CN113736766B - 胶原蛋白水解酶及其编码基因、制备方法和用途 - Google Patents
胶原蛋白水解酶及其编码基因、制备方法和用途 Download PDFInfo
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- CN113736766B CN113736766B CN202111291737.XA CN202111291737A CN113736766B CN 113736766 B CN113736766 B CN 113736766B CN 202111291737 A CN202111291737 A CN 202111291737A CN 113736766 B CN113736766 B CN 113736766B
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- collagen
- hydrolase
- collagen hydrolase
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Abstract
本发明公开了一种胶原蛋白水解酶,其氨基酸序列如SEQ ID NO.1所示。本发明还公开了胶原蛋白水解酶基因、胶原蛋白水解酶基因重组载体、胶原蛋白水解酶基因工程菌、胶原蛋白水解酶的制备方法和胶原蛋白水解酶的用途。本发明的胶原蛋白水解酶具有能够降解骨胶原蛋白,并提高小分子量骨胶原蛋白肽的得率。
Description
技术领域
本发明涉及生物技术领域。更具体地说,本发明涉及一种胶原蛋白水解酶及其编码基因、制备方法和用途。
背景技术
畜禽骨中含有丰富得骨胶原蛋白,是制备功能性骨胶原蛋白肽的重要原料,利用价值巨大。畜禽骨胶原蛋白经酶解制备的小分子量骨胶原蛋白肽,具有抗氧化、抗衰老、促进矿物质吸收、抗骨质疏松等活性。同时,小分子量骨胶原蛋白肽具有溶解性好、易吸收等特点,吸收效果比胶原蛋白更佳。蛋白酶水解法是当前制备小分子量骨胶原蛋白肽最常用的方法,具有成本低、操作简单、条件温和等优点。但是由于胶原蛋白独特的(G-X-Y)n重复单元序列和致密的三螺旋结构,只有极少部分蛋白酶具有催化骨胶原蛋白水解的能力,目前市售的胶原蛋白酶及生产菌株较少。有研究表明,Clostridium histolyticum、Bacillussp.,Candida albicans和Vibrio vulnificus等具有产生胶原蛋白水解酶的潜力,但是这些菌株大部分为致病菌,在产胶原蛋白水解酶时,病原菌也产生相应的毒素,并不适合应用于骨源食品的制备。
发明内容
本发明的一个目的是提供一种胶原蛋白水解酶及其编码基因、制备方法和用途,该胶原蛋白水解酶能够降解骨胶原蛋白,并提高小分子量骨胶原蛋白肽的得率。
为了实现根据本发明的这些目的和其它优点,提供了一种胶原蛋白水解酶,其氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种胶原蛋白水解酶基因,其编码上述胶原蛋白水解酶。
优选的是,所述的胶原蛋白水解酶基因,其DNA序列如SEQ ID NO.2所示,cDNA序列如SEQ ID NO.4所示。
本发明还提供了一种胶原蛋白水解酶基因重组载体,其包含上述胶原蛋白水解酶基因。
本发明还提供了一种胶原蛋白水解酶基因工程菌,其包含上述胶原蛋白水解酶基因重组载体。
本发明还提供了一种上述胶原蛋白水解酶的制备方法,包括如下步骤:
S1、提取宿主菌株Rhizopusoryzae CGMCC3.17463的RNA序列,通过反转录获得宿主菌株的cDNA序列,以宿主菌株的cDNA序列为模板进行PCR扩增,获得胶原蛋白水解酶的cDNA序列,然后将胶原蛋白水解酶的cDNA序列连接到毕赤酵母表达载体pPIC9上,获得胶原蛋白水解酶基因重组载体;
S2、将胶原蛋白水解酶基因重组载体转化到毕赤酵母Gs115宿主细胞中,获得胶原蛋白水解酶基因工程菌株;
S3、将胶原蛋白水解酶基因工程菌株活化后,30℃条件下培养2-3d,在甲醇诱导下使胶原蛋白水解酶基因工程菌株表达生产出胶原蛋白水解酶的粗酶液;
S4、将粗酶液进行浓缩纯化,即得纯化后的胶原蛋白水解酶。
优选的是,所述的胶原蛋白水解酶的制备方法,步骤S1中,以宿主菌株的cDNA序列为模板进行PCR扩增,获得胶原蛋白水解酶的cDNA序列,具体包括以下步骤:
S1a、设计并合成胶原蛋白水解酶的特异性引物对RoAPA_F和RoAPA_R,其中, RoAPA_F的序列如SEQ ID NO.5所示, RoAPA_R的序列如SEQ ID NO.6所示;
S1b、以反转录获得的宿主菌株的cDNA为模板,以RoAPA_F和RoAPA_R为引物,进行PCR扩增,获取胶原蛋白水解酶的cDNA序列。
优选的是,所述的胶原蛋白水解酶的制备方法,步骤S1中,以宿主菌株的cDNA序列为模板进行PCR扩增,获得胶原蛋白水解酶的cDNA序列的条件为:97℃ 3 min;95℃ 30 s,63℃ 30 s,72℃ 1 min,32个循环;72℃ 10 min。
本发明还提供了一种上述胶原蛋白水解酶的用途,用于降解胶原蛋白或者用于制备小分子量胶原蛋白肽。
本发明至少包括以下有益效果:
第一、本发明发掘了一种新的具有胶原蛋白水解活性的胶原蛋白水解酶,并通过生物酶工程的方法将其编码基因导入工程菌株,实现该胶原蛋白水解酶的异源表达,促进骨胶原蛋白水解酶的工业化生产。
第二、在酶法制备骨胶原蛋白肽的过程中,使用本发明所提供的胶原蛋白水解酶能够提高小分子量骨胶原蛋白肽的得率。
本发明的其它优点、目标和特征将部分通过下面的说明体现,部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明
图1为本发明的其中一种技术方案所述胶原蛋白水解酶RoAPA在不同pH条件下相对酶活力曲线图;
图2为本发明的其中一种技术方案所述胶原蛋白水解酶RoAPA的pH稳定性曲线图;
图3为本发明的其中一种技术方案所述胶原蛋白水解酶RoAPA在不同温度下的相对酶活力曲线图;
图4为本发明的其中一种技术方案所述胶原蛋白水解酶RoAPA的温度稳定性曲线图;
图5为本发明的其中一种技术方案所述不同抑制剂对RoAPA催化活性的影响;
图6为本发明的其中一种技术方案使用RoAPA水解骨胶原蛋白的SDS-PAGE分析图。
具体实施方式
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
应当理解,本文所使用的诸如“具有”、“包含”以及“包括”术语并不配出一个或多个其它元件或其组合的存在或添加。
试验材料和试剂
1、菌株及载体:宿主菌株为米根霉(Rhizopusoryzae),可以从中国普通微生物菌种保藏管理中心获得(CGMCC),编号为CGMCC 3.17463;用于蛋白异源表达的工程菌株为毕赤酵母 (Pichia pastoris GS115),购买自生工生物工程(上海)股份有限公司,毕赤酵母表达载体pPIC9购自于Invitrogen公司;
2、酶类及其它生化试剂:内切酶购自TaKaRa公司,连接酶购自Invitrogen公司,其它都为国产试剂(均可从生化试剂公司购买得到);
3、大肠杆菌培养基:1%酵母提取物,2%蛋白胨,1.34% YNB,0.000049< Biotin,1%甘油(v/v);
4、BMGY培养基;1%酵母提取物,2%蛋白胨,1.34% YNB,0.000049< Biotin,1%甘油(v/v)。
5、BMMY培养基:除以0.5%甲醇代替甘油,其余成份均与BMGY相同,pH为4.0。
注:以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
实施例
1、胶原蛋白水解酶RoAPA的DNA序列的获得
提取宿主菌株Rhizopusoryzae CGMCC3.17463的DNA序列,置于-20℃温度下保存。设计用于胶原蛋白水解酶RoAPA基因克隆的特异性引物RoAPA_F和RoAPA_R,引物RoAPA_F和RoAPA_R的序列分别为SEQ ID NO.5和SEQ ID NO.6,以宿主菌株RhizopusoryzaeCGMCC3.17463的DNA序列为模板进行PCR扩增,其中,扩增条件为:97℃ 3 min;95℃ 30 s,63℃ 30 s,72℃ 1 min,32个循环;72℃ 10 min。得到一段约1252 bp的DNA序列,将该DNA序列回收后送至上海美吉生物医药科技有限公司测序,其基因序列见SEQ ID NO.2,为胶原蛋白水解酶RoAPA的DNA序列,对应的氨基酸序列为SEQ ID NO.1。
2、胶原蛋白水解酶RoAPA的cDNA序列的获得
提取宿主菌株Rhizopusoryzae CGMCC3.17463的RNA序列,再通过反转录得到宿主菌株Rhizopusoryzae CGMCC3.17463的cDNA序列。设计克隆引物RoAPA_F和RoAPA_R,引物RoAPA_F和RoAPA_R的序列分别为SEQ ID NO.5和SEQ ID NO.6,以宿主菌株RhizopusoryzaeCGMCC3.17463的cDNA序列为模板进行PCR扩增,扩增得到胶原蛋白水解酶RoAPA的cDNA序列,将扩增得到的序列送至上海美吉生物科技有限公司测序,长度为1194 bp,其基因序列见SEQ ID NO.4。
分析胶原蛋白水解酶RoAPA的DNA序列与胶原蛋白水解酶RoAPA的cDNA序列信息,胶原蛋白水解酶RoAPA的DNA序列全长为1252 bp,有1个内含子,长度为58bp,碱基序列如SEQ ID NO.3所示。胶原蛋白水解酶RoAPA的cDNA序列推导出的氨基酸序列经软件预测发现N端的21个氨基酸为蛋白的信号肽序列。经Blast比对发现,胶原蛋白水解酶RoAPA蛋白序列与数据库中已发布的蛋白酶序列最高相似度仅有78.6%,与现有晶体结构报道相关酶最高序列相似度仅为51.3%,以上结果表明从宿主菌株Rhizopusoryzae CGMCC3.17463中分离克隆得到的蛋白酶编码基因具有较高的新颖性。
3、重组工程菌株的制备
(1)重组工程菌株的制备
以测序正确的胶原蛋白水解酶RoAPA的cDNA为模板,设计合成了分别带有EcoR I和Not I限制性酶切位点的引物RoAPA_F和RoAPA_R,引物RoAPA_F和RoAPA_R的序列分别为SEQ ID NO.5和SEQ ID NO.6,其中,引物RoAPA_F序列(CGGAATTCATGAAATTCACTCTTGTCTCTT)划线部分为EcoR I限制性酶切位点,引物RoAPA_R序列(TTGCGGCCGCTTATTTGTTTTGGTCAACAGAAGC)划线部分为Not I限制性酶切位点。以胶原蛋白水解酶RoAPA的cDNA为模板,以RoAPA_F和RoAPA_R为引物进行PCR扩增,然后利用EcoR I 和 Not I酶切PCR产物,获得扩增后的胶原蛋白水解酶RoAPA的cDNA序列,将扩增后的胶原蛋白水解酶RoAPA的cDNA序列连接到毕赤酵母表达载体pPIC9上,获得重组表达载体pPIC9-RoAPA。即将胶原蛋白水解酶RoAPA的cDNA序列插入到上述表达载体的信号肽序列的下游,与信号肽形成正确的阅读框架,构建成毕赤酵母表达载体pPIC9-RoAPA,然后转化到大肠杆菌培养基中的大肠杆菌感受态细胞Trans1中。将阳性转化子进行DNA测序,将测序正确的转化子用于制备大量的重组质粒。用限制性内切酶Bgl II进行线性化表达质粒载体DNA序列,电击转化毕赤酵母GS115感受态细胞,并在30℃温度下培养2-3天,挑取在MD平板上生长的转化子进行进一步的表达实验,具体操作请参考毕赤酵母表达操作手册。并且以同样的方式构建含胶原蛋白水解酶RoAPA信号肽序列的cDNA序列的表达载体,并转化。
(2)高胶原蛋白水解酶活性转化子的筛选
用灭菌的牙签从长有转化子的MD板上挑取多个单菌落,按照编号点到另一个MD平板上,将MD平板置于30℃培养箱中培养1-2天,至菌落长出。按编号依次从MD平板上挑取转化子分别对应接种于装有3 mL BMGY培养基的离心管中,在温度30℃、转速220 rpm的条件下摇床培养48 h;将摇床培养48 h的菌液3000×g离心15 min,去上清,离心管中再加入1mL含有0.5%甲醇的BMMY培养基,在温度30℃、转速220 rpm的条件下诱导培养;诱导培养48h后,3000×g离心5 min,取上清用于酶活性检测,从中筛选出高胶原蛋白水解酶活性的转化子,具体操作请参考毕赤酵母表达操作手册。
4、重组胶原蛋白水解酶RoAPA的制备
(1)重组工程菌株pPIC9-RoAPA的表达
筛选出酶活较高的转化子,接种于300 mL BMGY液体培养基内,在温度30℃,转速220 rpm条件下摇床振荡培养48 h;摇床振荡培养后5000 rpm离心5 min,弃上清,再向菌体加入100 mL含有0.5%甲醇的BMMY液体培养基,在温度30℃,转速220 rpm条件下诱导培养72h。诱导培养期间,间隔24 h补加一次甲醇溶液以补偿甲醇的损失,使甲醇浓度保持在0.5%左右;诱导培养72 h后12000×g离心10 min,收集上清发酵液,检测酶活性并进行SDS-PAGE蛋白电泳分析。
(2)纯化获取重组胶原蛋白水解酶RoAPA
收集摇瓶表达的重组工程菌株胶原蛋白水解酶上清液,通过10 kDa膜包进行浓缩,同时用低盐缓冲液置换其中的培养基,然后用10 kDa超滤管进一步的浓缩。浓缩能稀释到一定倍数的重组胶原蛋白水解酶RoAPA,再通过离子交换层析进行纯化,从而得到重组胶原蛋白水解酶RoAPA。具体地,取重组胶原蛋白水解酶RoAPA浓缩液2.0 mL经预先用20 mMTris-HCl(pH 7.5)平衡过的HiTrap Q Sepharose XL阴离子柱,然后用0.1 mol/L的NaCl进行线性梯度洗脱,对分步收集的洗脱液检测酶活性和进行蛋白浓度的测定。
5、对胶原蛋白水解酶RoAPA的部分性质进行分析
采用福林酚试剂显色法对本发明制备的胶原蛋白水解酶RoAPA进行活性分析。具体方法如下:将胶原蛋白水解酶RoAPA与1 mL的反应体系反应10 min后,加入1 mL三氯乙酸(0.4mol/L)终止反应,其中,1 mL的反应体系的pH为3.0,温度为30℃,并且含有500 µL适当的稀释酶液,500 µL底物;终止反应后,将该反应体系12000 rpm离心3 min,吸500 µL上清液加入2.5 mL碳酸钠(0.4 mol/L),再加入500 µL福林酚试剂,在40℃的温度下显色20min,冷却后在紫外波长680 nm条件下测定OD值。蛋白酶活性单位定义:在一定条件下,每分钟分解底物酪蛋白生成l µmol酪氨酸所需的酶量为1个活性单位(U)。
(1)胶原蛋白水解酶RoAPA的最适pH及pH稳定性的检测
经过本发明纯化获得的胶原蛋白水解酶RoAPA在不同的pH条件下进行酶促反应以测定其最适pH值。所用缓冲液pH为2.0-3.0的甘氨酸-盐酸缓冲液,pH为3.0-8.0的柠檬酸-磷酸氢二钠系列缓冲液及pH为8.0-10.0的Tris-HCl系列缓冲液。纯化获得的胶原蛋白水解酶RoAPA在不同pH的缓冲体系中,温度为55℃条件下测定的最适pH结果如图1所示:温度为55℃条件下,胶原蛋白水解酶RoAPA的最适pH为3.0,在pH为3.0-4.0范围内,该酶能够维持较高的酶活力。
将酶液在不同pH值的缓冲液中于30 ℃下处理60 min,再测定酶活性以研究酶的pH稳定性。结果如图2所示,结果表明:胶原蛋白水解酶RoAPA的pH在3.0-6.0之间能够维持90%以上的酶活力,说明该酶在酸性条件下具有良好的pH稳定性。
(2)胶原蛋白水解酶RoAPA最适反应温度以及热稳定性的检测
经过纯化获得的胶原蛋白水解酶RoAPA在pH为3.0的条件下,测定不同温度(30-70℃)下的酶活性,如图3所示:该酶的最适反应温度为55 ℃,并且在60℃时依然具有80%以上的酶活力。经过纯化获得的胶原蛋白水解酶RoAPA分别在50 ℃、55 ℃和60 ℃条件下处理不同时间,再在55 ℃下进行酶活性测定。结果如图4所示,胶原蛋白水解酶RoAPA在60 ℃条件下处理30 min能使蛋白完全失活。综上表明,该胶原蛋白水解酶RoAPA在50-60℃条件下具有高效的蛋白质水解活性,且60 ℃孵育30 min能彻底灭活该蛋白酶。这意味着本发明的蛋白酶在食品、医药等领域具有重要的应用价值。
(3)不同金属离子/试剂对胶原蛋白水解酶RoAPA活性的影响
为了测定不同金属离子对胶原蛋白水解酶RoAPA活性的影响,在测定胶原蛋白水解酶RoAPA的催化活性前,向反应体系中加入Mn2+、Cu2+、Cr3+、Zn2+、K+、Na+、Ca2+、Mg2+、Ni2+、Co2 +、Pb2+、Fe3+等不同的金属离子,控制其终浓度为3 mM,调整溶液pH至3.0;然后在55℃进行蛋白酶活性测定。结果表明,Mn2+和Cu2+对胶原蛋白水解酶RoAPA有明显的激活作用,Pb2+和Fe3+对胶原蛋白水解酶RoAPA蛋白水解活性有明显的抑制作用,其它金属离子对胶原蛋白水解酶RoAPA活性基本没有影响。同时,我们分析了添加不同类型蛋白酶抑制剂(空白对照CK,2mM半胱氨酸蛋白酶抑制剂E-64,2 mM 丝氨酸蛋白酶抑制剂PMSF,5 mM 金属蛋白酶抑制剂EDTA,和0.05 mM天冬氨酸蛋白酶抑制剂pepstatin A)对胶原蛋白水解酶RoAPA的活性的影响,结果如图5所示。研究表明,胶原蛋白水解酶RoAPA能够特异性的被Pepstatin A所抑制,Pepstatin A能特异性地结合天冬氨酸蛋白酶的催化口袋但不被切割,从而抑制了催化残基的活性,进一步证明了胶原蛋白水解酶RoAPA属于天冬氨酸蛋白酶家族。
6、胶原蛋白水解酶RoAPA在制备小分子量骨胶原蛋白肽中的应用
(1)骨胶原蛋白的酶解
准确称取5.0 g骨胶原蛋白并加入1000 mL水配置成0.5%(w/v)的骨胶原蛋白溶液;调整溶液pH至3.5,按照5000U/g的添加量加入胶原蛋白水解酶RoAPA,并搅拌均匀,在50℃条件下进行酶解反应,待反应结束后,沸水浴加热10 min使胶原蛋白水解酶RoAPA彻底失活。取0.25 mL骨胶原蛋白酶解液与等体积10% TCA混合,震荡摇匀,10000 g、4 ℃离心20min。吸取上清液50 μL依次顺序加入96孔板,然后依次加入200 μL BCA工作液,室温孵育2h后。放置酶标仪中,于562 nm波长下测定吸光度。研究发现,随着酶解时间的延长,水解度逐渐变大,至4 h左右到达平台期,水解度不再随时间延长而增加。以上结果表明胶原蛋白水解酶RoAPA在酸性条件下能催化骨胶原蛋白水解。将骨胶原蛋白水解物进行聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,结果显示骨胶原蛋白经胶原蛋白水解酶RoAPA水解,分子量明显降低,骨胶原蛋白的特征条带(α1和α2带)随着反应时间的延长逐渐消失(图6)。以上结果表明,胶原蛋白水解酶RoAPA能催化骨胶原蛋白水解产生小分子肽,具有工业化制备骨胶原蛋白肽的应用潜力。
(2)骨胶原蛋白酶解产物的分子量分布测定
采用安捷伦HPLC1260-II系统(Agilent Technologies Inc., California, USA)测定骨胶原蛋白水解产物分子量分布。TSK gel G2000 SWXL色谱柱(7.8×300 mm, TOSOH,Tokyo, Japan);柱温:40℃;流动相:A为添加0.1%的三氟乙酸混合的45%(v/v)乙腈溶液;等梯度洗脱;流速:0.5 mL/min;进样体积:10 μL,于214 nm波长下测定响应值。以Gly-Sar(146 Da)、Gly-Gly-Tyr-Arg(451 Da)、Bacitracin(1422 Da)、Aprotinin(6511 Da)和Cytochrome C(12327 Da)为标准品,建立保留时间(X)和分子量对数(Y)之间的标准曲线(Y=-3.9331X+27.517, R2=0.987)。表1是胶原蛋白水解酶RoAPA水解骨胶原蛋白制备骨胶原蛋白肽的分子量分布,由此可以看出与胃蛋白酶和胰蛋白酶相比,经胶原蛋白水解酶RoAPA水解获得的胶原蛋白肽在分子量分布上更有优势,分子量小于1000Da的总和与分子量小于2000 Da的总和均最高。分析骨胶原蛋白酶解产物游离氨基酸含量发现,骨胶原蛋白经胶原蛋白水解酶RoAPA水解产生的游离氨基酸约占6.53 %,低于胃蛋白酶和胰蛋白酶解产物中游离氨基酸的比率,说明骨胶原蛋白的RoAPA水解物主要以肽的形式存在。
表 1 酶解法制备骨胶原蛋白肽分子量分布
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
<110> 中国农业科学院农产品加工研究所
<120> 胶原蛋白水解酶及其编码基因、制备方法和用途
<130> 2021
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 397
<212> PRT
<213> Rhizopus oryzae CGMCC3.17463
<400> 1
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Ala Ile Ala Lys Tyr Gln Lys His Ala Ile Asn Pro Leu Lys Asn Thr
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Pro Ser Gly Ser Ser Ser Thr Glu Gly Thr Gly Val Val Pro Val Thr
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Asp Tyr Gly Asn Asp Ile Glu Tyr Tyr Gly Asp Val Gln Ile Gly Thr
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Pro Pro Gln Asn Phe Lys Ile Asn Phe Asp Thr Gly Ser Ser Asp Leu
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Trp Val Ala Ser Thr Leu Cys Ala Ser Cys Thr Ser His Thr Arg Tyr
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Asn Pro Asn Lys Ser Ser Thr Tyr Val Lys Asp Gly Arg Pro Trp Ser
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Ile Ser Tyr Gly Asp Gly Ser Thr Ala Ser Gly Ile Leu Ala Tyr Asp
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Thr Val Thr Leu Gly Gly Leu Ala Ile Lys Lys Gln Thr Ile Glu Leu
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Ala Gln Lys Glu Ser Ser Ser Phe Ala Ser Asp Pro Ile Asp Gly Leu
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Leu Gly Leu Gly Phe Asn Thr Ile Thr Thr Val Arg Gly Ile Lys Thr
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Pro Val Asp Asn Leu Ile Ser Gln Gly Leu Ile Thr Ser Pro Ile Tyr
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Gly Val Ser Leu Gly Lys Ala Ser Asn Gly Gly Gly Gly Glu Tyr Leu
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Phe Gly Gly Tyr Asn Lys Ser Lys Phe Thr Gly Thr Leu Lys Thr Val
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Pro Val Asp Asn Ser Gln Gly Phe Trp Gly Ile Thr Val Ser Asp Leu
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Lys Val Gly Thr Lys Ser Tyr Gly Thr Phe Asp Gly Ile Leu Asp Thr
275 280 285
Gly Thr Thr Leu Leu Leu Phe Pro Thr Ala Tyr Ala Asn Lys Val Ala
290 295 300
Thr Ala Tyr Gly Ala Thr Ala Asn Gly Asp Gly Thr Tyr Asn Ile Asn
305 310 315 320
Cys Asn Thr Ser Gly Phe Lys Pro Leu Glu Phe Thr Ile Asn Gly Ala
325 330 335
Thr Phe Tyr Val Pro Thr Asn Ser Leu Ile Phe Gln Lys Ser Gly Ser
340 345 350
Arg Cys Tyr Ala Ser Phe Gly Ser Ser Asn Ile Pro Phe Ala Ile Leu
355 360 365
Gly Asp Thr Phe Leu Lys Asn Asn Tyr Val Val Phe Asn Gln Gln Val
370 375 380
Pro Glu Val Gln Ile Ala Ala Ser Val Asp Gln Asn Lys
385 390 395
<210> 2
<211> 1252
<212> DNA
<213> Rhizopus oryzae CGMCC3.17463
<400> 2
atgaaattca ctcttgtctc ttcttgtgtg gcactggttg tcatggctct ttctgttgaa 60
gcagctccta atggcaagaa actttccatt gctttaaagc aaaatactga atacaagcct 120
agtgctcccg ctgctgttgc aaaggccatt gccaagtatc aaaagcatgc tattaatcct 180
ctcaaaaaca ctccttctgg atcttcctct actgaaggta ctggtgttgt acctgtcact 240
gattacggaa atgatattga atattacggt gatgttcaaa tcggtactcc tcctcaaaac 300
ttcaagatta actttgatac cggttcctcc gatttatggg ttggtaagta caattctctt 360
tttttgttta aaatattata taactaaact attattatta gcctctactt tgtgtgcttc 420
ttgtaccagt catactcgtt acaatcccaa caaatcaagc acttatgtca aggatggtcg 480
tccatggtct atctcttacg gtgatggatc tactgctagc ggtattttag cttacgatac 540
tgttacttta ggtggccttg ctatcaagaa acaaactatt gaattagctc aaaaagaatc 600
cagcagtttc gcttctgatc ctattgatgg tcttctcggt cttggtttca ataccattac 660
cactgttaga ggtatcaaga ctcctgttga taacttgatc agtcaaggtt taattacttc 720
tcctatttat ggtgtttctc tcggtaaggc cagcaatggt ggaggtggtg aatacctctt 780
tggtggttac aataagtcca agttcactgg tactttaaag actgttcctg ttgataactc 840
tcaaggtttc tggggtatta ctgtcagtga tcttaaggtt ggtaccaaga gctatggtac 900
tttcgatggc atccttgata ccggtaccac tcttttactt ttccctactg cctatgccaa 960
caaggtcgcc actgcttatg gtgctactgc taatggtgat ggtacttaca acatcaactg 1020
taacacttct ggtttcaagc ctcttgaatt cactatcaat ggtgctactt tctatgttcc 1080
taccaactct ttgatcttcc aaaagagtgg atccagatgt tatgcttcat tcggttcatc 1140
caacattcct ttcgctattc ttggtgatac tttcttgaag aacaactatg ttgtattcaa 1200
ccaacaagtc cctgaagttc aaatcgctgc ttctgttgac caaaacaaat aa 1252
<210> 3
<211> 58
<212> DNA
<213> Rhizopus oryzae CGMCC3.17463
<400> 3
gtaagtacaa ttctcttttt ttgtttaaaa tattatataa ctaaactatt attattag 58
<210> 4
<211> 1194
<212> DNA
<213> Rhizopus oryzae CGMCC3.17463
<400> 4
atgaaattca ctcttgtctc ttcttgtgtg gcactggttg tcatggctct ttctgttgaa 60
gcagctccta atggcaagaa actttccatt gctttaaagc aaaatactga atacaagcct 120
agtgctcccg ctgctgttgc aaaggccatt gccaagtatc aaaagcatgc tattaatcct 180
ctcaaaaaca ctccttctgg atcttcctct actgaaggta ctggtgttgt acctgtcact 240
gattacggaa atgatattga atattacggt gatgttcaaa tcggtactcc tcctcaaaac 300
ttcaagatta actttgatac cggttcctcc gatttatggg ttgcctctac tttgtgtgct 360
tcttgtacca gtcatactcg ttacaatccc aacaaatcaa gcacttatgt caaggatggt 420
cgtccatggt ctatctctta cggtgatgga tctactgcta gcggtatttt agcttacgat 480
actgttactt taggtggcct tgctatcaag aaacaaacta ttgaattagc tcaaaaagaa 540
tccagcagtt tcgcttctga tcctattgat ggtcttctcg gtcttggttt caataccatt 600
accactgtta gaggtatcaa gactcctgtt gataacttga tcagtcaagg tttaattact 660
tctcctattt atggtgtttc tctcggtaag gccagcaatg gtggaggtgg tgaatacctc 720
tttggtggtt acaataagtc caagttcact ggtactttaa agactgttcc tgttgataac 780
tctcaaggtt tctggggtat tactgtcagt gatcttaagg ttggtaccaa gagctatggt 840
actttcgatg gcatccttga taccggtacc actcttttac ttttccctac tgcctatgcc 900
aacaaggtcg ccactgctta tggtgctact gctaatggtg atggtactta caacatcaac 960
tgtaacactt ctggtttcaa gcctcttgaa ttcactatca atggtgctac tttctatgtt 1020
cctaccaact ctttgatctt ccaaaagagt ggatccagat gttatgcttc attcggttca 1080
tccaacattc ctttcgctat tcttggtgat actttcttga agaacaacta tgttgtattc 1140
aaccaacaag tccctgaagt tcaaatcgct gcttctgttg accaaaacaa ataa 1194
<210> 5
<211> 30
<212> DNA
<213> 人工合成
<400> 5
cggaattcat gaaattcact cttgtctctt 30
<210> 6
<211> 34
<212> DNA
<213> 人工合成
<400> 6
ttgcggccgc ttatttgttt tggtcaacag aagc 34
Claims (9)
1.胶原蛋白水解酶,其特征在于,其氨基酸序列如SEQ ID NO.1所示。
2.胶原蛋白水解酶基因,其特征在于,其编码权利要求1所述的胶原蛋白水解酶。
3.如权利要求2所述的胶原蛋白水解酶基因,其特征在于,其DNA序列如SEQ ID NO.2所示,cDNA序列如SEQ ID NO.4所示。
4.胶原蛋白水解酶基因重组载体,其特征在于,其包含权利要求2所述的胶原蛋白水解酶基因。
5.胶原蛋白水解酶基因工程菌,其特征在于,其包含权利要求4所述的胶原蛋白水解酶基因重组载体。
6.如权利要求1所述的胶原蛋白水解酶的制备方法,其特征在于,包括如下步骤:
S1、提取宿主菌株Rhizopusoryzae CGMCC3.17463的RNA序列,通过反转录获得宿主菌株的cDNA序列,以宿主菌株的cDNA序列为模板进行PCR扩增,获得胶原蛋白水解酶的cDNA序列,然后将胶原蛋白水解酶的cDNA序列连接到毕赤酵母表达载体pPIC9上,获得胶原蛋白水解酶基因重组载体;
S2、将胶原蛋白水解酶基因重组载体转化到毕赤酵母Gs115宿主细胞中,获得胶原蛋白水解酶基因工程菌株;
S3、将胶原蛋白水解酶基因工程菌株活化后,30℃条件下培养2-3d,在甲醇诱导下使胶原蛋白水解酶基因工程菌株表达生产出胶原蛋白水解酶的粗酶液;
S4、将粗酶液进行浓缩纯化,即得纯化后的胶原蛋白水解酶。
7.如权利要求6所述的胶原蛋白水解酶的制备方法,其特征在于,步骤S1中,以宿主菌株的cDNA序列为模板进行PCR扩增,获得胶原蛋白水解酶的cDNA序列,具体包括以下步骤:
S1a、设计并合成胶原蛋白水解酶的特异性引物对RoAPA_F和RoAPA_R,其中, RoAPA_F的序列如SEQ ID NO.5所示, RoAPA_R的序列如SEQ ID NO.6所示;
S1b、以反转录获得的宿主菌株的cDNA为模板,以RoAPA_F和RoAPA_R为引物,进行PCR扩增,获取胶原蛋白水解酶的cDNA序列。
8.如权利要求7所述的胶原蛋白水解酶的制备方法,其特征在于,步骤S1中,以宿主菌株的cDNA序列为模板进行PCR扩增,获得胶原蛋白水解酶的cDNA序列的条件为:97℃ 3min;95℃ 30 s,63℃ 30 s,72℃ 1 min,32个循环;72℃ 10 min。
9.如权利要求1所述的胶原蛋白水解酶的用途,其特征在于,用于降解胶原蛋白或者用于制备小分子量胶原蛋白肽。
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