CN113999802B - 一种可表达高活性乳糖酶的黄瓜类芽孢杆菌菌株 - Google Patents
一种可表达高活性乳糖酶的黄瓜类芽孢杆菌菌株 Download PDFInfo
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Abstract
本发明属于微生物领域，公开了该菌株为黄瓜类芽孢杆菌（Paenibacillus cucumis.），菌株编号BHNB003001，保藏日期为2021年10月11日，保藏单位为中国典型培养物保藏中心（CCTCC），地址为湖北省武汉市武昌区八一路299号，保藏编号为CCTCC NO：M 20211256。所筛选获得的黄瓜类芽孢杆菌菌株CCTCC NO：M 20211256所产的乳糖酶对酶活性适应温度和pH范围较广，在工业生产中有较大的优势。
Description
技术领域
本发明涉及微生物领域，具体讲是一种可表达高活性乳糖酶的黄瓜类芽孢杆菌菌株。
背景技术
黄瓜类芽孢杆菌（Paenibacillus cucumis.） 属于芽孢杆菌科、类芽孢杆菌属，是一类能产生抗力内生孢子的革兰氏阳性菌，细胞呈杆状且外层覆盖大量的吡啶二羧酸钙。其皮层位于核心和芽孢壳之间，含丰富的肽聚糖；核心是一种高度浓缩的、惰性的染色体；最外层的外壁为一层肽聚糖壁，且一层或多层的成分为蛋白质的芽孢衣。由于芽孢具有厚而含水量低的多层结构，所以折光性强，对染料不易着色，稳定性好，耐氧化、耐挤压、耐高温，能长期耐受 60 ℃高温，在温度 120 ℃下能存活 20 min，且耐酸碱，在胃酸环境中能保持活性，这可能与芽孢独具的高含量吡啶二羧酸有关。同时还具有广谱杆菌活性，能产生细菌素，抑制病原菌。
而其所产的乳糖酶在工业上主要用于乳制品工业。利用其水解作用分解乳制品中的乳糖，生产低乳糖或无乳糖乳制品，供乳糖不耐受的广大人群食用。葡萄糖是人体各部分代谢的能量来源，半乳糖则是人大脑和黏膜组织代谢时必需的结构糖，是婴幼儿脑发育的必要组织，与婴儿大脑的迅速成长有密切联系。再者，乳糖酶还可在人体内通过转糖苷作用生成低聚糖，这些低聚糖是一种低分子量，不粘稠的水溶性膳食纤维，它在肠道内作为增殖因子仅被双歧杆菌利用，却不会被腐败细菌利用，如此可大大减少肠道有害毒素的产生，对预防便秘和腹泻有很重要的作用。
乳糖酶在乳制品工业应用研究具有广阔的前景，但目前市场上销售的乳糖酶存在温度适应范围窄、热稳定性差等问题。
因此，筛选出一种能表达出温度适应范围广、热稳定性好、拥有高活性的乳糖酶的菌株对工业化生产具有重大意义。
发明内容
本发明所要解决的技术问题是，克服以上现有技术的缺点：提供一种能表达出温度适应范围广、热稳定性好、高活性的乳糖酶的菌株。
本发明的技术解决方案如下：一种可表达高活性乳糖酶的黄瓜类芽孢杆菌菌株，该菌株为黄瓜类芽孢杆菌（Paenibacillus cucumis.），菌株编号BHNB003001，保藏日期为2021年10月11日，保藏单位为中国典型培养物保藏中心（CCTCC），地址为湖北省武汉市武昌区八一路299号，保藏编号为CCTCC NO：M 20211256。
其表达乳糖酶酶活力适应温度范围为20-60℃。
其表达乳糖酶最适的酶反应温度为35℃。
其表达乳糖酶酶活力适应pH值范围为4-8。
其表达乳糖酶在pH 6.5时的酶活力最高。
其核苷酸测序序列如SEQ ID NO：1所示。
本发明的有益效果是：本发明的黄瓜类芽孢杆菌菌株CCTCC NO：M 20211256从海水中筛选获得。所筛选获得的黄瓜类芽孢杆菌菌株CCTCC NO：M 20211256产出的乳糖酶的酶活性适应温度和pH范围较广，在工业生产中有较大的优势。
本发明黄瓜类芽孢杆菌（Paenibacillus cucumis.）的保藏日期为2021年10月11日，保藏单位为中国典型培养物保藏中心（CCTCC），保藏编号为CCTCC NO：M 20211256。
附图说明
图1为本发明黄瓜类芽孢杆菌菌株CCTCC NO：M 20211256的系统发育树；
图2为温度对本发明黄瓜类芽孢杆菌菌株CCTCC NO：M 20211256菌株表达的乳糖酶酶活性影响的结果图；
图3为pH对本发明黄瓜类芽孢杆菌菌株CCTCC NO：M 20211256表达的乳糖酶酶活性影响的结果图。
具体实施方式
下面用具体实施例对本发明做进一步详细说明，但本发明不仅局限于以下具体实施例。
实施例
一、菌株培养、鉴定及构建系统发育树
1、菌株培养
LB培养基：氯化钠10g/L，蛋白胨10g/L，酵母浸粉5g/L，琼脂20g/L，纯水配制，添加20mg/ml X-gal。
YPD培养基（选择性培养基）：蛋白胨20g/L，酵母浸粉10g/L，葡萄糖40g/L,琼脂20g/L，纯水配制，添加20mg/ml X-gal。
LB深孔板：氯化钠10g/L，蛋白胨10g/L，酵母浸粉5g/L，纯水配制。
酶活力单位的定义：一个酶活单位（NLU）定义为在反应条件下释放1.30 μmol/min的邻硝基苯酚所需的酶量。
酶活测定方法：邻硝基苯酚β-D半乳糖苷（ONPG）溶于P-E-M缓冲液中（ pH值为6.5）配制成质量分数为0.25％的底物溶液。取5ml底物溶液加入1ml酶液中，30℃保温10min。加入2mL碳酸钠溶液显色，测定420nm光吸收值。计算水解产物邻硝基酚（ONP）的含量和酶活性。
培养：从海南采集了一些海水和沙土样本，并将采集到样本逐一进行培养，分别涂布于LB和YPD平板培养基中，对应的平板分别编号，贴上封口膜，将平板置于30℃恒温倒置培养48h，之后平板上挑出了显蓝色单菌落，将挑出的单菌落分别接到LB摇瓶中，置于震荡培养箱培养，培养条件为30℃，避光，转速为245r/min。培养24h后，取样，添加50%甘油保存于-80℃冰箱。分别取培养24h、48h、72h的菌液，测定酶活。
2、菌株鉴定
对该菌株进行16S rDNA的PCR扩增鉴定，具体步骤如下：
PCR扩增：通用引物27F和1492R进行PCR扩增，其中，
引物27F序列为：5′-AGAGTTTGATCCTGGCTCAG-3′；
引物1492R序列为： 5′-GGTTACCTTGTTACGACTT-3′。
PCR反应体系为：25×TAQ酶5μL，27F引物2μL，1492R引物2μL，菌液模板5μL，无菌水16μL。PCR扩增程序为：94℃预变性5min；94℃变性30s，56℃退火1s，72℃延伸60s，30个循环；72℃延伸5min，4℃终止反应。
凝胶电泳及PCR产物柱回收：PCR产物经1.5％琼脂糖凝胶电泳鉴定。利用EasyPurePCR Purification Kit纯化试剂盒（北京全式金生物技术有限公司）对目的PCR产物进行回收纯化，进行16S rRNA测序。对所测得的16S rDNA，运用NCBI中的Blast程序与数据库中的细菌16S rDNA序列进行相似性比对。
比对结果：菌株BHNB003001为黄瓜类芽孢杆菌（Paenibacillus cucumis.）的菌株。
3、构建菌株BHNB003001的系统发育树
获得菌株BHNB003001的16S rDNA后，用MEGA 5.1 软件的（Neighbor -Joining）构建该菌株的系统发育树（如图1所示），分析各菌株的进化关系。
分析结果：菌株BHNB003001为黄瓜类芽孢杆菌（Paenibacillus cucumis.）的菌株。
二、菌株保藏
筛选得到的黄瓜类芽孢杆菌，命名为黄瓜类芽孢杆菌（Paenibacillus cucumis），菌株编号BHNB003001，其保藏编号为CCTCC NO：M 20211256，保藏日期为2021年10月11日，保藏单位为中国典型培养物保藏中心（CCTCC），地址为湖北省武汉市武昌区八一路299号武汉大学。
三、关于乳糖酶酶活力的最适温度
在pH值为5.5条件下，分别在不同温度（20℃、25℃、30℃、33℃、35℃、37℃、40℃、50℃、60℃）下，测定酶活力，测定的相对酶活力变化曲线如图2所示，此乳糖酶的适应温度范围较广，在25-40℃之间相对酶活都在80%以上，最适的酶反应温度为35℃。
可见，本发明筛选得到的菌株BHNB003001所产的乳糖酶具有较广的适应温度范围，菌株BHNB003001在工业中具有良好的优势。
四、关于乳糖酶酶活力的最适PH
取酶液分别用pH为（4.0、4.5、5.0、5.5、6.0、6.5、6.8、 7.0、7.2、7.5、8.0）的缓冲液稀释，测定酶活力，相对酶活力变化曲线如图3所示，乳糖酶在pH 6.5时的酶活力最高，pH适应范围为4.0-8.0。
以上仅是本发明的特征实施范例，对本发明保护范围不构成任何限制。凡采用同等交换或者等效替换而形成的技术方案，均落在本发明权利保护范围之内。
序列表
<110> 宁波希诺亚海洋生物科技有限公司
<120> 一种可表达高活性乳糖酶的黄瓜类芽孢杆菌菌株
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1212
<212> DNA
<213> 黄瓜类芽孢杆菌菌株(Paenibacillus cucumis)
<400> 1
cggggggatc tatactgcaa gtcgagcgga cttgatgaga agcttgcttc tctgatggtt 60
agcggcggac gggtgagtaa cacgtaggca acctgccctc aagcttggga caactaccgg 120
aaacggtagc taataccgaa tacttgtttt cttcgcctga agaaaactgg aaagacggag 180
caatctgtca cttggggatg ggcctgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatgggcg aaagcctgac 360
ggagcaatgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gccagggaag 420
aacgcttggg agagtaactg ctctcaaggt gacggtacct gagaagaaag ccccggctaa 480
ctacgtgcca gcagccgcgg taatacgtag ggggcaagcg ttgtccggaa ttattgggcg 540
taaagcgcgc gcaggcggtc atgtaagtct ggtgtttaat cccggggctc aaccccggat 600
cgcactggaa actgcgtgac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660
tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg ggctgtaact 720
gacgctgagg cgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 780
gtaaacgatg agtgctaggt gttaggggtt tcgataccct tggtgccgaa gtttaacaca 840
ttaagcactc cgcctggggg agtacggtcg caagactgaa actcaaagga attgacgggg 900
acccgcacaa gcagtggagt atgtggttta attcgaagca acgcgaagaa ccctttacca 960
ggtcttgaca tccaactaaa cgaagcagag atgcatcagg tgccctttcg ggggaaagtt 1020
gagacagtgg ttgcatgggt tgtcgtcagc ttcgtgtcgt tgagatgttt gggtttaagt 1080
tcccgcaaac ggagcgcacc ctttattatt tttagttgcc agcaacttcc ggttggcacc 1140
ttcttaaaat tagactgccg ttgacgaacc ggcaggcaag cttgggggat tggacggttc 1200
aaaattcatt ca 1212
Claims (1)
1.一种可表达高活性乳糖酶的黄瓜类芽孢杆菌菌株，其特征在于：该菌株为黄瓜类芽孢杆菌（Paenibacillus cucumis），保藏日期为2021年10月11日，保藏单位为中国典型培养物保藏中心（CCTCC），保藏编号为CCTCC NO：M 20211256。
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| CN111630172A (zh) * | 2017-11-16 | 2020-09-04 | 拜耳作物科学有限合伙公司 | 基于类芽孢杆菌属的内生孢子呈现平台、产品和方法 |
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| CN111630172A (zh) * | 2017-11-16 | 2020-09-04 | 拜耳作物科学有限合伙公司 | 基于类芽孢杆菌属的内生孢子呈现平台、产品和方法 |
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