土壤中筛选出的菌；菌种描述：在PDA培养基上菌落生长较慢，28℃黑暗条件下5 d菌落直径50~55 mm，菌落边缘整齐，孢子很少，有香甜味；该菌株在以亚硝酸钠为唯一氮源的培养基中生长，对亚硝酸钠的耐受性及利用率较高，表明其具有较高的亚硝酸盐降解活性。
按照常规方法从Daldinia sp.HYTC-TB2的纯培养物提取基因组DNA，运用rDNA内转录间隔区（ITS）序列特定引物ITS1/ITS4，通过PCR和测序得到ITS序列；在GenBank数据库中进行同源性比对，发现HYTC-TB2与已知的Daldinia eschscholzii 的ITS序列的相似性为97%；结合其形态学特征，确定它是一株真菌新种，将其归入炭棒科（Xylariaceae），命名为Daldinia sp. HYTC-TB2，该菌保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，保藏名称Daldinia sp. HYTC-TB2，保藏编号：CCTCC M 2016067，保藏日期2016年2月22日。
将本发明的菌株Daldinia sp.HYTC-TB2接种于以亚硝酸钠为唯一氮源的液体培养基中，研究其对培养基中亚硝酸钠的利用率，结果表明：Daldinia sp.HYTC-TB2在以亚硝酸钠为氮源的液体培养基（pH 7.4）中，在28℃，120 rpm/min条件下进行液体发酵，其中亚硝酸钠浓度为1 g/L，每天取样测试亚硝酸钠含量变化；生长经过48 h的延滞期进入对数生长期，60 h后，处于稳定生长期，菌株在0～48 h处于亚硝态氮降解的延滞期；48 h后亚硝态氮浓度迅速下降，60 h降解率达99.8％，表明该菌具有较好的亚硝酸盐降解活性。
本发明的优点是：真菌新种Daldinia sp. HYTC-TB2具有产亚硝酸盐还原酶活性，用于生产亚硝酸还原酶，对亚硝酸钠的耐受性及利用率较高，表明其具有较高的亚硝酸盐降解活性。
附图说明
图1为菌种纯培养照片。
图2为菌种在不同浓度亚硝酸钠平板培养基上生长照片。
具体实施方式
下面给出具体实施例对本发明的技术方案进一步说明，这些实施例不能理解为是对技术方案的限制。
实施例1：菌株的分离筛选
从河边采污泥土样，用无菌水进行梯度稀释10-1、10-2、10-3，分别取各梯度稀释液0.2 ml涂布于以亚硝酸钠为氮源的查氏培养基（葡萄糖30 g/L、NaNO2 2.5 g/L、MgSO4·7H2O 0.5 g/L、KCl 0. 5 g/L、FeSO4·7H2O 0. 01 g/L、K2HPO4 1 g/L）平板上，28℃恒温培养箱中倒置培养；待菌落长出后，从菌落中间挑取少量菌丝，再次转接到以亚硝酸钠为氮源的查氏培养基平板上，直至获得纯培养物，命名为Daldinia sp.HYTC-TB2，保存于PDA斜面上。
实施例2：真菌Daldinia sp.HYTC-TB2的鉴定
1、形态学鉴定：菌株的形态学初步鉴定依据《真菌鉴定手册》；如图1所示，在PDA培养基上菌落生长较慢，28℃黑暗条件下5天菌落直径50~55 mm，菌落边缘整齐，孢子很少，有香甜味；该菌株在以亚硝酸钠为唯一氮源的培养基中生长，对亚硝酸钠的耐受性及利用率较高，表明其具有较高的亚硝酸盐降解活性。
2、菌株分子生物学鉴定：菌株的DNA提取、PCR扩增及扩增产物的序列分析：将培养4 d的新鲜菌体作为DNA提取材料，采用尿素提取法提取菌株基因组DNA，0.8%琼脂糖凝胶电泳检测纯度；以上述提取的总DNA为模板，用引物ITS1/ITS4进行PCR；PCR产物送至上海生工生物工程有限公司进行测序；测序结果如下：
1 ttaactcagg acatctaact ccaccctatg tgaacttacc gccgttgcct cggcgggccgcgttcgccct gtagtttact
81 acctggcggc gcgctacagg cccgccggtg gactgctaaa ctctgttata tatacgtatctctgaatgct tcaacttaat
161 aagttaaaac tttcaacaac ggatctcttg gttctggcat cgatgaagaacgcagcgaaa tgcgataagt aatgtgaatt
241 gcagaattca gtgaatcatc gaatctttga acgcacattg cgcccattagtattctagtg ggcatgcctg ttcgagcgtc
321 atttcaaccc ttaagcccct gttgcttagc gttgggaatc taggtctctagggcctagtt ccccaaagtc atcggcggag
401 tcggagcgta ctctcagcgt agtaatacca ttctcgcttt tgcagtagccccggcggctt gccgtaaaac ccttatatct
481 ttagtggttg acctcgaatc aggtaggaat acccgctgaa cttaagcata tcaaaaggc
在GenBank数据库中进行同源性比对，发现Daldinia sp.HYTC-TB2与已知的Daldinia eschscholzii 的ITS序列的相似性为97%；结合其形态学特征，确定它是一株真菌新种，将其归入炭棒科（Xylariaceae），命名为Daldinia sp. HYTC-TB2，该菌保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，保藏名称Daldinia sp. HYTC-TB2，保藏编号：CCTCC M 2016067，保藏日期2016年2月22日。
实施例3、Daldinia sp.HYTC-TB2在平板上对亚硝酸钠的耐受性测试：将Daldinia sp.HYTC-TB2接种在以亚硝酸钠为唯一氮源的固体平板上，其中亚硝酸钠浓度分别为1 g/L、5 g/L、10 g/L、30 g/L，结果表明该菌在亚硝酸钠浓度为30 g/L的培养基上生长，说明该菌的亚硝酸钠耐受性较高。
实施例4、液体培养基中亚硝酸钠的降解率测试：将二块Daldinia sp.HYTC-TB2菌饼（直径为5 mm）接种于200 ml发酵培养基（配方：葡萄糖30 g/L、NaNO2 1 g/L、MgSO4·7H2O0.5 g/L、KCl 0. 5 g/L、FeSO4·7H2O 0. 01 g/L、K2HPO4 1 g/L）中，28℃，120 rpm，摇床培养；每天取样，利用盐酸萘乙二胺法检测亚硝酸钠的含量。
亚硝酸钠标准曲线的制作：分别吸取400 μl、200 μl、100 μl、50 μl、25 μl 0.1mmol·L-1的NaNO2溶液，加去离子水至1000 μl，则NaNO2终浓度为40 μmol·L-1、20 μmol·L-1、10 μmol·L-1、5 μmol·L-1、2.5 μmol·L-1；每个样品中分别加入40 μl浓度的对氨基苯磺酸，混匀避光静置5 min；然后分别加入20 μl 浓度为0.2%（W/V）的盐酸萘乙二胺溶液，混匀避光静置15 min；取200 μl样品反应液于96孔板中，用酶标仪在波长540 nm处测定其吸光值，分别为1.179075、0.658675、0.360775、0.188475和0.101175，标准曲线方程为y =0.0286x + 0.0548。
将每天取的样品稀释到原来的千分之一，用盐酸萘乙二胺法进行检测，结果表明：0~3 d 培养基中亚硝酸钠含量基本未变；第4 d时亚硝酸钠的利用率为25%，第5 d 时亚硝酸钠的利用率达到67.5%；第6 d时培养基中的亚硝酸钠已利用完全；由此表明本发明的Daldinia sp. HYTC-TB2具有较高的降解亚硝酸盐的能力。
实施例5、亚硝酸盐还原酶活的测定：将二块Daldinia sp.HYTC-TB2菌饼（直径为5mm）接种于200 mL发酵培养基（配方：葡萄糖30 g/L、NaNO2 1 g/L、MgSO4·7H2O 0.5 g/L、KCl 0. 5 g/L、FeSO4·7H2O 0. 01 g/L、K2HPO4 1 g/L）中，28℃，120 rpm，摇床培养5 d，过滤收集菌体，并用去离子水漂洗两次，用pH7.4缓冲液漂洗一次；每克湿菌体加入10 ml于4℃预冷的缓冲液，冰浴超声波破碎菌体1 h，每超声5 秒停10 秒，超声后取出12 000 rpm离心10 min，取上清即为真菌Daldinia sp. HYTC-TB2的胞内总蛋白样品液。
酶催化反应体系为250 μl，先取125 μl 0.1 mol·L-1 pH 7.4的Tris-Cl缓冲液、15 μl 0.1 mol·L-1的 NaCl溶液、12.5 μl 0.1 mol·L-1的NaNO2溶液和7.5 μl 0.1 mol·L-1的甲基紫精溶液于1.5 ml离心管中，混匀，30℃预热5 min；再加入50 μl粗蛋白样品液，30℃预热5 min；加入40 μl 0.1 mol·L-1的Na2S2O4溶液（Na2S2O4溶于0.1 mol·L-1的NaHCO3溶液中），此时酶催化反应被启动，置于30℃水浴锅中，并计时；分别在0 min、30 min取反应液10 μl，加990 μl水，剧烈震荡至蓝色完全褪去，酶催化反应终止；在该条件下，每分钟降解1 μmol NaNO2所需的酶量定义为1个酶活力单位；用盐酸萘乙二胺法测定样品中亚硝酸钠的含量变化，结果表明Daldinia sp. HYTC-TB2发酵5 d后胞内总蛋白亚硝酸盐还原酶酶活达到5.6 U/mg总蛋白；由此表明本发明的真菌Daldinia sp. HYTC-TB2具有产亚硝酸盐还原酶活性。
序列表
< 110 ＞淮阴师范学院
< 120 ＞一株亚硝酸盐降解菌
< 160 > l
< 210 > ITS
< 211 > 539
< 212 > DNA
< 222 > 人工序列
< 400 > ITS
TTAACTCAGGACATCTAACTCCACCCTATGTGAACTTACCGCCGTTGCCTCGGCGGGCCGCGTTCGCCCTGTAGTTTACTACCTGGCGGCGCGCTACAGGCCCGCCGGTGGACTGCTAAACTCTGTTATATATACGTATCTCTGAATGCTTCAACTTAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCTTAAGCCCCTGTTGCTTAGCGTTGGGAATCTAGGTCTCTAGGGCCTAGTTCCCCAAAGTCATCGGCGGAGTCGGAGCGTACTCTCAGCGTAGTAATACCATTCTCGCTTTTGCAGTAGCCCCGGCGGCTTGCCGTAAAACCCTTATATCTTTAGTGGTTGACCTCGAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAAAGGC
Claims (1)
1. 一株亚硝酸盐降解菌，其特征是：该亚硝酸盐降解菌保藏于中国典型培养物保藏中心，保藏名称：Daldinia sp. HYTC-TB2，保藏编号：CCTCC NO: M 2016067，保藏日期：2016年2月22日。
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