CN104928216B - 一株拟无枝酸菌及其应用 - Google Patents

一株拟无枝酸菌及其应用 Download PDF

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CN104928216B
CN104928216B CN201510356936.2A CN201510356936A CN104928216B CN 104928216 B CN104928216 B CN 104928216B CN 201510356936 A CN201510356936 A CN 201510356936A CN 104928216 B CN104928216 B CN 104928216B
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amycolatopsis
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张改云
王爽
王李娜
周渊
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CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION
Third Institute of Oceanography SOA
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Abstract

一株拟无枝酸菌及其应用,涉及微生物技术领域。所述拟无枝酸菌(Amycolatopsis sp.)WP1,保藏中心登记入册编号:CGMCC No.10738。所述拟无枝酸菌(Amycolatopsis sp.)WP1可在制备过氧化氢酶生物催化剂、蛋白酶生物催化剂、木聚糖酶生物催化剂、磷酸酶生物催化剂等多种生物催化剂中应用。具有过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶活性,具有开发为过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶等多种生物催化剂的潜能,对过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶的研究和利用具有重要的价值。

Description

一株拟无枝酸菌及其应用
技术领域
本发明涉及微生物技术领域,具体是涉及一株具有多种生物酶活性的海洋拟无枝酸菌(Amycolatopsis sp.)WP1。
背景技术
海洋微生物在漫长的演化过程中依靠体内特有的、强大的极端酶系适应其独特的栖息环境,这些极端酶系既是微生物赖以生存的保证,同时也为人类提供了巨大的财富。
放线菌是最具经济价值和生物技术价值的原核生物,除了能够产生丰富的次级代谢产物,放线菌还是生物催化剂的主要来源,目前已从放线菌中分离得到一系列具有重要工业用途的酶,如蛋白酶、纤维素酶、脂肪酶、木聚糖酶、果胶酶、淀粉酶、葡萄糖氧化酶、脂肪氧化酶、肌醇六磷酸酶和过氧化酶等,他们在洗涤、食品、酿造、皮革、纺织、动物饲料、饮料、造纸和烘培等领域的应用已取得令人瞩目的成果。对放线菌产酶能力的研究结果表明海洋放线菌具有生产多种生物酶的潜能,从放线菌中获得酶制剂是一个充满前景的发展方向。
目前,世界各国开始积极探索海洋放线菌的开发利用潜力,从中开发和利用具有新颖生物活性的产品,包括了化工小分子的生产、生物活性物质的发现与利用、生物酶的高值利用等。
随着中国经济的飞速发展,人民生活水平的提高,人们对生物酶产业的需求越来越大。因此深入开展海洋放线菌产生物酶的相关研究已成为当今的一个研究热点,同时也具有重要的应用价值和科学意义。
发明内容
本发明的目的在于提供一株具有多种生物酶活性的海洋拟无枝酸菌(Amycolatopsis sp.)WP1及其应用。
所述拟无枝酸菌(Amycolatopsis sp.)WP1已于2015年4月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮编:100101,保藏中心登记入册编号:CGMCC No.10738。
所述拟无枝酸菌(Amycolatopsis sp.)WP1,从西南印度洋63.50E,27.89S,2945m深的沉积物样品中分离筛选得到。菌株分离后经菌落形态鉴定,该菌在高氏一号培养基上28℃培养7天后,菌落呈圆形,表面和边缘不光滑,中部有褶皱状凸起,白色,不透明,产孢子,无晕圈,菌落直径为2~3mm。
所述拟无枝酸菌(Amycolatopsis sp.)WP1为革兰氏阳性菌,氧化酶试验阴性,过氧化氢酶实验阳性,硫化氢产生试验和吲哚产生试验阴性,V-P试验阳性。
由于所述拟无枝酸菌(Amycolatopsis sp.)WP1具有过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶活性,因此所述拟无枝酸菌(Amycolatopsis sp.)WP1可在制备过氧化氢酶生物催化剂、蛋白酶生物催化剂、木聚糖酶生物催化剂、磷酸酶生物催化剂等多种生物催化剂中应用。
所述拟无枝酸菌(Amycolatopsis sp.)WP1通过16S rDNA序列比对分析,与已知典型种Amycolatopsis magusensis KT2025T,Amycolatopsis palatopharyngis 1BDZ T和Amycolatopsis marina MS392AT的16S rDNA序列的同源性最高,分别为97.8%,97.3%和97.2%,与其他典型种的16S rDNA序列相似性均低于97%。通过结合菌体形态特征、生长条件、生理生化鉴定结果确定所述拟无枝酸菌(Amycolatopsis sp.)WP1属于假诺卡氏菌科(Pseudonocardiaceae)拟无枝酸菌属(Amycolatopsis)。
所述拟无枝酸菌(Amycolatopsis sp.)WP1可在高氏一号培养基上生长,其生长温度为10~45℃,最适生长温度为30~35℃,pH生长为5~11,最适pH为8~9,盐度生长为0~8%,最适盐度生长为0~3%。
所述高氏一号培养基配方为可溶性淀粉20g,NaCl 0.5g,KNO31g,K2HPO4·3H2O0.5g,MgSO4·7H2O 0.5g,FeSO4·7H2O 0.01g,琼脂18g,蒸馏水1000ml,pH 7.4~7.6。
本发明所述拟无枝酸菌(Amycolatopsis sp.)WP1具有过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶活性,具有开发为过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶等多种生物催化剂的潜能,对过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶的研究和利用具有重要的价值。
附图说明
图1为本发明所述的拟无枝酸菌(Amycolatopsis sp.)WP1在高氏一号培养基上的宏观形态。
图2为本发明所述的拟无枝酸菌(Amycolatopsis sp.)WP1在高氏一号培养基上的扫描电镜照片。
图3为本发明所述的拟无枝酸菌(Amycolatopsis sp.)WP1及部分相关菌株依据16S rDNA序列构建的系统发育树。
具体实施方式
下面结合具体实施例对本发明做进一步描述,但本发明的保护范围并不仅限于此。
实施例1 拟无枝酸菌(Amycolatopsis sp.)WP1的分离
1.生物材料及培养基:用于分离海洋放线菌的生物材料为采自西南印度洋63.50E,27.89S,2945m深的沉积物样品。用于放线菌分离所用的培养基为海水琼脂培养基,配方为:人工海水1L,琼脂粉15g,pH 7.2~7.6;人工海水配方为:NaCl 24.477g、MgCl2·6H2O4.981g、Na2SO43.917g、CaCl2·2H2O 1.102g、KCl 0.664g、NaHCO30.192g、KBr 0.096g、H3BO30.026g、SrCl20.024g、NaF 0.0039g及蒸馏水1000mL。
2.样品处理:称取1g沉积物样品,溶于45ml人工海水中(内加10颗左右玻璃珠),28℃摇床中200rpm振荡2h,取200μl涂布至含终浓度为50μg/ml制霉菌素和50μg/ml萘啶酮酸的海水琼脂培养基上。
3.放线菌培养:将涂布悬浊液的分离培养基置于28℃恒温培养箱中倒置培养,培养14天左右开始挑取培养基上生长的单菌落进行转接纯化,根据菌株的形态特征和培养特征,去除重复菌株,对已纯化菌株,接种于海水琼脂培养基中,于28℃培养箱中培养后,置于4℃冰箱保存备用。
实施例2 拟无枝酸菌(Amycolatopsis sp.)WP1的生物学性状及生理生化和分子鉴定
1、形态特征
将拟无枝酸菌(Amycolatopsis sp.)WP1在高氏一号培养基上用无菌接种环划线接种,28℃培养7天,菌落呈圆形、表面和边缘不光滑、中部有褶皱状凸起、不透明、产生白色孢子、无晕圈,直径2~3mm。
2、生理生化特征
参照《放线菌快速鉴定与系统分类》(阮继生,黄英.放线菌快速鉴定与系统分类[M].北京:科学出版社,2011)中所述的方法,对菌株WP1的生理生化特征如碳源利用、H2S产生、吲哚产生、明胶液化、生长温度、盐度及pH耐受性等指标进行测定。测定结果表明:拟无枝酸菌(Amycolatopsis sp.)WP1可利用柠檬酸,不能利用葡萄糖、甘露醇、肌醇、山梨醇、鼠李糖、蔗糖、蜜二糖和阿拉伯糖;不能产生硫化氢和吲哚,可使明胶液化,生长温度为10~45℃(最适30~35℃),生长盐度为0~8%(最适0~3%),生长pH为5~11(最适8~9)。
3、分子鉴定
用热破壁法提取拟无枝酸菌(Amycolatopsis sp.)WP1的基因组DNA,采用国际细菌鉴定通用引物(27F:5’-AGAGTTTGATCCTGGCTCAG-3’和1492R:5’-GGTTACCTTGTTACGACTT-3’),以基因组DNA为模板进行PCR扩增,然后利用胶回收试剂盒回收纯化PCR产物,之后进行克隆、转化,筛选阳性克隆,经扩大培养后委托上海生工生物工程技术服务有限公司进行测序。测序结果显示:拟无枝酸菌(Amycolatopsis sp.)WP1的16S rDNA序列长度为1482bp,其序列如SEQ ID NO:1所示,将测序结果与GenBank中的16S rDNA序列进行同源性比对,然后用软件构建系统发育树(如图1所示),以确定菌株的种属关系。同源性分析结果表明,该序列与拟无枝酸菌属成员的16S rDNA序列的同源性最高,相似性为93.9~97.8%。结合菌体形态特征、生长条件、生理生化鉴定结果确定拟无枝酸菌(Amycolatopsis sp.)WP1属于假诺卡氏菌科(Pseudonocardiaceae)拟无枝酸菌属(Amycolatopsis),为拟无枝酸菌(Amycolatopsis sp.)WP1(Amycolatopsis sp.WP1)。
实施例3 拟无枝酸菌(Amycolatopsis sp.)WP1产过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶活性测定
1、酶活检测培养基及方法
1)过氧化氢酶活性检测:用竹签挑取部分拟无枝酸菌(Amycolatopsis sp.)WP1菌体于玻片上,滴加10%过氧化氢溶液,立即有气泡产生即为有过氧化氢酶活性。
2)蛋白酶活性检测:用竹签沾取适量拟无枝酸菌(Amycolatopsis sp.)WP1菌体,划线于蛋白酶活性检测培养基(胰蛋白胨1%,酵母提取物0.5%,NaCl 1%,脱脂奶粉1%)28℃培养72h,有透明水解圈出现的为有活性,否则为没有活性。
3)木聚糖酶活性测定:用竹签沾取适量拟无枝酸菌(Amycolatopsis sp.)WP1菌体,划线于木聚糖酶活性检测培养基(木聚糖3%,MgSO4·7H2O 0.05%,NaCl 0.05%,KNO30.1%,K2HPO40.05%,琼脂2%,刚果红0.04%,pH 8.0)。
4)磷酸酶活性测定:用竹签沾取适量拟无枝酸菌WP1菌体,划线于LB培养基上(胰蛋白胨1%,酵母提取物0.5%,氯化钠1%),28℃培养两天,待菌落长出后,将浸在1mol/LTris-HCl溶液(pH 8.5,含2mg/mL pNPP底物)中的滤纸片贴在菌落上,28℃培养3min,若滤纸片变黄则为有活性。
2、酶活检测结果
测试结果发现,拟无枝酸菌(Amycolatopsis sp.)WP1具有过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶活性,具有开发为过氧化氢酶、蛋白酶、木聚糖酶和磷酸酶等多种生物催化剂的潜能。

Claims (4)

1.拟无枝酸菌(Amycolatopsis sp.)WP1在制备过氧化氢酶生物催化剂、蛋白酶生物催化剂、木聚糖酶生物催化剂或磷酸酶生物催化剂中应用,所述拟无枝酸菌(Amycolatopsissp.)WP1,已于2015年4月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏中心登记入册编号:CGMCC No.10738。
2.如权利要求1所述应用,其特征在于所述拟无枝酸菌(Amycolatopsis sp.)WP1在高氏一号培养基上生长,其生长温度为10~45℃,pH生长为5~11,盐度生长为0~8%。
3.如权利要求2所述应用,其特征在于所述拟无枝酸菌(Amycolatopsis sp.)WP1在高氏一号培养基上生长,其生长温度为30~35℃,pH生长为8~9,盐度生长为0~3%。
4.如权利要求2所述应用,其特征在于所述高氏一号培养基配方为可溶性淀粉20g,NaCl 0.5g,KNO3 1g,K2HPO4·3H2O 0.5g,MgSO4·7H2O 0.5g,FeSO4·7H2O 0.01g,琼脂18g,蒸馏水1000ml,pH 7.4~7.6。
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