CN110923220B - 一种酶组合物、制备酶组合物的方法及应用 - Google Patents
一种酶组合物、制备酶组合物的方法及应用 Download PDFInfo
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- CN110923220B CN110923220B CN201911295038.5A CN201911295038A CN110923220B CN 110923220 B CN110923220 B CN 110923220B CN 201911295038 A CN201911295038 A CN 201911295038A CN 110923220 B CN110923220 B CN 110923220B
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- lysostaphin
- acetylglucosaminidase
- leu
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- bacillus subtilis
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Abstract
本发明公开了一种酶组合物、制备酶组合物的方法及应用,属于基因工程技术领域。一种酶组合物,包括溶葡萄球菌酶和乙酰葡萄糖胺酶。溶葡萄球菌酶和乙酰葡萄糖胺酶编码基因按照枯草芽孢杆菌密码子偏好性优化后与枯草芽孢杆菌分泌表达质粒连接,构建重组表达质粒。然后将重组表达质粒电转化入枯草芽孢杆菌中,获得重组枯草芽孢杆菌,发酵产生的溶葡萄球菌酶与乙酰葡萄糖胺酶,用于溶葡萄球菌酶和乙酰葡萄糖胺酶的产品制备和杀菌剂的应用。本发明产生的粗酶可以直接用于专一性金黄色葡萄球菌生物膜的降解和活菌细胞的杀灭。本发明提供的方法操作简单、易于实现,具有很好的应用前景。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种酶组合物、制备酶组合物的方法及应用。
背景技术
自然界中99%的细菌以生物膜的形式存在,细菌生物膜的复杂结构增加了致病菌对抗生素的抵抗作用,是导致慢性感染性疾病治疗失败的重要原因。金黄色葡萄球菌是一种重要的临床致病菌,常见于植入医疗器械的染等的并发症,造成的严重血液和深部组织感染可以导致病人死亡,严重威胁着人类健康。金黄色葡萄球菌容易形成细胞群落而黏附在内植医疗器械上,菌落的表面包裹着一层自身合成的多糖、脂质和蛋白质等形成的生物被膜。此膜可以保护细菌不被宿主免疫系统识别和清除,而抗生素类药物由于生物被膜的阻碍无法接触到致病菌,也达不到预期的杀菌效果。抗生素的长期使用也造成了金黄色葡萄球菌的耐药菌越来越常见,新的金黄色葡萄球菌杀菌剂的开发是一个热点领域。
溶葡萄球菌酶(Lysostaphin)是一种专一性水解金黄色葡萄球菌的酶,其商业化酶制剂已经问世,市场需求潜力很大。溶葡萄球菌酶能够专一性水解金黄色葡萄球菌的细胞壁肽聚糖的“甘氨酰五肽”键,造成细胞溶胀裂解死亡,从而实现杀灭效果。由于作用靶点具有特殊性,所以溶葡萄球菌酶的长期使用不仅不会产生耐药性,而且能够有效杀灭已经产生其他抗生素抗药性的金黄色葡萄球菌菌株,例如耐甲氧西林和万古霉素的金葡菌。但是由于生物膜的空间阻隔作用,溶葡萄球菌酶对形成生物膜的金黄色葡萄球菌杀灭效果很差。
乙酰葡萄糖胺酶(dispersin B,Dsp B)是一种β-N-乙酰葡萄糖胺酶,能够专一性降解聚β-(1,6)-N-乙酰葡萄糖胺底物(PNAG),这种大分子是金黄色葡萄球菌等多种细菌的生物膜多糖的主要组成成份。已有报道证明乙酰葡萄糖胺酶作用于金黄色葡萄球菌生物膜,能够有效降解生物膜多糖组分,充分破坏生物膜结构,同时释放出来游离金黄色葡萄球菌细胞。而游离出的金黄色葡萄球菌细胞能够迁移到新的位置重新定植,反而会促进病原金黄色葡萄球菌的侵袭扩展。因此乙酰葡萄糖胺酶的使用引起争议,使用范围受到很大限制。
目前报道的表达系统如大肠杆菌和毕赤酵母,还没有食品级的表达系统高水平生产溶葡萄球菌酶的报道。而乙酰葡萄糖胺酶的商业化酶制剂在国内还没有产品问世,其主要原因是缺乏高水平表达乙酰葡萄糖胺酶的重组工程菌。
溶葡萄球菌酶和乙酰葡萄糖胺酶各自单独用于金黄色葡萄球菌杀灭,均有各自的缺点。将溶葡萄球菌酶和乙酰葡萄糖胺酶联合使用作用于金黄色葡萄球菌生物膜防治是否能够获得更好的杀灭效果,值得探索。
发明内容
针对现有耐药性金黄色葡萄球菌的抗菌剂缺乏的问题,本发明提供了一种胞外分泌溶葡萄球菌酶和乙酰葡萄糖胺酶的重组枯草芽孢杆菌,通过发酵制备的重组酶,实现金黄色葡萄球菌生物膜的降解和活菌细胞的杀灭,简化生产过程。
一种酶组合物,包括溶葡萄球菌酶和乙酰葡萄糖胺酶,优选的,溶葡萄球菌酶氨基酸序列如SEQ ID NO.2所示,乙酰葡萄糖胺酶氨基酸序列如SEQ ID NO.3所示。
为增强发酵产生的粗酶液杀灭金黄色葡萄球菌的效果,优选的,所述的溶葡萄球菌酶的浓度≥7.5mg/L,所述乙酰葡萄糖胺酶的浓度≥8.35mg/L,更为优选的,所述的溶葡萄球菌酶的浓度范围为7.5~30mg/L,所述乙酰葡萄糖胺酶的浓度范围为8.35~33.4mg/L。
更为优选的,酶组合物在制备杀菌剂中的应用以及一种基于酶组合物的杀菌剂,该杀菌剂可有效杀死金黄色葡萄球菌。
本发明提供了一种重组枯草芽孢杆菌,包含有能够胞外分泌表达溶葡萄球菌酶和乙酰葡萄糖胺酶的基因序列,优选的,溶葡萄球菌酶基因序列如SEQ ID NO.4所示,乙酰葡萄糖胺酶基因序列如SEQ ID NO.5所示。
枯草芽孢杆菌作为无内毒素,遗传背景清晰,发酵工艺较为成熟的优势。通过重组枯草芽孢杆菌发酵生产溶葡萄球菌酶和乙酰葡萄糖胺酶,是生产食品级和医药级的溶葡萄球菌酶和乙酰葡萄糖胺酶制剂的最佳选择。
优选的,枯草芽孢杆菌外源导入重组表达载体,重组表达载体包含溶葡萄球菌酶基因和乙酰葡萄糖胺酶基因,两个基因的N端均具有表达信号肽的序列,引导新合成的蛋白质向分泌通路转移,两个基因各自由一个p43启动子启动表达。
本发明还提供了一种制备酶组合物的方法,酶组合物包括溶葡萄球菌酶和乙酰葡萄糖胺酶,使用重组枯草芽孢杆菌进行发酵,发酵产物分离去除菌体后获得粗酶液即为所述酶组合物,该产物分离简单,所得粗酶液即可有效降解生物膜多糖组分,杀灭病原菌,抑制了金黄色葡萄球菌的侵袭扩展。
优选的,制备的酶组合物在制备杀菌剂中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明通过重组枯草芽孢杆菌,发酵制备粗酶液,溶葡萄球菌酶能够专一性水解金黄色葡萄球菌的细胞壁肽聚糖的“甘氨酰五肽”键,造成细胞溶胀裂解死亡,但是由于生物膜的空间阻隔作用,溶葡萄球菌酶对形成生物膜的金黄色葡萄球菌杀灭效果很差。乙酰葡萄糖胺酶能够专一性降解金黄色葡萄球菌生物膜上多糖组分,充分破坏生物膜结构,同时释放出游离金黄色葡萄球菌细胞,溶葡萄球菌酶和乙酰葡萄糖胺酶协同后可有效杀灭金黄色葡萄球菌,杀灭效果强于两种酶纯化后单独使用的杀灭效果。
附图说明
图1为实施例1中pHP13-p43dual质粒;
图2为重组质粒pHP13-p43dual MCS-Dispersin-MCS-Lys图谱;
图3实施例2中,重组枯草芽孢杆菌菌落PCR的琼脂糖凝胶电泳图;
图4为实施例3中重组枯草芽孢杆菌表达溶葡萄球菌酶和乙酰葡萄糖胺酶胞外上清粗酶液电泳图;
图5为实施例5重组溶葡萄球菌酶和乙酰葡萄糖胺酶纯化后电泳图;
图6为实施例6发酵液粗酶和水解抑制金黄色葡萄球菌生物膜形成结果。
具体实施方式
实施例1构建重组表达质粒
(1)溶葡萄球菌酶编码基因的核苷酸序列(Genbank号:KF724949.1)和乙酰葡萄糖胺酶编码基因的氨基酸序列(Genbank号:WP_005566076)根据上述两个参考序列按照枯草芽孢杆菌密码子偏好性优化后全基因合成基因序列。
将优化序列的两个基因的N端融合枯草芽孢杆菌的α淀粉酶的信号肽序列,溶葡萄球菌酶c末端融合6×His标签基因序列,乙酰葡萄糖胺酶c末端融合S-tag标签基因序列。两个基因融合不同的标签,便于分别纯化两种蛋白。
(2)pHP13-p43dual载体为本实验室自主构建的枯草芽孢杆菌双启动子表达载体,复制起点为rep60,带有氯霉素抗性基因,带有两组多克隆位点,每组多克隆位点前面都带有p43启动子。
(3)根据pHP13-p43dual载体的酶切位点,设计两个酶的表达引物序列如下,带有酶切位点(斜体)和信号肽序列(下划线):
上游引物1 Dispersin-F:
下游引物1 Dispersin-R:
上游引物2 Lysostaphin-F:
下游引物2 Lysostaphin-R:
(3)将上述设计的引物,以合成溶葡萄球菌酶和乙酰葡萄糖胺酶编码基因为模板扩增基因片段。扩增条件:使用Superpfu Mix试剂,扩增两个目的基因片段,PCR扩增体系如下:
表1基因PCR扩增条件
PCR的反应条件为:95℃预变性4min;95℃变性30s,60℃退火30s,68℃延伸3min,循环数30;72℃延伸10min。
(4)如图1所示,表达质粒pHP13-p43dual经SalI和HindIII双酶切线性化后与同样双酶切之后的溶葡萄球菌酶扩增基因片段,通过T4连接酶连接,构建重组质粒pHP13-p43dual MCS-Lys,测序验证,确认重组质粒构建成功。
pHP13-p43dual MCS-Lys再经BamHI和XhoI双酶切线性化后与同样双酶切之后的乙酰葡萄糖胺酶扩增基因片段,通过T4连接酶连接,构建重组质粒pHP13-p43dual MCS-Dispersin-MCS-Lys,测序验证,确认重组质粒构建成功。如图2所示。
实施例2构建重组枯草芽孢杆菌株
将构建好的重组表达质粒pHP13-p43dual MCS-Dispersin-MCS-Lys,采用电转化原生质体的方法转入枯草芽孢杆菌(168,ATCC56765)中。
具体如下:
(1)接种枯草芽孢杆菌168菌株于3ml LB培养基中,过夜培养。
(2)取2.6ml过夜培养物接入40ml(LB+0.5M山梨醇)中,37℃,200rpm培养至OD600=0.85~0.95。
(3)将菌液冰水浴10min,然后5 000g,5min,4℃离心收集菌体。
(4)用50ml预冷的电转培养基(0.5M山梨醇,0.5M甘露醇,10%葡萄糖),重新吹悬菌体,5 000g,5min,4℃离心去上清,如此漂洗4次。
(5)将洗涤后的菌体吹悬于1ml电转培养基中,每EP管分装120μl。
(6)将60μl感受态细胞中加入50ng DNA(1-8μl),冰上孵育2min,加入预冷的电转杯(1mm)中,电击一次。电转仪设置:2.0kv,1mm,电击1次。(电击结果:时间常数=4.5~5.0ms,如果时间常数<4.2,则需要增加电转培养基的漂洗次数或者提高感受态的稀释倍数来获得更高的转化效率。
(7)电击完毕取出杯子并立即加入1ml RM(LB培养基+0.5M山梨醇+0.38M甘露醇),37℃,200rpm,复苏3h后,涂板,37℃,过夜培养。
(8)采用Dispersin-F/R,Lysostaphin-F/R引物进行菌落PCR,筛选阳性转化子。
菌落PCR扩增条件如下:
表2菌落PCR扩增条件
PCR的反应条件为:95℃预变性4min;95℃变性30s,55℃退火30s,72℃延伸1min,循环数30;72℃延伸10min。
PCR产物用琼脂糖凝胶电泳检测,结果见图3。结果显示,1-5号克隆都检测到有约800bp和1100bp目的条带出现,则验证重组枯草芽孢杆菌已经构建成功。
实施例3摇瓶发酵生产溶葡萄球菌酶和乙酰葡萄糖胺酶
(1)将实施例2成功构建的重组枯草芽孢杆菌单菌落接种于种子培养基中,于37℃、220rpm下培养10h,备用。
种子培养基:包括以下质量浓度的组分:蛋白胨1%,酵母粉0.5%,葡萄糖0.5%,pH 7.0。
(2)将上述培养好的重组枯草芽孢杆菌的种子液按体积5%接种于液体发酵培养基中,于37℃、220rpm条件下发酵72h,12 000rpm离心10min,固液分离,获取发酵液上清液,即为重组溶葡萄球菌酶和乙酰葡萄糖胺酶粗酶液。
发酵培养基包含以下质量浓度的组分:葡萄糖15g/L、豆粕30g/L、淀粉1g/L,K2HPO4·3H2O 12.5g/L、KH2PO4 2.5g/L以及MgSO4·7H2O 1.5g/L,pH 7.0。
实施例4重组溶葡萄球菌酶和乙酰葡萄糖胺酶产量的测定
(1)上述实施例3中得到的发酵液粗酶液,SDS-PAGE检测蛋白的表达情况,结果见图4。利用凝胶成像系统的灰度测定软件对图4中重组溶葡萄球菌酶和乙酰葡萄糖胺酶的条带进行分析计算,蛋白Marker条带作为参照。
(2)经过测定和计算,溶葡萄球菌酶和乙酰葡萄糖胺酶产量可达到750mg/L和835mg/L。结果证明溶葡萄球菌酶和乙酰葡萄糖胺酶,在重组枯草芽孢杆菌实现了胞外分泌高表达。
实施例5重组溶葡萄球菌酶和乙酰葡萄糖胺酶的纯化
(1)20ml镍亲和层析介质置于砂芯分液漏斗中,用100mM Na2HPO4/NaH2PO4(pH7.0)的缓冲液冲洗2个主体积,以平衡介质。
(2)取100ml上述实施例3中得到的发酵液粗酶液,0.22μm微孔滤膜过滤除去不溶颗粒后,滤清液加入到平衡好的20ml镍亲和层析介质中,轻轻搅拌混匀,静置20min,以保证充分亲和吸附。
(3)打开分液漏斗,释放含有未吸附杂蛋白的流穿液,单独收集。
(4)分批加入50ml的100mM,200mM,500mM和1M的咪唑溶液洗脱,并单独分批收集每次的洗脱液。
(5)将每批洗脱液置于透析袋中,在100mM Na2HPO4/NaH2PO4(pH7.0)的缓冲液体系中透析,除去咪唑。合并样品为溶葡萄球菌酶的纯化样品。
(6)步骤3中的流穿液,置于透析袋中,在100mM Na2HPO4/NaH2PO4(pH 7.0)的缓冲液体系中透析,除去咪唑。
(7)透析好的样品,加入到平衡好的结合S-Tag融合蛋白的S-蛋白琼脂糖中,轻轻搅拌混匀,静置20min,以保证充分亲和吸附。
(8)释放含有未吸附杂蛋白的流穿液,用洗脱液(3M NaSCN、3M MgCl2和0.2M柠檬酸,pH 2.0)将结合蛋白从S-蛋白琼脂糖上洗脱下来。洗脱液置于透析袋中,在100mMNa2HPO4/NaH2PO4(pH 7.0)的缓冲液体系中透析,除去多余离子。
(9)SDS-PAGE分别检测两次洗脱样品中纯化后目的蛋白的纯度,结果如图5所示。
(10)Bradford方法测定上述两种纯化蛋白的浓度。
步骤如下:纯化酶稀释50倍,取0.5mL,加入2.5mL考马斯亮蓝,反应3分钟,用紫外分光光度计检测595nm波长下的吸光度。蛋白质浓度与吸光值关系标准曲线如图6。
根据测定OD595数值,利用标准曲线公式计算纯化酶的蛋白含量。
纯化的溶葡萄球菌酶的蛋白含量为205mg/L,纯化的乙酰葡萄糖胺酶的蛋白含量为197mg/L。
实施例6溶葡萄球菌酶和乙酰葡萄糖胺酶水解金黄色葡萄球菌生物膜形成的测定
采用微量板半定量法检测两种酶对金黄色葡萄球菌生物膜形成的影响。
(1)固体LB培养基平板挑金黄色葡萄球菌ATCC29213单菌落,入3mL TSB培养基(50ml离心管)中,37℃剧烈振荡培养8小时;TSB培养基购买于青岛海博生物科技有限公司。
(2)取菌液用TSB培养基(单独添加1%葡萄糖)适当稀释后,分光光度计测OD600;
(3)步骤2中菌液稀释至OD600为0.01;
(4)在1ml稀释后的菌液中加入100μl不同稀释度的酶样品,混匀。
(5)于96孔板(聚苯乙烯)中每孔小心加入100μl混匀的菌液,勿使液滴挂壁及溅出,封口膜封闭96孔板边缘;
(6)37℃静置培养20h;
(7)吸出菌液,用水清洗96孔板2次,吹干;
(8)加入150μl 0.1%结晶紫染色20min;
(9)吸出结晶紫,用生理盐水彻底清洗至溶液无色,吸出残水,吹干;
(10)200μl 33%醋酸静置溶解,于酶标仪上振荡混匀,590nm波长测定吸光度值。
实验分为空白对照组和样品组,
空白对照组:不含有质粒的枯草芽孢杆菌发酵上清液
样品组1:1:25稀释度的粗酶(60μg/ml)
样品组2:1:5稀释度的纯化溶葡萄球菌酶(40μg/ml)
样品组3:1:5稀释度的纯化乙酰葡萄糖胺酶(40μg/ml)
样品组4:1:50稀释度的粗酶(30μg/ml)
样品组5:1:10稀释度的纯化溶葡萄球菌酶(20μg/ml)
样品组6:1:10稀释度的纯化乙酰葡萄糖胺酶(20μg/ml)
样品组7:1:250稀释度的粗酶(6μg/ml)
样品组8:1:20纯化溶葡萄球菌酶(10μg/ml)
样品组9:1:20纯化乙酰葡萄糖胺酶(10μg/ml)
样品组10:1:32纯化溶葡萄球菌酶与纯化乙酰葡萄糖胺酶混合(5μg/ml)
结果见图6,根据图6中的结果,无论是纯化溶葡萄球菌酶还是纯化乙酰葡萄糖胺酶单独使用,即使在1:5稀释倍数的高浓度(约40μg/ml)情况下,样品组2和3降解生物膜的效果都低于样品组10中较低浓度(蛋白含量均为为5μg/ml)下两种酶混合使用的效果。样品组7中1:250稀释度的粗酶(两种目的酶蛋白含量估算均为6μg/ml)也产生了很好的降解抑制生物膜的效果。而不含有质粒的枯草芽孢杆菌发酵上清液没有抑制生物膜的活性。上述结果证明溶葡萄球菌酶和乙酰葡萄糖胺酶在降解抑制金葡菌生物膜的过程中,产生了良好的协同效应。
对于既有协同作用的两种酶,在工程菌中进行重组表达的时候,经常会同时导入到一个受体菌中进行表达,这样可以通过一次发酵同时获得两种蛋白,简化生产步骤。本发明中枯草芽孢杆菌极强的分泌能力,同时保证了两种蛋白的高效分泌表达,同时含有两种酶的粗酶液无需要纯化,两种酶同时使用,表现出良好的抑制金葡菌生物膜的活性。
序列表
<110> 杭州师范大学
<120> 一种酶组合物、制备酶组合物的方法及应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7208
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tatattttat aggattggtt tttaggaaat ttaaactgca atatatcctt gtttaaaact 60
tggaaattat cgtgatcaac aagtttattt tctgtagttt tgcataattt atggtctatt 120
tcaatggcag ttacgaaatt acacctcttt actaattcaa gggtaaaatg gccttttcct 180
gagccgattt caaagatatt atcatgttca tttaatctta tatttgtcat tattttatct 240
atattatgtt ttgaagtaat aaagttttga ctgtgtttta tatttttctc gttcattata 300
accctcttta atttggttat atgaattttg cttattaacg attcattata accacttatt 360
ttttgtttgg ttgataatga actgtgctga ttacaaaaat actaaaaatg cccatatttt 420
ttcctcctta taaaattagt ataattatag cacgaaaagg atctaggtga agatcctttt 480
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 540
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 600
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 660
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 720
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 780
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 840
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 900
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 960
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 1020
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 1080
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 1140
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 1200
ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 1260
ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 1320
cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 1380
ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 1440
taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 1500
tatgttgtgt ggaattgtga gcggataaca atttcacgcc aagcttggct gcagttactt 1560
gatggtaccc cacaagacac ccaaggtgtt ggtggacttg ttccaggttc tgactggcaa 1620
gtagattctt tgaccggagt taccggtgta accgacccag acgtgaccgt cttgcttcat 1680
gacctcgtcg tagtggatgg tttgaccagc cttcaagaca ccggattgtg gcatggatct 1740
gaatggaccg gtggttctgg tgatgatgtc ggtgtttggg gtgaaggaag cggactcgga 1800
cttgtacaag gtaccgtact tgttggtctt ccaaccggtg tttggggttg gggtgacggt 1860
accaccagcc ttaccgtaac cagcggactt caagaatggc attgggtctt gagcggtgga 1920
gttggagaag gagttgacca ttctttggaa gtgcaagtgt ggagcggtgg agtaaccggt 1980
ggaaccggac caaccgatga tttgaccagc cttgacgtag tcaccgacct tgacgttgta 2040
cttggacaag tgcatgtacc attgtctgtg gacaccgtcg ttctcgatca aaccgatttg 2100
gttaccacca ccgtagttgg accaaccagc ctcgacgatc ttaccggagg agatagcctt 2160
gactggggta ccgatgttca tgaagaagtc aacaccgtag tgcataccac cgttgatacc 2220
caatgggtat ggaccgtaac cgtaaccctt cttgtagttg ttcaaccatt gagcggagtg 2280
ctcgtgggta gcagcgtcga cacaggaaac agctcctccg ctatcacttt atattttaca 2340
taatcgcgcg ctttttttca cgcccatttc taaaaatgta aaataaatgt aagaattttc 2400
agccattaca gctttggcaa aaaaataagt aaaccaatga tacacgaaat cacggcaaaa 2460
acgcaaacag ccccggcggc agcgtccttg gccgctttag caagagggtg atgtttgtca 2520
gttattaaat caaccgtatg ttcaatggct gtatttaaaa gttcaagcga aaacatacca 2580
cctatcaatg accatgatta ctgataggtg gtatgttttc gcttgaactt ttaaatacag 2640
ccattgaaca tacggttgat ttaataactg acaaacatca ccctcttgct aaagcggcca 2700
aggacgctgc cgccggggct gtttgcgttt ttgccgtgat ttcgtgtatc attggtttac 2760
ttattttttt gccaaagctg taatggctga aaattcttac atttatttta catttttaga 2820
aatgggcgtg aaaaaaagcg cgcgattatg taaaatataa agtgatagcg gaggggatcc 2880
atgaactaca tcaagaaaat cattctgagc ctgttcctgc tgggtctgtt tagcgtgctg 2940
aactgctgcg ttaagggcaa cagcatttac ccgcagaaga ccagcaccaa acaaaccggt 3000
ctgatgctgg acatcgcgcg tcacttttat agcccggaag ttatcaagag cttcatcgat 3060
accatcagcc tgagcggtgg caacttcctg cacctgcact ttagcgacca cgagaactac 3120
gcgatcgaaa gccacctgct gaaccagcgt gcggagaacg cggtgcaagg caaggatggc 3180
atctacatta acccgtatac cggtaaaccg tttctgagct accgtcagct ggacgatatc 3240
aaggcgtatg cgaaggcgaa aggcatcgag ctgattccgg aactggacag cccgaaccac 3300
atgaccgcga ttttcaagct ggtgcaaaaa gatcgtggtg ttaagtacct gcagggcctg 3360
aaaagccgtc aagtggacga tgagatcgac attaccaacg cggatagcat cacctttatg 3420
cagagcctga tgagcgaagt tatcgacatt ttcggtgata ccagccaaca ctttcacatc 3480
ggtggcgatg agttcggcta tagcgtggag agcaaccacg aatttattac ctacgcgaac 3540
aagctgagct atttcctgga aaagaaaggt ctgaagaccc gtatgtggaa cgacggcctg 3600
atcaaaaaca ccttcgagca gattaacccg aacatcgaaa ttacctactg gagctatgac 3660
ggtgataccc aagacaaaaa cgaggcggcg gaacgtcgtg atatgcgtgt tagcctgccg 3720
gagctgctgg cgaagggctt taccgtgctg aactacaaca gctactatct gtatatcgtt 3780
ccgaaagcga gcccgacctt cagccaggat gcggcgtttg cggcgaagga tgtgattaaa 3840
aactgggacc tgggtgtttg ggatggccgt aacaccaaga accgtgttca gaacacccac 3900
gaaattgcgg gtgcggcgct gagcatttgg ggcgaggatg cgaaggcgct gaaagatgaa 3960
accatccaaa agaacaccaa aagcctgctg gaggcggtga ttcacaaaac caacggtgac 4020
gaacaccacc accaccacca ctaactcgag taaccgggaa ttcactggcc gtcgttttac 4080
aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 4140
ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 4200
gcagcctgaa tggcgaatgg cgactaacgg ggcaggttag tgacattaga aaaccgactg 4260
taaaaagtac agtcggcatt atctcatatt ataaaagcca gtcattaggc ctatctgaca 4320
attcctgaat agagttcata aacaatcctg catgataacc atcacaaaca gaatgatgta 4380
cctgtaaaga tagcggtaaa tatattgaat tacctttatt aatgaatttt cctgctgtaa 4440
taatgggtag aaggtaatta ctattattat tgatatttaa gttaaaccca gtaaatgaag 4500
tccatggaat aatagaaaga gaaaaagcat tttcaggtat aggtgttttg ggaaacaatt 4560
tccccgaacc attatatttc tctacatcag aaaggtataa atcataaaac tctttgaagt 4620
cattctttac aggagtccaa ataccagaga atgttttaga tacaccatca aaaattgtat 4680
aaagtggctc taacttatcc caataaccta actctccgtc gctattgtaa ccagttctaa 4740
aagctgtatt tgagtttatc acccttgtca ctaagaaaat aaatgcaggg taaaatttat 4800
atccttcttg ttttatgttt cggtataaaa cactaatatc aatttctgtg gttatactaa 4860
aagtcgtttg ttggttcaaa taatgattaa atatctcttt tctcttccaa ttgtctaaat 4920
caattttatt aaagttcatt tgatatgcct cctaaatttt tatctaaagt gaatttagga 4980
ggcttacttg tctgctttct tcattagaat caatcctttt ttaaaagtca atattactgt 5040
aacataaata tatattttaa aaatatccca ctttatccaa ttttcgtttg ttgaactaat 5100
gggtgcttta gttgaagaat aaaagaccac attaaaaaat gtggtctttt gtgttttttt 5160
aaaggatttg agcgtagcga aaaatccttt tctttcttat cttgatacta tatagaaaca 5220
acatcatttt tcaaaattag gtcaaagcct tgtgtatcaa gggtttgatg gttctttgac 5280
aggtaaaaac tccttctgct attattaagg tgtcgaatca aaataataga atgctagaga 5340
actagctcag aaggagtttt tttgttgatt tattcatctg aaaatgatta tagcatcctc 5400
gaagataaaa ccgcaacagg taaaaagcgg gattggaagg ggaaaaagag acggacgaac 5460
ctcatggcgg agcattacga agcgttagag agtaagattg gggcacctta ctatggcaaa 5520
aaggctgaaa aactaattag ttgtgcagag tatctttcgt ttaagagaga cccggagacg 5580
ggcaagttaa aactgtatca agcccatttt tgtaaagtga ggttatgtcc gatgtgtgcg 5640
tggcgcaggt cgttaaaaat tgcttatcac aataagttga tcgtagagga agccaataga 5700
cagtacggct gcggatggat ttttctcacg ctgacgattc gaaatgtaaa gggagaacgg 5760
ctgaagccac aaatttctgc gatgatggaa ggctttagga aactgttcca gtacaaaaaa 5820
gtaaaaactt cggttcttgg atttttcaga gctttagaga ttaccaaaaa tcatgaagaa 5880
gatacatatc atcctcattt tcatgtgttg ataccagtaa ggaaaaatta ttttgggaaa 5940
aactatatta agcaggcgga gtggacgagc ctttggaaaa aggcgatgaa attggattac 6000
actccaattg tcgatattcg tcgagtgaaa ggtaaagcta agattgacgc tgaacagatt 6060
gaaaacgatg tgcggaacgc aatgatggag caaaaagctg ttctcgaaat ctctaaatat 6120
ccggttaagg atacggatgt tgtgcgcggt aataaggtga ctgaagacaa tctgaacacg 6180
gtgctttact tggatgatgc gttggcagct cgaaggttaa ttggatacgg tggcattttg 6240
aaggagatac ataaagagct gaatcttggt gatgcggagg acggcgatct ggtcaagatt 6300
gaggaagaag atgacgaggt tgcaaatggt gcatttgagg ttatggctta ttggcatcct 6360
ggcattaaaa attacataat caaataaaaa aagcagacct ttagaaggcc tgctttttta 6420
actaacccat ttgtattgtg ttgaaatatg ttttgtatgg tgcactctca gtacaatctg 6480
ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 6540
acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 6600
catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 6660
acgcctattt ttataggtta atgtcatgat aataatggtt tcttagcgat tcacaaaaaa 6720
taggcacacg aaaaacaagt taagggatgc agtttatgca tcccttaact tacttattaa 6780
ataatttata gctattgaaa agagataaga attgttcaaa gctaatattg tttaaatcgt 6840
caattcctgc atgttttaag gaattgttaa attgattttt tgtaaatatt ttcttgtatt 6900
ctttgttaac ccatttcata acgaaataat tatacttttg tttatctttg tgtgatattc 6960
ttgatttttt tctacttaat ctgataagtg agctattcac tttaggttta ggatgaaaat 7020
attctcttgg aaccatactt aatatagaaa tatcaacttc tgccattaaa agtaatgcca 7080
atgagcgttt tgtatttaat aatcttttag caaacccgta ttccacgatt aaataaatct 7140
cattagctat actatcaaaa acaattttgc gtattatatc cgtacttatg ttataaggta 7200
tattacca 7208
<210> 2
<211> 247
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Leu Leu Asp Gly Thr Pro Gln Asp Thr Gln Gly Val Gly Gly Leu Val
1 5 10 15
Pro Gly Ser Asp Trp Gln Val Asp Ser Leu Thr Gly Val Thr Gly Val
20 25 30
Thr Asp Pro Asp Val Thr Val Leu Leu His Asp Leu Val Val Val Asp
35 40 45
Gly Leu Thr Ser Leu Gln Asp Thr Gly Leu Trp His Gly Ser Glu Trp
50 55 60
Thr Gly Gly Ser Gly Asp Asp Val Gly Val Trp Gly Glu Gly Ser Gly
65 70 75 80
Leu Gly Leu Val Gln Gly Thr Val Leu Val Gly Leu Pro Thr Gly Val
85 90 95
Trp Gly Trp Gly Asp Gly Thr Thr Ser Leu Thr Val Thr Ser Gly Leu
100 105 110
Gln Glu Trp His Trp Val Leu Ser Gly Gly Val Gly Glu Gly Val Asp
115 120 125
His Ser Leu Glu Val Gln Val Trp Ser Gly Gly Val Thr Gly Gly Thr
130 135 140
Gly Pro Thr Asp Asp Leu Thr Ser Leu Asp Val Val Thr Asp Leu Asp
145 150 155 160
Val Val Leu Gly Gln Val His Val Pro Leu Ser Val Asp Thr Val Val
165 170 175
Leu Asp Gln Thr Asp Leu Val Thr Thr Thr Val Val Gly Pro Thr Ser
180 185 190
Leu Asp Asp Leu Thr Gly Gly Asp Ser Leu Asp Trp Gly Thr Asp Val
195 200 205
His Glu Glu Val Asn Thr Val Val His Thr Thr Val Asp Thr Gln Trp
210 215 220
Val Trp Thr Val Thr Val Thr Leu Leu Val Val Val Gln Pro Leu Ser
225 230 235 240
Gly Val Leu Val Gly Ser Ser
245
<210> 3
<211> 381
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Asn Tyr Ile Lys Lys Ile Ile Leu Ser Leu Phe Leu Leu Gly Leu
1 5 10 15
Phe Ser Val Leu Asn Cys Cys Val Lys Gly Asn Ser Ile Tyr Pro Gln
20 25 30
Lys Thr Ser Thr Lys Gln Thr Gly Leu Met Leu Asp Ile Ala Arg His
35 40 45
Phe Tyr Ser Pro Glu Val Ile Lys Ser Phe Ile Asp Thr Ile Ser Leu
50 55 60
Ser Gly Gly Asn Phe Leu His Leu His Phe Ser Asp His Glu Asn Tyr
65 70 75 80
Ala Ile Glu Ser His Leu Leu Asn Gln Arg Ala Glu Asn Ala Val Gln
85 90 95
Gly Lys Asp Gly Ile Tyr Ile Asn Pro Tyr Thr Gly Lys Pro Phe Leu
100 105 110
Ser Tyr Arg Gln Leu Asp Asp Ile Lys Ala Tyr Ala Lys Ala Lys Gly
115 120 125
Ile Glu Leu Ile Pro Glu Leu Asp Ser Pro Asn His Met Thr Ala Ile
130 135 140
Phe Lys Leu Val Gln Lys Asp Arg Gly Val Lys Tyr Leu Gln Gly Leu
145 150 155 160
Lys Ser Arg Gln Val Asp Asp Glu Ile Asp Ile Thr Asn Ala Asp Ser
165 170 175
Ile Thr Phe Met Gln Ser Leu Met Ser Glu Val Ile Asp Ile Phe Gly
180 185 190
Asp Thr Ser Gln His Phe His Ile Gly Gly Asp Glu Phe Gly Tyr Ser
195 200 205
Val Glu Ser Asn His Glu Phe Ile Thr Tyr Ala Asn Lys Leu Ser Tyr
210 215 220
Phe Leu Glu Lys Lys Gly Leu Lys Thr Arg Met Trp Asn Asp Gly Leu
225 230 235 240
Ile Lys Asn Thr Phe Glu Gln Ile Asn Pro Asn Ile Glu Ile Thr Tyr
245 250 255
Trp Ser Tyr Asp Gly Asp Thr Gln Asp Lys Asn Glu Ala Ala Glu Arg
260 265 270
Arg Asp Met Arg Val Ser Leu Pro Glu Leu Leu Ala Lys Gly Phe Thr
275 280 285
Val Leu Asn Tyr Asn Ser Tyr Tyr Leu Tyr Ile Val Pro Lys Ala Ser
290 295 300
Pro Thr Phe Ser Gln Asp Ala Ala Phe Ala Ala Lys Asp Val Ile Lys
305 310 315 320
Asn Trp Asp Leu Gly Val Trp Asp Gly Arg Asn Thr Lys Asn Arg Val
325 330 335
Gln Asn Thr His Glu Ile Ala Gly Ala Ala Leu Ser Ile Trp Gly Glu
340 345 350
Asp Ala Lys Ala Leu Lys Asp Glu Thr Ile Gln Lys Asn Thr Lys Ser
355 360 365
Leu Leu Glu Ala Val Ile His Lys Thr Asn Gly Asp Glu
370 375 380
<210> 4
<211> 741
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gctgctaccc acgagcactc cgctcaatgg ttgaacaact acaagaaggg ttacggttac 60
ggtccatacc cattgggtat caacggtggt atgcactacg gtgttgactt cttcatgaac 120
atcggtaccc cagtcaaggc tatctcctcc ggtaagatcg tcgaggctgg ttggtccaac 180
tacggtggtg gtaaccaaat cggtttgatc gagaacgacg gtgtccacag acaatggtac 240
atgcacttgt ccaagtacaa cgtcaaggtc ggtgactacg tcaaggctgg tcaaatcatc 300
ggttggtccg gttccaccgg ttactccacc gctccacact tgcacttcca aagaatggtc 360
aactccttct ccaactccac cgctcaagac ccaatgccat tcttgaagtc cgctggttac 420
ggtaaggctg gtggtaccgt caccccaacc ccaaacaccg gttggaagac caacaagtac 480
ggtaccttgt acaagtccga gtccgcttcc ttcaccccaa acaccgacat catcaccaga 540
accaccggtc cattcagatc catgccacaa tccggtgtct tgaaggctgg tcaaaccatc 600
cactacgacg aggtcatgaa gcaagacggt cacgtctggg tcggttacac cggtaactcc 660
ggtcaaagaa tctacttgcc agtcagaacc tggaacaagt ccaccaacac cttgggtgtc 720
ttgtggggta ccatcaagta a 741
<210> 5
<211> 1170
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaactaca tcaagaaaat cattctgagc ctgttcctgc tgggtctgtt tagcgtgctg 60
aactgctgcg ttaagggcaa cagcatttac ccgcagaaga ccagcaccaa acaaaccggt 120
ctgatgctgg acatcgcgcg tcacttttat agcccggaag ttatcaagag cttcatcgat 180
accatcagcc tgagcggtgg caacttcctg cacctgcact ttagcgacca cgagaactac 240
gcgatcgaaa gccacctgct gaaccagcgt gcggagaacg cggtgcaagg caaggatggc 300
atctacatta acccgtatac cggtaaaccg tttctgagct accgtcagct ggacgatatc 360
aaggcgtatg cgaaggcgaa aggcatcgag ctgattccgg aactggacag cccgaaccac 420
atgaccgcga ttttcaagct ggtgcaaaaa gatcgtggtg ttaagtacct gcagggcctg 480
aaaagccgtc aagtggacga tgagatcgac attaccaacg cggatagcat cacctttatg 540
cagagcctga tgagcgaagt tatcgacatt ttcggtgata ccagccaaca ctttcacatc 600
ggtggcgatg agttcggcta tagcgtggag agcaaccacg aatttattac ctacgcgaac 660
aagctgagct atttcctgga aaagaaaggt ctgaagaccc gtatgtggaa cgacggcctg 720
atcaaaaaca ccttcgagca gattaacccg aacatcgaaa ttacctactg gagctatgac 780
ggtgataccc aagacaaaaa cgaggcggcg gaacgtcgtg atatgcgtgt tagcctgccg 840
gagctgctgg cgaagggctt taccgtgctg aactacaaca gctactatct gtatatcgtt 900
ccgaaagcga gcccgacctt cagccaggat gcggcgtttg cggcgaagga tgtgattaaa 960
aactgggacc tgggtgtttg ggatggccgt aacaccaaga accgtgttca gaacacccac 1020
gaaattgcgg gtgcggcgct gagcatttgg ggcgaggatg cgaaggcgct gaaagatgaa 1080
accatccaaa agaacaccaa aagcctgctg gaggcggtga ttcacaaaac caacggtgac 1140
gaacaccacc accaccacca ctaactcgag 1170
<210> 6
<211> 110
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggatccatgg tttctatacg tcgttcattt gargcttatg tcgacgatat gaatatatta 60
ctgttcttat tcctgctgag cagaaggaga ttgctgctac ccacgagcac 110
<210> 7
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctcgagttac ttgatggtac cccacaa 27
<210> 8
<211> 115
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtcgacatgg tttctatacg tcgttcattt gargcttatg tcgacgatat gaatatatta 60
ctgttcttat tcctgctgag cagaaggaga ttatgaacta catcaagaaa atcat 115
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gaattcctcg agttagtggt ggtggtggtg 30
Claims (1)
1.酶组合物在制备用于杀灭金黄色葡萄球菌的杀菌剂中的应用,所述酶组合物由溶葡萄球菌酶和β-N-乙酰氨基葡萄糖苷酶组成,
制备酶组合物的方法包括以下步骤:
将重组枯草芽孢杆菌进行发酵,发酵产物分离去除菌体后获得粗酶液即为所述酶组合物,
所述的重组枯草芽孢杆菌,由枯草芽孢杆菌外源导入重组表达载体,所述重组表达载体包含溶葡萄球菌酶基因和β-N-乙酰氨基葡萄糖苷酶基因,两个基因的N端均具有表达信号肽的序列,两个基因各自由一个p43启动子启动表达,
所述重组表达载体的序列如SEQ ID No.1所示。
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Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase;Nanna等;《Viruses》;20180818;第10卷;第2页倒数第1段至第3页第1段、第3页第3段、第12页倒数第1段至第13页第1段 * |
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