CN114591936B - 一种突变型褐藻胶裂解酶及其应用 - Google Patents
一种突变型褐藻胶裂解酶及其应用 Download PDFInfo
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- CN114591936B CN114591936B CN202210319319.5A CN202210319319A CN114591936B CN 114591936 B CN114591936 B CN 114591936B CN 202210319319 A CN202210319319 A CN 202210319319A CN 114591936 B CN114591936 B CN 114591936B
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Abstract
本发明公开了一种褐藻胶裂解酶突变体E135G,其在pH6.0‑9.0范围内酶活超过50%,具有良好pH稳定性;突变体E135G抑制铜绿假单胞菌生物膜生成的效率为63.9%,比野生型Alys1高8.5%;破解铜绿假单胞菌生物膜的效率为77.6%,比野生型Alys1高22.1%,能与抗生素左氧氟沙星具有良好的协同作用;在高血糖的存在下,突变体E135G仍对生物膜具有破除作用。
Description
技术领域
本发明属于生物技术领域,尤其涉及突变型褐藻胶裂解酶及其应用。
背景技术
褐藻胶是一种酸性的水溶性多糖,广泛存在于褐藻、龙须菜、海带等海洋藻类植物中,我国沿海地区如山东、福建等省份是其主要产区。褐藻胶存在于褐藻细胞壁及细胞间质中,由β-D-甘露糖醛酸(M)和α-L-古洛糖醛酸(G)两个异构体残基通过1,4糖苷键共价连接,以均匀或随机的方式排列成的线性多糖。分为同源多糖醛酸M残基(PolyM)、G残基(PolyG)以及杂多糖醛酸(PolyMG或PolyGM)。褐藻胶广泛应用于医药、食品、工业等领域。研究表明,低聚褐藻寡糖具有清除自由基、通过促进促进生物体免疫功能实现其抗肿瘤作用、降低血糖及减少脂质作用、促进植物根系生长、提高植物的抗逆性等多种生物活性,并且具有较好的抗病原菌活性。
目前,褐藻寡糖的制备方法分为物理降解法、化学降解法和生物酶解法。其中,生物酶解法具有专一高效,反应过程温和,操作简单,在制备褐藻寡糖方面更具优势。由于从野生菌中分离提取褐藻胶裂解酶过程较为繁琐,且产量低,通过基因工程的手段实现褐藻胶裂解酶的异源表达成为主流研究方向。
铜绿假单胞菌(Pseudomonas aeruginosa,PA)是临床感染中常见的革兰阴性条件致病菌,具有高度适应性、侵入性和产毒性。近年来,生物膜的形成已经被认定为大多数微生物引发感染和耐药的关键因素。到目前为止,抑制生物被膜的形成或提高抗生素的疗效已被提出多种方法:使用群体感应抑制剂、凝集素抑制剂、铁螯合、生物被膜分散剂、具有固有抗生物被膜特性的聚合物、阶段疗法、电化学支架和疫苗策略纳米颗粒等。
褐藻胶是其生物膜的关键基质多糖,通过防止药物渗透、结合和排斥带电抗生素以及提供针对天然宿主免疫的保护,对外来分子建立了一种天然的物理屏障,并为结合的细菌提供营养支持。同时,褐藻胶作为一个关键致病因子,通过干扰巨噬细胞和中性粒细胞的吞噬,促进了铜绿假单胞菌菌落的形成,加重了肺部感染。大多数专门设计用于杀菌的抗生素没有能力根除。传统治疗细菌感染的方法直接靶向病原菌,但生物膜的存在使抗菌药物的有效浓度提升到了危险水平。此外,包裹在生物膜中的病原菌还可能产生代谢休眠细胞或分子持久性机制对抗药物治疗,这也是导致生物膜类感染在临床治疗停顿一段时间后复发的原因。
目前对褐藻胶的降解主要有三种方法:物理法、化学法和生物法。然而通过物理法和化学法降解褐藻胶后,不仅降解效率低,产物分子量难以控制,还能够对环境造成污染。褐藻胶经过生物法(褐藻胶裂解酶)进行降解,可以定向生产特定分子量及无毒副作用的寡糖。并且该法与另外两种方法相比,具有环保、反应条件温和、产物单一及生物活性高等优点。
目前已知的褐藻胶裂解酶,大多都是单一底物特异性酶,应用范围狭窄。目前已知只有具有广泛底物特异性的双功能褐藻胶裂解酶,能够降解铜绿假单胞菌产生的生物膜,且与抗生素具有协同作用。而具有单一底物特异性的褐藻胶裂解酶不能有效降解生物膜,且无法与抗生素协同作用。这证实了褐藻胶裂解酶广泛的底物特异性会影响药物应用。本发明的目的是提供一种双功能褐藻胶裂解酶突变体及应用,通过定点突变得到能够降解铜绿假单胞菌生物膜的双功能褐藻胶裂解酶,为细菌性肺部感染提供了新的治疗思路,改善多药耐药细菌感染,将有助于褐藻胶裂解酶作为抗生物膜剂的进一步开发。
发明内容
由于正常人体细胞不含褐藻胶,褐藻胶裂解酶是一种较低毒性的分解人体内铜绿假单胞菌生物膜的优良选择。定向改造后的产酶菌株适合于分离出活性高和稳定性好的新型酶,具有较大的发展前景。褐藻胶裂解酶在医药领域的应用是抗生物膜剂的制备。这一应用对细菌性肺部感染提供了新的治疗方法,改善多药耐药细菌感染,将有助于褐藻胶裂解酶作为抗生物膜剂的进一步开发。因此,提供一种新型的褐藻胶裂解酶基因并实现重组表达,研究其性质,对于海洋资源的开发利用和医药卫生的深入研究具有重要意义。
一方面,本发明提供了一种突变型褐藻胶裂解酶,所述突变型褐藻胶裂解酶的氨基酸序列如SEQ ID No.1所示。
另一方面,本发明还提供了编码上述突变型褐藻胶裂解酶的基因;优选的,所述基因的序列如SEQ ID No.2所示。
另一方面,本发明还提供了包含上述编码基因的重组载体,所述重组载体优选为重组表达载体,如pET系列的载体,例如pET-28a;又如,pRSFDuet-1载体。
另一方面,本发明还提供了包含上述重组载体的重组菌株,优选的,所述重组菌株为大肠杆菌,如,大肠杆菌Rosetta agami(DE3)。
另一方面,本发明还提供了上述突变型褐藻胶裂解酶、基因、载体或菌株在降解底物中的应用,所述底物选自褐藻胶、PolyG(聚α-L-古洛糖醛酸)或PolyM(聚β-D-甘露糖醛酸)。
另一方面,本发明还提供了上述突变型褐藻胶裂解酶、基因、载体或菌株在抑制或降解微生物生物膜中的用途。优选的,所述生物膜为微生物以海藻酸盐为基质形成的生物膜。
另一方面,本发明还提供了上述突变型褐藻胶裂解酶、基因、载体或菌株在制备用于抑制或降解微生物生物膜的制剂中的用途。
在一个实施方式中,所述微生物为铜绿假单胞菌。
另一方面,本发明还提供了上述突变型褐藻胶裂解酶、基因、载体或菌株在抑制铜绿假单胞菌中的应用,或在制备用于抑制铜绿假单胞菌的制剂中的用途。
在一个实施方式中,本发明还提供了上述突变型褐藻胶裂解酶、基因、载体或菌株在制备用于治疗或缓解由铜绿假单胞菌引起的疾病的药物中的用途。
在一个实施方式中,所述药物中还含有抗生素;优选的,所述抗生素选自左氧氟沙星或头孢克肟。
另一方面,本发明还提供了一种处理铜绿假单胞菌生物膜的方法,所述方法包括利用上述突变型褐藻胶裂解酶、基因、载体或菌株对所述生物膜进行处理的步骤。
另一方面,本发明还提供了一种抑制铜绿假单胞菌的方法,所述方法包括利用上述突变型褐藻胶裂解酶、基因、载体或菌株对铜绿假单胞菌进行处理的步骤。
在一个实施方式中,所述抑制铜绿假单胞菌的方法是在葡萄糖存在的情况下抑制铜绿假单胞菌;所述葡萄糖的浓度为5mg/dl-200mg/dl,例如,10mg/dl、20mg/dl、40mg/dl、50mg/dl、60mg/dl、70mg/dl、80mg/dl、90mg/dl、100mg/dl、110mg/dl、120mg/dl、130mg/dl、150mg/dl或180mg/dl;上述mg/dl为毫克/分升的意思。
另一方面,本发明还提供了上述突变型褐藻胶裂解酶、基因、载体或菌株在制备用于治疗或缓解在葡萄糖存在的情况下由铜绿假单胞菌引起的疾病的药物中的用途;优选的,所述葡萄糖的浓度为5mg/dl-200mg/dl,例如,10mg/dl、20mg/dl、40mg/dl、50mg/dl、60mg/dl、70mg/dl、80mg/dl、90mg/dl、100mg/dl、110mg/dl、120mg/dl、130mg/dl、150mg/dl或180mg/dl。
在一个实施方式中,所述突变型褐藻胶裂解酶的适用温度为10℃-50℃,例如,20℃、30℃、35℃、40℃或45℃。
在一个实施方式中,所述突变型褐藻胶裂解酶的适用pH为4-9,例如,5、6、6.5、7、7.5或8。
另一方面,本发明还提供了一种降解褐藻胶的方法,所述方法包括利用上述突变型褐藻胶裂解酶、基因、载体或菌株对褐藻胶进行处理的步骤。
本发明获得的褐藻胶裂解酶突变体E135G与Alys1相比从偏好PolyM的双功能裂解酶变成偏好PolyG的双功能裂解酶,且PolyG酶活较Alys1提高了1.06倍;突变体E135G在pH6.0-9.0范围内酶活超过50%,具有良好pH稳定性;突变体E135G抑制铜绿假单胞菌生物膜生成的效率为63.9%,比Alys1高8.5%;破解铜绿假单胞菌生物膜的效率为77.6%,比Alys1高22.1%,能与抗生素左氧氟沙星具有良好的协同作用;在高血糖的存在下,突变体E135G仍对生物膜具有破除作用。
附图说明
图1.PCR扩增片段电泳图。
图2.重组酶的SDS-PAGE图。
图3.Alys1和突变体E135G的最适温度。
图4.Alys1和突变体E135G的热稳定性。
图5.Alys1(左)和突变体E135G(右)的最适pH。
图6.Alys1(左)和突变体E135G(右)的pH稳定性。
图7.Alys1和突变体E135G的最适底物。
图8.结晶紫法测定褐藻胶裂解酶Alys1和突变体对生物膜的破除能力。
图9.荧光染色法测定褐藻胶裂解酶Alys1和突变体对生物膜的破除能力;其中,左.Alys1破膜效果;中.E135G破膜效果;右.阴性对照(PBS处理)。
图10.Ca2+与Zn2+对褐藻胶裂解酶Alys1和突变体E135G酶活的影响。
图11.不同浓度的葡萄糖对褐藻胶裂解酶Alys1和突变体E135G的稳定性影响。
图12.不同葡萄糖浓度下酶对生物膜的降解能力。
图13.褐藻胶裂解酶单独对成膜的作用。
图14.两种抗生素对成膜的作用。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
突变蛋白E135G的获得
本发明中的褐藻胶裂解酶基因alys1分离自威海食用海参肠道的Tamlanasp.s12,全长1017bp,编码355个氨基酸(Genbank号OBQ55419),第1-23位氨基酸为信号肽(Met1-Cys23)。
本实施方式中,通过对褐藻胶裂解酶基因alys1的关键氨基酸位点的分析,将第135位的E突变为G,进而对突变蛋白的活性进行了研究。突变后得到的E135G蛋白变成了偏好polyG的双功能褐藻胶裂解酶,对polyG催化活性高于野生型Alys1。去除信号肽后,E135G蛋白的氨基酸序列如SEQ IDNo.1所示,其核酸序列如SEQ ID No.2所示。
本实施方式中,E135G代表突变型蛋白,Alys1代表野生型蛋白。
具体的,设计含有突变位点的引物,以重组质粒pET28a-Alys1为模板,通过全质粒突变法将Alys1进行单点突变,转入Rosetta(DE3)中,获得突变体重组菌E135G。具体步骤如下:
正反引物如下:
E135G-F:5'CATAGTGATGAAGGACACGGGAATGAACCTC 3'
E135G-R:5'GAGGTTCATTCCCGTGTCCTTCATCACTATG 3'
PCR扩增体系:10xPfu缓冲液5μL,pET28a-alys1质粒1μL,dNTP Mix 5μL,突变上游引物1μL,突变下游引物1μL,Pfu DNA聚合酶1.2μL,ddH2O补至50μL。PCR反应参数:95℃预变性3min;95℃变性30s,60℃退火60s,68℃延伸14min,18个循环;68℃保温1min;4℃保存。往PCR产物中加入1μL的Dpn I酶,混合均匀,在37℃条件下保温1h,消化原始模板。取10μL消化产物加入到200μL DH5α感受态细胞中,轻微吹打混匀后冰浴30min;42℃热激90s,于冰上放置2min;加入800μL的LB培养基,37℃、180r/min培养1h;涂布在含100μg/mL卡那霉素的LB平板上,37℃培养24h。挑取克隆子通过pcr验证有条带之后(图1),送去测序公司进行测序验证;图1中,泳道M:DNA marker;泳道1:Alys1的PCR片段;泳道2:突变体E135G的PCR片段。
重组突变体E135G的表达
提取重组质粒Pet28a-E135G,将其转入Rosetta(DE3)感受态细胞中,挑取单菌落于LB(Kana+)液体培养基中,37℃200r/min培养过夜,使其OD600达到0.6-0.7,加入IPTG(终浓度1mM)于20℃200r/min诱导20h。离心收集菌体,冰水浴破碎,离心得到上清液用于实验。
目的蛋白的纯化
所用的缓冲体系见如下:
结合缓冲液:50mmol/L NaH2PO4:300mmol/L NaCl;10mmol/L咪唑;pH 8.0。梯度洗脱缓冲液:50mmol/L NaH2PO4:300mmol/L NaCl;10-250mmol/L咪唑;pH 8.0。用镍柱纯化重组目的蛋白。操作步骤:将上清液加到镍柱上进行纯化,使用磷酸盐缓冲液作为平衡缓冲液,配置不同浓度梯度的咪唑对镍柱进行洗脱,收集250mM咪唑缓冲液冲洗的洗脱蛋白,用SDS-PAGE检测蛋白纯度,得到一条单一蛋白条带(见图2);图2中,泳道M:Protein marker;泳道1:经过纯化的Alys1;泳道2:经过纯化的突变体E135G。
重组突变体E135G的酶学性质测定:
最适温度:将取50μL酶E135G与褐藻胶等比例混合后,突变体E135G在不同温度下水浴反应30min,加入150μL的DNS终止反应,煮沸5min显色,测定在540nm处吸光度。结果如图3所示,突变体E135G的最适温度为30℃,低于Alys1的35℃。
热稳定性:分别测定酶在不同温度下水浴1h后的残余活性。图4所示,突变体E135G和Alys1在60℃几乎全部失活。
最适pH值测定:将酶分别加入到由不同pH缓冲液:(pH为4.0-6.0的Na2HPO4-CitricAcid缓冲液,pH为6.0-7.5的Na2HPO4-NaH2PO4缓冲液和pH 7.5-9.0的Na2HPO4-K2HPO4缓冲液)配置的浓度为0.5%的褐藻胶底物中在最适温度下进行反应,采用DNS法测酶活。图5中,两种酶在Na2HPO4-NaH2PO4中酶活最高,其中Alys1最适pH为7.0,突变体E135G最适pH为6.0。pH稳定性测定:将酶分别加入到以上三种缓冲液体系中,并于4℃保存24h,用DNS法测酶活。图6中,Alys1和突变体E135G在pH6.0-9.0时残余酶活达到50%以上,说明酶具有良好的pH稳定性。
重组酶最适底物测定
用最适pH缓冲液配置0.2%褐藻胶、PG和PM底物溶液。按照底物与酶液9:1的比例混合后反应5min,在235nm的波长下测其吸光度。如图7所示,Alys1和突变体E135G分别偏好PolyM和偏好PolyG的底物。突变体E135G对PolyG的活性是Alys1的1.06倍。
重组酶的Tm值测定
将纯化后的酶冻干,放入差示热重分析仪上进行DSC分析。以1.0℃为间隔从0.0℃到130.0℃收集数据。Alys1和突变体E135G的Tm值分别为56℃和41.5℃。说明突变体E135G比Alys1更不稳定。
铜绿假单胞菌生物膜构建
将铜绿假单胞菌接种于TSA平板上,在37℃培养箱中培养24h后形成单菌落。挑取一单菌落接种于含有TSB培养液(3mL)的试管中,放入37℃、200rpm/min的摇床中振荡培养过夜。用TSB培养液将菌悬液稀释至浓度约为0.5麦氏浓度(1.5×108个细菌/ml),在96孔板的每个孔中加入200μL稀释后的菌液,37℃培养48h待生物膜形成。
生物膜降解测定
用无菌生理盐水(0.9%NaCl)将形成生物膜的96孔板冲洗3次;往96孔板的每个孔中分别加入100μL待测酶液作实验组,以100μL的PBS缓冲液作空白对照组,每组重复三孔,在37℃下继续培养48h。
通过结晶紫染色法测定生物膜剩余量。用无菌生理盐水漂洗三次,每孔加入100μL甲醇对生物膜进行固定,15min后吸出甲醇,自然晾干后每孔加入100μL结晶紫溶液(0.1%)进行染色,30min后吸出染液,用无菌生理盐水冲洗多次,自然晾干后每孔加入100μL乙酸溶液(33%)用来溶解结晶紫染料,5min后立即用酶标仪在波长600nm条件下测量每孔的吸光度。图8所示为突变体E135G降解生物膜能力显著。
荧光法定性探究酶降解生物膜能力
将盖玻片平铺在6孔板中,每孔加入1.5mL的铜绿假单胞菌菌液(0.5麦氏浓度),37℃培养48h。将孔内液体吸出后,用无菌生理盐水漂洗3次。继续往每孔加入1.5mL待测酶液,每组重复3孔,37℃下培养48h。PBS在实验期间用作空白对照组。重复上面的漂洗过程,漂洗完成后,用4%多聚甲醛进行固定,30min后用PBS漂洗三次。每孔加入1.5mL的5-(4,6-二氯三嗪基)氨基荧光素(10μg/mL),25℃下避光孵育2h后继续用PBS漂洗三次。取出玻片并用甘油封片(甘油:PBS=1:1),荧光显微下使用油镜进行观察,最后进行拍照。图9中,两种酶与空白组相比对生物膜都具有破除作用,突变体E135G的破除效果比Alys1明显,与之前结晶紫染色结果一致。
金属离子对褐藻胶裂解酶及突变体稳定性影响
酶液在10mM不同金属离子存在下,4℃孵育4小时后,加入等体积0.5%褐藻胶,在37℃下水浴30min,再加入三倍酶体积的DNS,于沸水中煮沸5min,在540nm的波长下测定吸光度。图10中,钙离子和锌离子都对两种酶具有抑制作用,其中突变体E135G对金属离子更敏感,酶活抑制效果更为明显。
葡萄糖对褐藻胶裂解酶及突变体稳定性影响
利用pH 6.0的Na2HPO4-NaH2PO4缓冲液体系配制不同浓度葡萄糖溶液(50、70、90、110、130、200mg/dL)。将酶液与葡萄糖溶液按照1:1的比例混合后,在37℃水浴1h,再加入等体积的0.5%褐藻胶,37℃水浴30min,反应后加入三倍酶体积的DNS,沸水浴5min,在540nm的波长下测定吸光度。将酶液换成灭活酶液测定空白葡萄糖溶液标准曲线。图11中,随着葡萄糖浓度的升高,酶活降低越明显,其中Alys1受葡萄糖抑制作用更明显。
酶在葡萄糖的存在下对生物膜的降解能力
按照构建生物膜的步骤将100μL待测酶液换成50μL酶液和50μL葡萄糖溶液,按照结晶紫法测定OD值。图12中,随着葡萄糖浓度的增长,两种酶对生物膜的降解作用逐渐下降,Alys1下降程度最为显著。突变体E135G在葡萄糖浓度低于180mg/dL时,对生物膜仍然具有一定的破除效果。这一结果与在不同葡萄糖浓度下对酶活影响一致。说明突变体E135G可能为高血糖感染者提供新的治疗方法。
酶与抗生素联合使用对生物膜成膜影响
最小抑菌浓度测定
药物选取头孢克肟和左氧氟沙星,将2种抗菌药物以PBS缓冲液倍比稀释成12个浓度,分别为2048、1024、512……1μg/mL。两种酶液以PBS缓冲液倍比稀释成8个浓度,分别为150、75、37.5……1.17μg/mL。按照构建生物膜的步骤将200μL菌液换成100μL菌液和100μL抗生素溶液或酶液,37℃培养48h后使用结晶紫法测定吸光度。能够产生显著抑制成膜效果的最小药液或酶液浓度值即为MIC。
图13-14中,随着酶液或药液浓度降低,抑制成膜效果减弱。两种酶与两种抗生素对铜绿假单胞菌生物膜的MIC如表1所示。突变体E135G的MIC更低,效果更好。
表1.2种褐藻胶裂解酶和2种抗生素对铜绿假单胞菌生物膜的MIC(μg/mL)
酶与抗生素组合的抑菌浓度测定
将稀释好的抗菌药物按棋盘法设计,根据最小抑菌浓度的测定,选取最高浓度为单药MIC的8倍,选择其中7个连续的药物浓度梯度作为联合试验梯度,横向、纵向分别是抗生素和酶的降梯度方向。96孔板中每孔加入100μL浓度为0.5麦氏浊度的菌悬液,再取相应梯度浓度的药物各50μL两两组合加入相应孔中(第8列和第8行为空白对照)。37℃培养48h后使用结晶紫法测定吸光度。确定能够产生显著抑制成膜效果的最小药酶联合浓度值,计算FIC指数。
FIC=MICA(联用)/MICA(单用)+MICB(联用)/MICB(单用)
FIC指数的判定标准如下:当FIC≤0.5时,两种抗菌药物联合使用后发挥协同作用;当0.5<FIC≤1.0,两种抗菌药物联合使用后起相加主要;当1.0<FIC≤2.0,两种抗菌药物联合使用后表现为无相关作用;当FIC>2.0时,则两种抗菌药物显示出拮抗作用。
如表2-3所示,突变体E135G与左氧氟沙星联用产生协同作用,其余情况均为相加作用。这表明褐藻胶裂解酶与适当抗生素联用会产生更好的抑制生物膜形成的效果。
表2.左氧氟沙星和2种褐藻胶裂解酶对铜绿假单胞菌生物膜的体外联合实验(μg/mL)
表3.头孢克肟和2种褐藻胶裂解酶对铜绿假单胞菌生物膜的体外联合实验(μg/mL)
本发明获得的褐藻胶裂解酶突变体E135G与Alys1相比从偏好PolyM的双功能裂解酶变成偏好PolyG的双功能裂解酶,且PolyG酶活较Alys1提高了1.06倍;突变体E135G在pH6.0-9.0范围内酶活超过50%,具有良好pH稳定性;突变体E135G抑制铜绿假单胞菌生物膜生成的效率为63.9%,比Alys1高8.5%;破解铜绿假单胞菌生物膜的效率为77.6%,比Alys1高22.1%,能与抗生素左氧氟沙星具有良好的协同作用;在高血糖的存在下,突变体E135G仍对生物膜具有破除作用。
上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 山东大学
<120> 一种突变型褐藻胶裂解酶及其应用
<130> 11
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 332
<212> PRT
<213> Artificial Sequence
<220>
<223> E135G
<400> 1
Val Asn Lys Ala Lys Lys Thr Thr Glu Asn Ala Ile Val Lys Thr Glu
1 5 10 15
Glu Ser Lys Val Tyr Pro Ser Asp Val Ile Pro Phe Met Asp Gln Trp
20 25 30
Lys Ile Leu Leu Gly Asn Gly Glu Ser Ser Lys Asp Leu Lys Asn Tyr
35 40 45
Glu His Glu Asp Phe Phe Tyr Val Ala Asn Asp Gln Asp Thr Asn Trp
50 55 60
Val Val Tyr Lys Thr Pro Asn Ser Gly Ile Thr Ser Arg Thr Ser Ser
65 70 75 80
Asn Thr Arg Thr Glu Leu Gly Gln Lys Ala His Trp Thr Pro Glu Thr
85 90 95
Gly Gly Lys Leu Thr Gly Thr Leu Lys Val Met His Val Ser Thr Ser
100 105 110
Gly Asp Ala Arg Val Ala Ala Ser Phe Ser Thr Val Val Gly Gln Ile
115 120 125
His Ser Asp Glu Gly His Gly Asn Glu Pro Leu Lys Ile Phe Tyr Lys
130 135 140
Lys Phe Pro Gly His Thr Lys Gly Ser Val Phe Trp Asn Tyr Glu Ile
145 150 155 160
Asn Thr Lys Gly Asp Asn Ser Lys Arg Trp Asp Tyr Ser Thr Thr Val
165 170 175
Trp Gly Tyr Asp Met Ser Val Val Gly Glu Thr Pro Thr Thr Phe Pro
180 185 190
Glu Glu Pro Lys Asp Gly Ile Ala Leu Gly Glu Thr Phe Thr Tyr Glu
195 200 205
Ile Asn Val Tyr Gln Gly Ile Met Tyr Leu Thr Phe Thr Ser Glu Gly
210 215 220
His Glu Thr Val Lys Phe Thr Lys Asp Leu Thr Lys Ser Asp Phe Ala
225 230 235 240
Thr Lys Ala Asp Ile Pro Lys Gln Ile Trp Thr Leu Tyr Ala Ser Ile
245 250 255
Gly Arg Asp Gly Val Glu Arg Glu Thr Ala Tyr Ser Asn Glu Met Gln
260 265 270
Tyr Phe Lys Gln Gly Ala Tyr Asn Gln Ser Asn Gly Lys Asn Pro Glu
275 280 285
Asp Asn Met Val Trp Ser Thr Gly Ser Asp His Phe Asn Gly Asp Ile
290 295 300
Ala Lys Gln Tyr Gln Asn Gly Ala Tyr Thr Glu Val Trp Phe Lys Glu
305 310 315 320
Gly Thr Val Ser Pro Gly Thr Pro Pro Asp Met Glu
325 330
<210> 2
<211> 996
<212> DNA
<213> Artificial Sequence
<220>
<223> E135G
<400> 2
gttaataagg caaagaaaac taccgaaaac gccattgtga aaaccgaaga aagcaaggtt 60
tatcctagtg atgtgattcc ttttatggac caatggaaaa ttcttttggg taatggcgaa 120
tcgtcgaaag atttaaaaaa ctacgagcat gaagatttct tttatgtagc gaacgatcaa 180
gatactaatt gggtggttta taaaacccca aattcaggaa ttacttctag aacctctagt 240
aacacgagaa ccgaattagg tcagaaagcc cattggactc cagaaacggg aggcaaacta 300
acaggaacct taaaagtaat gcatgtttct acaagtggag atgcacgtgt agcggcttct 360
ttttcaaccg tcgttggaca aattcatagt gatgaaggac acgggaatga acctctaaaa 420
atattctaca agaaatttcc tggccacacg aaaggctctg ttttttggaa ctatgaaatc 480
aatacgaaag gcgataactc gaaacgatgg gattattcta ccacggtttg gggttacgat 540
atgtcggtag ttggagaaac cccaaccacc ttccccgaag aaccaaagga cgggattgct 600
ttaggagaaa cctttacata cgaaatcaac gtttatcaag gcatcatgta tttaacattt 660
acaagtgaag gccacgaaac ggtaaaattc accaaagatt taacaaaatc agattttgct 720
acaaaagccg atattccaaa acaaatatgg actttatacg ctagtattgg tcgtgacggt 780
gtcgagcgtg aaacggctta ctcaaacgaa atgcaatatt ttaaacaagg ggcttacaac 840
caatcgaatg gtaaaaaccc ggaagacaat atggtttgga gtacaggatc cgatcatttt 900
aatggtgata ttgcgaaaca atatcaaaac ggagcctaca cagaagtatg gtttaaagaa 960
gggactgtaa gtcctggcac acctcccgat atggag 996
Claims (13)
1.一种突变型褐藻胶裂解酶,所述突变型褐藻胶裂解酶的氨基酸序列如SEQ ID No.1所示。
2.编码权利要求1所述突变型褐藻胶裂解酶的基因。
3.包含权利要求2所述基因的重组载体。
4.包含权利要求3所述重组载体的重组菌株。
5.权利要求1所述的突变型褐藻胶裂解酶,或权利要求2所述的基因,或权利要求3所述的载体,或权利要求4所述的菌株在降解底物中的应用,所述底物选自褐藻胶、PolyG(聚α-L-古洛糖醛酸)或PolyM(聚β-D-甘露糖醛酸)。
6.权利要求1所述的突变型褐藻胶裂解酶,或权利要求2所述的基因,或权利要求3所述的载体,或权利要求4所述的菌株在抑制或降解微生物生物膜的形成中的用途,所述用途为非疾病治疗目的的用途。
7.权利要求1所述的突变型褐藻胶裂解酶,或权利要求2所述的基因,或权利要求3所述的载体,或权利要求4所述的菌株在抑制铜绿假单胞菌中的应用,所述应用为非疾病治疗目的的应用。
8.权利要求1所述的突变型褐藻胶裂解酶,或权利要求2所述的基因,或权利要求3所述的载体,或权利要求4所述的菌株在制备用于治疗或缓解由铜绿假单胞菌引起的疾病的药物中的用途。
9.根据权利要求8所述的用途,其特征在于,所述药物中还含有抗生素。
10.一种处理铜绿假单胞菌生物膜的方法,所述方法为非疾病治疗目的的方法,所述方法包括利用权利要求1所述的突变型褐藻胶裂解酶,或权利要求2所述的基因,或权利要求3所述的载体,或权利要求4所述的菌株对所述生物膜进行处理的步骤。
11.一种抑制铜绿假单胞菌的方法,所述方法为非疾病治疗目的的方法,所述方法包括利用权利要求1所述的突变型褐藻胶裂解酶,或权利要求2所述的基因,或权利要求3所述的载体,或权利要求4所述的菌株对铜绿假单胞菌进行处理的步骤。
12.根据权利要求11所述的方法,其特征在于,所述抑制铜绿假单胞菌的方法是在葡萄糖存在的情况下抑制铜绿假单胞菌。
13.权利要求1所述的突变型褐藻胶裂解酶,或权利要求2所述的基因,或权利要求3所述的载体,或权利要求4所述的菌株在制备用于治疗或缓解在葡萄糖存在的情况下由铜绿假单胞菌引起的疾病的药物中的用途。
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CN1514000A (zh) * | 2003-07-10 | 2004-07-21 | 中国海洋大学 | 一种褐藻胶裂解酶基因algL及其制备方法和应用 |
WO2009121848A2 (en) * | 2008-03-31 | 2009-10-08 | Umc Utrecht Holding B.V. | Use of pseudomonas aeruginosa alkaline protease (apra) and inhibitors thereof in medicine and agriculture |
CN106636049A (zh) * | 2016-12-27 | 2017-05-10 | 江南大学 | 一种分泌性能提高的碱性果胶酶突变体 |
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CN1514000A (zh) * | 2003-07-10 | 2004-07-21 | 中国海洋大学 | 一种褐藻胶裂解酶基因algL及其制备方法和应用 |
WO2009121848A2 (en) * | 2008-03-31 | 2009-10-08 | Umc Utrecht Holding B.V. | Use of pseudomonas aeruginosa alkaline protease (apra) and inhibitors thereof in medicine and agriculture |
CN106636049A (zh) * | 2016-12-27 | 2017-05-10 | 江南大学 | 一种分泌性能提高的碱性果胶酶突变体 |
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重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究;张潘潘;许延吉;王之可;刘晓;李素霞;;中国生物工程杂志(第05期);全文 * |
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