CN115094051A - 一种来源于海科贝特氏菌的重组溶菌酶及其制备方法和应用 - Google Patents
一种来源于海科贝特氏菌的重组溶菌酶及其制备方法和应用 Download PDFInfo
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- CN115094051A CN115094051A CN202210365829.6A CN202210365829A CN115094051A CN 115094051 A CN115094051 A CN 115094051A CN 202210365829 A CN202210365829 A CN 202210365829A CN 115094051 A CN115094051 A CN 115094051A
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Abstract
本发明属于生物化学技术领域,具体涉及一种来源于海科贝特氏菌(Cobetia marina)的重组溶菌酶及其制备方法和应用,所述重组溶菌酶的氨基酸序列如SEQ ID NO.2所示,其编码基因的的核苷酸序列如SEQ ID NO.1所示。本发明所获得的重组溶菌酶具有良好的酶学性质,其可以应用于制备饲料添加剂、食品添加剂。
Description
技术领域
本发明属于生物化学技术领域,具体涉及一种来源于海科贝特氏菌(Cobetia marina)的重组溶菌酶及其制备方法和应用。
背景技术
溶菌酶(EC 3.2.1.17),也称为胞壁酸酶或N-乙酰胞壁质聚糖水解酶(N-acetylmuramide glycanhydrolase),催化在肽聚糖中的N-乙酰胞壁酸和N-乙酰-D-葡糖胺残基之间的和在壳糊精中的N-乙酰-D-葡糖胺残基之间的1,4-β-键的水解,使肽聚糖骨架结构断裂后造成细胞壁破裂,最终导致细菌溶解。溶菌酶还可与带负电荷的病毒蛋白直接结合,与DNA、RNA、脱辅基蛋白形成复盐,使病毒失活。因此,溶菌酶具有抗菌、消炎、抗病毒等重要作用。
直接杀菌机制:一般人认为,溶菌酶能切断肽聚糖中N-乙酰葡萄糖胺和N-乙酰胞壁酸之间的β-1,4糖苷键,破坏肽聚糖支架,从而细菌细胞在内部渗透压的作用下胀裂而死亡。非溶菌机制:有报道称溶菌酶并不是直接作用微生物。如Brooks等(1991)提出一种观点,溶菌酶在正常生理渗透压平衡下溶菌酶并不杀灭敏感细菌,其发挥作用是在微生物被其它物质如卵清蛋白或昆虫血淋巴中多肽(Boman, H . G. ,1991),或动物血清中的补体(Flescher,E.,1991)杀死后,参与去除细胞壁的作用。Hisham R.等(1996)首次提供了一些遗传证据,即:溶菌酶的抗菌活性依赖于其胞壁质酶活性,抗菌活性是由于其结构因素所致。
溶菌酶作为高等有机体组织及体液中最强大的抗菌剂之一,是生物机体对抗外源病原菌侵袭的重要防御因子,广泛存在于人和动物的多种组织、分泌液如眼泪、唾液中,某些植物、微生物中也广泛存在。根据其来源的不同,可以分为c型溶菌酶,g型溶菌酶,i型溶菌酶,植物源溶菌酶,微生物源溶菌酶和噬菌体溶菌酶;根据其结构可将溶菌酶分类为五种不同的糖苷水解酶(GH)家族:母鸡蛋白溶菌酶(GH22),鹅蛋白溶菌酶(GH23),噬菌体T4溶菌酶(GH24),鞘氨醇单胞菌鞭毛蛋白(GH73)以及Chalaropsis溶菌酶(GH25)。
溶菌酶是生物体内重要的非特异性免疫因子,其作为一种天然蛋白质,能在胃肠道内作用于营养物质被消化和吸收,对人体无毒性,也不会在体内残留,是一种安全性很高的食品保鲜剂、营养保健品和药品。溶菌酶可用于各种加工食品或饮料制作中,集药理、保健和防腐三种功能于一体。
与其它抗菌因子相比,溶菌酶具有活性稳定、抗菌谱广、安全性高等优点,因此可广泛应用于食品、医药、饲料和科研等领域。在食品领域,溶菌酶可用作高安全性的食品防腐剂,具有一定的保健作用,可以选择性地、有目的地杀灭微生物而不作用于食物中的其他物质,保证食品原有营养成分不受损失;在乳制品中用作添加因子对肠道中腐败性微生物有特殊的杀灭作用,同时直接或间接地促进肠道中双歧杆菌的增值,是婴儿食品中的抗菌蛋白;在食品软包装中将溶菌酶固定化在食品包装材料上,生产出有抗拒功效的食品包装材料,以达到抗菌保鲜功能。在医药领域,溶菌酶能够参与粘多糖代谢,可作为对抗微生物感染的酶类抗菌药。在内服和外用药配合使用下,溶菌酶对急性炎症如急性咽炎、急性喉炎、急性中耳炎等疗效明显,对慢性鼻炎、慢性咽喉炎及扁平疣等也有辅助治疗的作用。另外,将溶菌酶与抗生素合用还有助于预防龋齿。在饲料领域,溶菌酶具有替代抗生素的作用,能提升饲料的喂养效率、降低细菌的易感性和增加病原体脱落,最大限速地减少养殖生产损失。猪进食含溶菌酶的食物,可以有效改善小肠形态,从而降低弯曲杆菌胃肠道中的疾病发病率,降低料肉比和腹泻率,提升猪的日增重和采食量, 提高仔猪的健康水平。在不添加抗生素的情况下,鸡饲料中添加溶菌酶还可以改善禽类生长性能,提高血清中的溶菌酶含量和免疫功能,促进更多的家禽抵御疾病侵害,更易成活和健康生长。
近年来,由于抗生素滥用导致“超级细菌”的出现和畜牧产品抗生素残留等食品安全问题日益突出,人们迫切需要开发新型杀菌剂。而溶菌酶作为一种天然杀菌蛋白,能有效杀灭各种有害微生物,更重要的是,溶菌酶具有与抗生素不同的抗菌机制,在抑杀病原体的同时不会导致细菌耐药性、对人体无毒副作用,因此与其相关的科研和应用已成为学术热点。作为抗生素的替代品,溶菌酶被WHO和许多国家认定为无毒、无害、安全的应用于食品和饲料添加剂领域,2010年被我国卫生部批准为食品添加剂。
由于溶菌酶具有上述多种用途,市场上对溶菌酶的需求量很大。目前,人们主要从鸡蛋清来制取溶菌酶。然而鸡蛋清来源的溶菌酶制取成本较高,而且浪费了鸡蛋原材料与人类的食物需求是有冲突的。且蛋清溶菌酶仅对革兰氏阳性菌有作用,限制了其应用范围。利用微生物生产溶菌酶,成本较低,节省材料,对环境污染较小,容易实现规模化生产,因而开发潜力巨大。近年来国内外对多种来源的溶菌酶进行了广泛的研究,但有关发酵方法制备微生物溶菌酶的研究报道较少,导致此类非蛋清溶菌酶在实际工业化大生产上应用有限。
随着基础研究得到的生物学信息不断充实,科研人员运用丰富的、关键的生物学信息,使用基因工程技术手段,构建了多种不同来源溶菌酶异源(原核及真核)表达系统,不同程度地提高了溶菌酶的生物学性能。但对温度、pH、培养基组成等表达条件的控制不够精确,不同重组溶菌酶表达量及活性差异显著。广泛发掘溶菌酶资源,同时开展溶菌酶真核表达研究,探索重组溶菌酶表达量及活性如何提高、分离纯化过程如何简化、生产成本如何降低等问题,以期获得适用于规模化生产的高产、高效、广谱的溶菌酶工程菌,是动物源溶菌酶基因工程未来的发展方向。
本发明从海洋细菌海科贝特氏菌(Cobetia marina)中克隆获得一个新的溶菌酶基因,实现了其在毕氏酵母菌株中的高水平表达;所表达的重组溶菌酶具有广泛的温度适用范围和pH适用范围,及良好的温度耐受性、pH耐受性和蛋白酶抗性,其在食品、医药和饲料等领域的有很好的应用价值。
发明内容
本发明的目的在于提供一种来源于海科贝特氏菌(Cobetia marina)的重组溶菌酶及其制备方法和应用,实现溶菌酶的产业化生产及应用推广。
为此,本发明提供了以下技术方案,并获得了良好的技术效果:
本发明首先提供了一种来源于海科贝特氏菌(Cobetia marina)的重组溶菌酶,所述重组溶菌酶的氨基酸序列如 SEQ ID NO.2所示,其编码基因的核苷酸序列如SEQ IDNO.1所示。
进一步的,上述重组溶菌酶具有如下理化性质:
①发酵产品水平可达到461700U/mL,比活力为24300U/mg;
②最适pH为5.5-7.5,其中最高点为7.0;
③最适反应温度为30-65℃,其中最高点为60℃;
④在50-75℃下处理3min后,重组溶菌酶的残余酶活力均能维持在90%以上;在80-85℃下处理3min后,重组溶菌酶的残余酶活力均能维持在70%以上;在90℃下处理3min后,重组溶菌酶的残余酶活力均能维持在57.9%;
⑤在pH2.0下处理1hr,重组溶菌酶仍能保持73.3%以上残余酶活力;在pH3.0-8.0下处理1hr,重组溶菌酶均能保持90%以上的残余酶活力;在pH9.0下处理1hr,重组溶菌酶仍能保持73.5%的残余酶活力;
⑥胰蛋白酶或胃蛋白酶分别处理2hr后,均仍有90%以上的残余酶活力。
本发明还提供了一种高效重组表达载体,所述重组表达载体携带上述的编码基因。
本发明还提供了一种重组基因工程菌株,所述重组基因工程菌株包含上述的重组表达载体,所述重组基因工程菌株以毕氏酵母GS115为宿主细胞。
本发明还提供了上述一种重组溶菌酶的制备方法,包括以下步骤:
1)从海科贝特氏菌(Cobetia marina)中提取基因组DNA;
2)以提取的基因组DNA为模板进行PCR扩增获得PCR扩增产物;
3)PCR扩增产物用限制性内切酶EcoR I和Not I进行分步酶切,然后与经同样酶酶切的pPIC9k表达载体相连接形成重组表达质粒;
4)将重组表达质粒转化至大肠杆菌DH5α中,扩大培养,然后从大肠杆菌DH5α抽提重组表达质粒;
5)将抽提的重组表达质粒用限制性内切酶Bgl II 进行线性化,电击转化至毕氏酵母GS115感受态细胞,经培养后筛选获得重组菌株;
6)重组菌株经发酵培养表达溶菌酶基因,收获表达产物重组溶菌酶。
本发明还提供了上述一种重组溶菌酶在制备饲料添加剂、食品添加剂中的应用。
本发明的有益效果如下:
本发明获得了一种新的溶菌酶编码基因,并实现了其在毕氏酵母菌株中的高效重组表达,本发明还通过酶学性质检验对所述重组溶菌酶的最适作用温度、最适作用pH值、pH稳定性、热稳定性及比活力进行了分析,结果证明本发明的重组溶菌酶具有很好的pH稳定性、良好的热稳定性以及抗蛋白酶水解能力,能够很好的满足和适应食品、医药和饲料行业对该产品的应用要求。
附图说明
图1:海科贝特氏菌(Cobetia marina)溶菌酶基因电泳图;泳道M为Marker,泳道1为克隆的溶菌酶的DNA。
图2:重组溶菌酶质粒LY/pPIC9k酶切后电泳图;泳道M为Maker,泳道1为酶切后重组质粒。
图3:重组毕氏酵母溶菌酶发酵液电泳图;泳道M为Maker,泳道1和2均为重组毕氏酵母溶菌酶的发酵取样。
图4:重组溶菌酶最适反应温度分析。
图5:重组溶菌酶温度耐受性分析。
图6:重组溶菌酶最适反应pH分析。
图7:重组溶菌酶pH耐受性分析。
图8:重组溶菌酶蛋白酶抗性分析。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
以下实施例中所用到的实验材料及实验方法如下:
1. 菌株和载体
海科贝特氏菌(Cobetia marina)CGMCC 1.8624购自中国普通微生物菌种保藏管理中心;大肠杆菌JM109、DH5α及表达载体pET28a(+)均购自安诺伦(北京)生物科技有限公司;毕氏酵母(Pichia pastoris)GS115及表达载体pPIC9k均购自美国英杰生命技术有限公司;藤黄微球菌(Micrococcus luteus)CICC10680购自中国工业微生物菌种保藏管理中心。
2. 酶类及其他生化试剂
PTM1:30mM硫酸铜,0.54mM碘化钠,17.6mM硫酸锰,0.80mM钼酸钠,0.32mM硼酸,2.4mM氯化钴,0.18mM氯化锌,0.24mM硫酸亚铁,1.6mM生物素,0.19M硫酸。
限制性内切酶EcoR I和Not I、DNA Maker、Protein Maker、T4连接酶、Primescript double strand cDNA synthesis kit均购自宝日医生物技术(大连)有限公司;pfu DNA合成酶购自富酶泰斯生物技术(深圳)有限公司;Ezup Column BacteriaGenomic DNA Purification Kit、SanPreP Column Plasmid Mini-Preps Kit、SanPrePColumn DNA Gel Extraction Kit、胶回收试剂盒和PCR产物回收试剂盒均购自生物工程(上海)股份有限公司;RNA抽提试剂盒RNeasy Mini Kit(cat. nos. 74104)购自凯杰企业管理(上海)有限公司;琼胶购自美国英杰生命技术有限公司;ATCC Medium 2(MarineMedium 2216)培养基购自美国典型培养物保藏中心。
其他常规试剂均为国产或进口。
3. 培养基
发酵基本培养基:26.2ml/L磷酸,0.80g/L硫酸钙,18.7g/L硫酸钾,15.5g/L硫酸镁,4.17g/L氢氧化钾,40g/L葡萄糖。
除发酵基本培养基及ATCC Medium 2(Marine Medium 2216)培养基,以下实施例中使用的其他培养基均参照美国英杰生命技术有限公司毕氏酵母操作手册进行配制。
4. 实验方法
本发明中所用到的生物化学技术均为本领域中的常规技术。在以下实施例中,除非特殊说明,所有实验操作均按照以下实验手册或文献中的相关章节或部分进行,包括:[美]J.莎姆布鲁克等,分子克隆实验指南;赵永芳等,生物化学技术原理及其应用(第二版);朱检等,生物化学实验[M]。
本发明中所有相关的酶活、酶活力、酶活性均是指溶菌酶活性,均采用《GB1886.257-2016 食品安全国家标准 食品添加剂溶菌酶》中的方法。
实施例1 Cobetia marina基因组DNA的提取
挑取海科贝特氏菌(Cobetia marina)CGMCC 1.8624冻干管菌种,接种于ATCCMedium 2(Marine Medium 2216)培养基中,28℃培养15-18h(至菌浓OD600nm达到0.6以上)后,取1.5mL菌体培养物于一灭菌Ep管中,12000rpm离心1min,弃去上清液,收集菌体。用Ezup Column Bacteria Genomic DNA Purification Kit按照其操作步骤提取基因组DNA,然后置于4℃保存备用。
实施例2 Cobetia marina溶菌酶基因的克隆
对数据库中Cobetia来源的溶菌酶基因序列进行对比分析,设计上、下游引物LY-F和LY-R。上、下游引物分别含有EcoRI和NotI酶切位点,交由上海生工合成,引物序列如下:
LY-F:5’-acggaattcATGCCTGCACATGCCCCAGCGCAC-3’(小写部分为为引入EcoR I的酶切位点及改变引物的GC含量和退火温度所补充的碱基)
LY-R:5’-acgaccgcggccgcTTATGCGATGTGGCCGCCAGC-3’(小写部分为为引入Not I的酶切位点及改变引物的退火温度所补充的碱基)
以实施例1中所获得的基因组DNA为模板,用pfu DNA合成酶及引物LY-F和LY-R进行PCR扩增,PCR扩增条件为:95℃ 3min;95℃ 20sec,55℃ 30sec,72℃ 1min12sec,30个循环;72℃ 6min13sec。PCR扩增产物进行1%琼脂糖凝胶电泳(见图1),并用胶回收试剂盒回收目标产物条带。然后用限制性内切酶EcoR I和Not I进行分步酶切,酶切产物用PCR回收试剂盒回收后,用T4连接酶与经过相同酶切的质粒pPIC9k片段连接,16℃连接过夜后,连接产物转化大肠杆菌DH5α感受态细胞,LB平板筛选得到阳性菌落(以100 ug/mL Amp为抗性)LY/pPIC9k/DH5α。用质粒提取试剂盒从阳性菌落的培养物中提取质粒LY/pPIC9k,取部分质粒用限制性内切酶EcoR I和Not I进行分步酶切,然后进行1%琼脂糖凝胶电泳(见图2),提取的质粒送上海英骏生物技术有限公司进行测序。由此获得溶菌酶的编码基因LY,其基因序列如SEQ ID NO.1所示,相应的氨基酸序列如SEQ ID NO.2所示。
实施例3 溶菌酶毕氏酵母重组工程菌株的构建
将实施例2所制备的LY/pPIC9k/DH5α接种到LB液体培养基中,37℃过夜培养,然后用质粒抽提试剂盒提取获得质粒LY/pPIC9k,用限制性内切酶Bgl II 酶切,胶回收纯化大片段,即得到酵母转化所需含突变基因的线性DNA。然后用电转化法转化毕赤酵母菌株GS115,经筛选(以G418为抗性,浓度为100 ug/mL)和鉴定获得重组毕赤酵母菌株LY/pPIC9k/GS115阳性克隆子。
实施例4 毕氏酵母发酵制备重组溶菌酶
取实施例3所构建的重组毕氏酵母菌株LY/pPIC9k/GS115阳性克隆子,接种于150ml YPD培养液中,30℃、250rpm振荡培养至OD600nm=0.3~0.5(约20hr),然后分别接种于3L发酵基本培养基(26.2ml/L磷酸,0.80g/L硫酸钙,18.7g/L硫酸钾,15.5g/L硫酸镁,4.17g/L氢氧化钾,40g/L葡萄糖)中,于5L发酵罐中进行发酵。
在起始菌体生长阶段,过程中用25%的氨水调节pH,使其维持在6.5-6.6,并且以4.0ml/hr的速度流加PTM1(30mM硫酸铜、0.54mM碘化钠、17.6mM硫酸锰、0.80mM钼酸钠、0.32mM硼酸、2.4mM氯化钴、0.18mM氯化锌、0.24mM硫酸亚铁、1.6mM生物素、0.19M硫酸),进行连续流加补料。搅拌并通气培养20-24hr,在菌体生长过程中溶氧逐渐下降至低于100%,直至碳源耗尽,溶氧又逐渐上升至高于80%,此时菌湿重可达到90-95g/L。
进入碳源饲喂阶段,以25ml/hr的速度流加用蒸馏水配置的含有25%(w/v)葡萄糖和12ml/L PTM1的溶液,持续流加4-6hr,并调节通气量,使溶氧维持在20%上下,到该阶段的末期,菌湿重可达到170-185g/L。
进入诱导阶段,以10-15ml/hr的速度流加含有12ml/L PTM1的甲醇,使培养基中甲醇的终浓度最高不要超过0.3%(v/v),并调节通气量搅拌转速,使溶氧维持在20%上下。发酵到达185hr时,菌湿重可达到300-325g/L,重组溶菌酶的表达水平(以发酵液上清的酶活力表示)可达到461700U/mL,比活力可达到24300U/mg,这说明Cobetia marina溶菌酶基因在毕氏酵母中得到了高效表达。
收集重组超氧化物歧化酶上清液进行SDS-PAGE分析,结果如图3所示。
实施例5 重组溶菌酶的酶学性质表征
将实施例4所制备的重组溶菌酶在磷酸氢二钠-柠檬酸(pH7.0,50mM)缓冲体系及不同温度下(20℃-80℃)进行酶促反应,以测定最适反应温度。结果表明,重组溶菌酶的最适反应温度为30-65℃(图4),其中最高点为60℃。在不同温度下(50℃-90℃)处理酶3min后测定残余酶活性,以进行热稳定性研究。结果表明,在50-75℃下处理3min后,重组溶菌酶的残余酶活力均能维持在90%以上;在80-85℃下处理3min后,重组溶菌酶的残余酶活力均能维持在70%以上;在90℃下处理3min后,重组溶菌酶的残余酶活力均能维持在57.9%(图5)。以上结果表明重组溶菌酶有广泛的温度适用范围和很好的耐热性。
在不同的pH下进行酶促反应以测定其最适pH。所用缓冲液的pH范围为3.0-9.0(pH3.0-8.0范围内采用50mM Na2HPO4-C6H8O7缓冲液,pH8.0-9.0采用50mM Gly-NaOH缓冲液)。溶菌酶在不同的pH的缓冲液中、60℃下测定酶活性,分析pH对酶活力的影响。结果表明溶菌酶的最适pH为5.5-7.5(见图6),其中最高点为7.0。将溶菌酶发酵液用不同pH值的缓冲液(pH2.0-10.0)稀释5倍后置于室温下处理1hr,然后再用pH7.0的缓冲液稀释适当倍数后测定残余酶活性以研究溶菌酶的pH稳定性。结果表明,在pH2.0下处理1hr,重组溶菌酶仍能保持73.3%以上残余酶活力;在pH3.0-8.0下处理1hr,重组溶菌酶均能保持90%以上的残余酶活力;在pH9.0下处理1hr,重组溶菌酶仍能保持73.5%的残余酶活力(见图7)。以上结果表明该溶菌酶具有很广泛的pH适用范围和很好的pH稳定性。
在重组溶菌酶溶液中加入0.05ml胰蛋白酶(0.1mg/ml,用pH7.0 1×PBS缓冲液配置)或胃蛋白酶(0.1mg/ml,用pH3.0甘氨酸-HCl缓冲液配置),于37℃处理2hr,用pH7.0的1×PBS缓冲液做适当稀释后再测定溶菌酶活性。经胰蛋白酶或胃蛋白酶分别处理2hr后,重组溶菌酶的残余酶活力均在90%以上(图8),说明该溶菌酶具有较好的抗蛋白酶水解能力。
在重组溶菌酶溶液中分别加入不同种类的金属离子(Na+、K+、Ca2+、Mg2+、Fe2+、Zn2+、Cu2+和Co2+)溶液,使酶溶液中的最终金属离子浓度分别为1mM、10mM和50mM,同时设置未添加金属离子的作为空白对照,在pH7.0、60℃下测定酶活力,分析金属离子对酶活力的影响。由表1的结果可知,Na+、K+和Ca2+对重组溶菌酶的酶活有激活作用,Mg2+、Fe2+和Cu2+对酶活没有显著影响,Zn2+和Co2+对酶活有抑制作用。
表1 金属离子对重组溶菌酶酶活力的影响
实施例6 重组溶菌酶对不同菌类的抑制作用
采用MIC法对实施例4所制备的重组溶菌酶的抑菌作用进行测定。直接取培养18-24h的菌落(革兰氏阳性、革兰氏阴性等共15 株菌株)调配成0.5麦氏比浊标准的菌悬液,用MH肉汤将上述菌悬液进行1∶100稀释后备用。在无菌试管中分别加入不同浓度的重组溶菌酶酶液及相应的菌液,将未加溶菌酶酶液的1管作为对照,置35℃普通空气孵箱中孵育16-24h后,以肉眼观察,无菌体生长管的最低溶菌酶浓度,即为受试菌的MIC。由表2的结果可知,本发明的重组溶菌酶对15种菌株均有不同程度的抑制作用。
表2 重组溶菌酶对不同菌类的抑制效果
实施例7 重组溶菌酶对肉鸡增益效果测试
肉鸡品种:白羽肉鸡,试验周期:42天,此次试验不分阶段,全程添加。试验分为2组,1个对照组(正常日粮),1个试验组(正常日粮+加溶菌酶(200g/吨饲料)),每组120只鸡,共计240只鸡。
饲养中期(21天)和结束后(42天),测定每组的最终总重量、总数量及饲料摄入量,计算平均单重和料重比。由表3的结果可知,重组溶菌酶对白羽肉鸡的生长性能有显著改善,21日龄和42日龄白羽肉鸡的平均单重增加和料重比降低均较为明显。
表3 重组溶菌酶对白羽肉鸡生长性能的影响
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建福大百特生物科技有限公司
<120> 一种来源于海科贝特氏菌的重组溶菌酶及其制备方法和应用
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 531
<212> DNA
<213> 人工序列
<400> 1
atgcctgcac atgccccagc gcactggctc gatgaggtcg agctggtggg cttcgatgag 60
cctgttgatc cggcggctga tgatgccagc aatctggcgg ccttcctgga caccattgcc 120
tatgccgagg gcacgccccg cttcagttcg attgagggct atgacgtgct ggtgggcggc 180
acgaccttcg acggcttcga tgatcatccc cgccagtcgg tatggctgaa gaagctcggc 240
atccacagca cggcggctgg ccggtatcag ttcctgataa ggacgtggga cgacctggcc 300
aatcgcttcc acctgtcgga cttctcgccg gcctctcagg atgaggcggc caagcagttg 360
atccgccagt gccgggcact ggggatggtg tatgacgggc gcatcgctga ggccatccac 420
gcctgtcggc gcatctgggc gagcctgccg ggggcaggtt acgggcagcg tgagcttgat 480
accgatgaac tgctgggcgt gtacgtgcgt gctggcggcc acatcgcata a 531
<210> 2
<211> 176
<212> PRT
<213> 人工序列
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Met Pro Ala His Ala Pro Ala His Trp Leu Asp Glu Val Glu Leu Val
1 5 10 15
Gly Phe Asp Glu Pro Val Asp Pro Ala Ala Asp Asp Ala Ser Asn Leu
20 25 30
Ala Ala Phe Leu Asp Thr Ile Ala Tyr Ala Glu Gly Thr Pro Arg Phe
35 40 45
Ser Ser Ile Glu Gly Tyr Asp Val Leu Val Gly Gly Thr Thr Phe Asp
50 55 60
Gly Phe Asp Asp His Pro Arg Gln Ser Val Trp Leu Lys Lys Leu Gly
65 70 75 80
Ile His Ser Thr Ala Ala Gly Arg Tyr Gln Phe Leu Ile Arg Thr Trp
85 90 95
Asp Asp Leu Ala Asn Arg Phe His Leu Ser Asp Phe Ser Pro Ala Ser
100 105 110
Gln Asp Glu Ala Ala Lys Gln Leu Ile Arg Gln Cys Arg Ala Leu Gly
115 120 125
Met Val Tyr Asp Gly Arg Ile Ala Glu Ala Ile His Ala Cys Arg Arg
130 135 140
Ile Trp Ala Ser Leu Pro Gly Ala Gly Tyr Gly Gln Arg Glu Leu Asp
145 150 155 160
Thr Asp Glu Leu Leu Gly Val Tyr Val Arg Ala Gly Gly His Ile Ala
165 170 175
<210> 3
<211> 33
<212> DNA
<213> 人工序列
<400> 3
acggaattca tgcctgcaca tgccccagcg cac 33
<210> 4
<211> 35
<212> DNA
<213> 人工序列
<400> 4
acgaccgcgg ccgcttatgc gatgtggccg ccagc 35
Claims (8)
1.一种来源于海科贝特氏菌(Cobetia marina)的重组溶菌酶,其特征在于:所述重组溶菌酶的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的重组溶菌酶,其特征在于:所述重组溶菌酶的编码基因的核苷酸序列如如SEQ ID NO.1所示。
3.根据权利要求1所述的重组溶菌酶,其特征在于:所述重组溶菌酶的理化性质为:
①发酵产品水平可达到461700U/mL,比活力为24300U/mg;
②最适pH为5.5-7.5,其中最高点为7.0;
③最适反应温度为30-65℃,其中最高点为60℃;
④在50-75℃下处理3min后,重组溶菌酶的残余酶活力均能维持在90%以上;在80-85℃下处理3min后,重组溶菌酶的残余酶活力均能维持在70%以上;在90℃下处理3min后,重组溶菌酶的残余酶活力均能维持在57.9%;
⑤在pH2.0下处理1hr,重组溶菌酶仍能保持73.3%以上残余酶活力;在pH3.0-8.0下处理1hr,重组溶菌酶均能保持90%以上的残余酶活力;在pH9.0下处理1hr,重组溶菌酶仍能保持73.5%的残余酶活力;
⑥胰蛋白酶或胃蛋白酶分别处理2hr后,均仍有90%以上的残余酶活力。
4.一种高效重组表达载体,其特征在于:所述重组表达载体携带权力要求2中所述的编码基因。
5.一种重组基因工程菌株,其特征在于:所述重组基因工程菌株包含如权利要求4所述的重组表达载体。
6.根据权利要求5所述的一种重组基因工程菌株,其特征在于:所述重组基因工程菌株以毕氏酵母GS115为宿主细胞。
7.一种重组溶菌酶的制备方法,其特征在于:包括以下步骤:
1)从海科贝特氏菌(Cobetia marina)中提取基因组DNA;
2)以提取的基因组DNA为模板进行PCR扩增获得PCR扩增产物;
3)PCR扩增产物用限制性内切酶EcoR I和Not I进行分步酶切,然后与经同样酶酶切的pPIC9k表达载体相连接形成重组表达质粒;
4)将重组表达质粒转化至大肠杆菌DH5α中,扩大培养,然后从大肠杆菌DH5α抽提重组表达质粒;
5)将抽提的重组表达质粒用限制性内切酶Bgl II 进行线性化,电击转化至毕氏酵母GS115感受态细胞,经培养后筛选获得重组菌株;
6)重组菌株经发酵培养表达溶菌酶基因,收获表达产物重组溶菌酶。
8.如权利要求1所述的重组溶菌酶在制备食品添加剂或饲料添加剂中的应用。
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