CN111647584A - 低温酸性蛋白酶PsAPA及其制备方法和应用 - Google Patents
低温酸性蛋白酶PsAPA及其制备方法和应用 Download PDFInfo
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- CN111647584A CN111647584A CN202010406280.1A CN202010406280A CN111647584A CN 111647584 A CN111647584 A CN 111647584A CN 202010406280 A CN202010406280 A CN 202010406280A CN 111647584 A CN111647584 A CN 111647584A
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- psapa
- protease
- acid protease
- sequence
- cdna sequence
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Abstract
本发明公开了一种低温酸性蛋白酶PsAPA及其制备方法和应用，PsAPA的氨基酸序列如SEQ ID NO.1所示；其DNA序列如SEQ ID NO.2所示，cDNA序列如SEQ ID NO.4所示；其制备方法为：S1、制备重组表达载体pPIC9‑PsAPA；S2、转化工程菌株Pichia pastoris Gs115；S3、对转入目的基因的工程菌株进行发酵表达，获得重组酸性蛋白酶粗酶液；S4、将粗酶液进行浓缩和纯化，从而得到纯化后的重组蛋白酶PsAPA；酸性蛋白酶PsAPA用于提取骨胶原蛋白的应用。本发明在骨胶原蛋白酶法提取过程中，使用酸性蛋白酶PsAPA能够提高骨胶原蛋白的提取率。
Description
技术领域
本发明涉及生物技术领域，更具体地，涉及一种低温酸性蛋白酶PsAPA及其制备方法和应用。
背景技术
畜禽骨中含有丰富的胶原蛋白，是制备功能食品的重要原料。骨胶原蛋白常见的提取方法有酸、碱、酶法等工艺，其中骨胶原蛋白的酶法提取最为高效。骨胶原蛋白在酸性溶液中有较高的溶解度，因此骨胶原蛋白提取通常是在低pH条件下进行的。提取过程中添加酸性蛋白酶能促进胶原蛋白端肽酶解，降低蛋白的交联程度，增加骨胶原蛋白提取效率。另外，为了防止胶原蛋白受热解螺旋，骨胶原蛋白提取通常是在低温条件下进行的；但是酶法提取过程中通常使用的胃蛋白酶为常温蛋白酶，在骨胶原蛋白低温提取过程中存在提取率低等问题。
发明内容
本发明的一个目的是解决至少上述问题，并提供至少后面将说明的优点。
本发明还有一个目的是提供一种酸性蛋白酶PsAPA，能够提高骨胶原蛋白的提取率。
本发明另一目的是提供一种酸性蛋白酶PsAPA的制备方法。
本发明的又一目的是提供一种酸性蛋白酶PsAPA在提取骨胶原蛋白上的应用。
为了实现根据本发明的这些目的和其它优点，酸性蛋白酶PsAPA的氨基酸序列如SEQ ID NO.1所示。
优选的是，所述酸性蛋白酶PsAPA的DNA序列如SEQ ID NO.2所示，所述酸性蛋白酶PsAPA的cDNA序列如SEQ ID NO.4所示。
优选的是，所述的酸性蛋白酶PsAPA的制备方法，包括以下步骤：
S1、首先提取Penicillium sp.XT7的总RNA序列，通过反转录获得Penicilliumsp.XT7的cDNA序列，以Penicillium sp.XT7的cDNA序列为模板扩增酸性蛋白酶PsAPA的cDNA序列，然后将酸性蛋白酶PsAPA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-PsAPA；
S2、将重组表达载体pPIC9-PsAPA转化到毕赤酵母宿主细胞中，获得重组工程菌株；
S3、将重组工程菌株在30℃条件下培养2-3d，在甲醇诱导下使重组工程菌株表达生产出粗酶液；
S4、将粗酶液进行浓缩纯化，从而得到纯化后的重组蛋白酶PsAPA。
4、如权利要求3所述的酸性蛋白酶PsAPA的制备方法，其特征在于，步骤S1中以Penicillium sp.XT7的cDNA为模板扩增酸性蛋白酶PsAPA的cDNA序列的具体包括以下步骤：
S1a、设计酸性蛋白酶PsAPA的特异性引物PsAPA_f核苷酸序列为：CGGAATTCATGGCCGCTGCTGCCCCAA，PsAPA_r核苷酸序列为：TTGCGGCCGCCTAAGCCTGCTTGGCGAAGCCAAG，并将设计好的PsAPA_f和PsAPA_r送至北京华大生物有限公司进行合成；
S1b、以反转录获得的Penicillium sp.XT7的cDNA为模板，以PsAPA_f，PsAPA_r为引物，进行PCR扩增以获取酸性蛋白酶PsAPA的cDNA序列。
优选的是，步骤S1中以Penicillium sp.XT7的cDNA序列为模板扩增酸性蛋白酶PsAPA的cDNA序列的条件为：95℃5min；94℃30s，60℃30s，72℃2min，35个循环；72℃10min。
优选的是，所述的酸性蛋白酶PsAPA的应用，所述酸性蛋白酶PsAPA用于骨胶原蛋白提取的应用。
本发明至少包括以下有益效果：
在酶法提取骨胶原蛋白的过程中，使用本发明所提供的酸性蛋白酶PsAPA能够提高骨胶原蛋白的提取率。
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酸性蛋白酶PsAPA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酸性蛋白酶PsAPA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酸性蛋白酶PsAPA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酸性蛋白酶PsAPA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骨胶原蛋白提取率对比图；
图6为本发明的其中一种技术方案使用酶法提取所得的骨胶原蛋白的SDS-PAGE分析图。
注：PSC代表胃蛋白酶辅助提取牛骨胶原蛋白，ESC代表酸性蛋白酶PsAPA处理后牛骨胶原蛋白。
具体实施方式
下面结合实施例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。
试验材料和试剂
1、菌株及载体：用于蛋白异源表达的工程菌株为毕赤酵母(Pichia pastorisGS115)，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及菌株GS115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、产酶培养基：向1L去离子水中加入30g/L麦麸，30g/L玉米芯粉，30g/L豆粕，5g/L大麦葡聚糖，5g/L(NH4)SO4，1g/L KH2PO4，0.5g/L MgSO4·7H2O，0.01g/L FeSO4·7H2O，0.2g/L CaCl2，在温度121℃，高压下蒸汽灭菌处理20min；
4、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)；
5、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)。
6、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、酸性蛋白酶PsAPA的DNA序列的获得
提取Penicillium sp.XT7的DNA序列，置于-20℃温度下保存。
设计克隆引物PsAPA_f和PsAPA_r，引物PsAPA_f和PsAPA_r的序列分别为SEQ IDNO.5和SEQ ID NO.6，以Penicillium sp.XT7的DNA序列为模板进行PCR扩增，其中，扩增条件为：95℃5min；94℃30s，60℃30s，72℃2min，35个循环；72℃10min。得到一段约1100bp的DNA序列，将该DNA序列回收后送至睿博生物技术有限公司测序，其基因序列见SEQ IDNO.2，为酸性蛋白酶PsAPA的DNA序列，对应的氨基酸序列为SEQ ID NO.1。
2、酸性蛋白酶PsAPA的cDNA序列的获得
提取Penicillium sp.XT7的RNA序列，再通过反转录得到Penicillium sp.XT7的cDNA序列。设计克隆引物PsAPA_f和PsAPA_r，引物PsAPA_f和PsAPA_r的序列分别为SEQ IDNO.5和SEQ ID NO.6，以Penicillium sp.XT7的cDNA序列为模板进行PCR扩增，扩增得到cDNA序列，将扩增得到的cDNA序列送至睿博生物技术有限公司测序，其基因序列见SEQ IDNO.4，其为酸性蛋白酶PsAPA的cDNA序列，对应的氨基酸序列为SEQ ID NO.1。
分析酸性蛋白酶PsAPA的DNA序列与PsAPA的cDNA序列信息，酸性蛋白酶PsAPA的DNA序列全长为1178bp，且含有1个内含子，内含子的碱基序列如SEQ ID NO.3所示，cDNA序列全长为1122bp。DNA序列表达出的氨基酸序列经软件预测N端不存在信号肽序列，所以酸性蛋白酶PsAPA的DNA序列表达出的氨基酸序列与cDNA序列表达出的氨基酸序列相同均为SEQ ID NO.1所示。经Blast比对证明从Penicillium sp.XT7中分离克隆得到的编码蛋白酶的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
以测序正确的酸性蛋白酶PsAPA的cDNA为模板，设计合成了分别带有EcoR I和NotI限制性酶切位点的引物PsAPA_f和PsAPA_r，引物PsAPA_f和PsAPA_r的序列分别为SEQ IDNO.5和SEQ ID NO.6，其中，引物PsAPA_f序列划线部分为EcoR I限制性酶切位点，引物PsAPA_r序列划线部分为Not I限制性酶切位点。以酸性蛋白酶PsAPA的cDNA为模板，以PsAPA_f和PsAPA_r为引物进行PCR扩增，然后利用EcoR I和Not I酶切PCR产物，获得扩增后的酸性蛋白酶PsAPA的cDNA序列，将扩增后的酸性蛋白酶PsAPA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-PsAPA。即将酸性蛋白酶PsAPA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-PsAPA，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PsAPA信号肽序列的cDNA序列的的表达载体，并转化。
(2)高蛋白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3mL BMGY培养基的离心管中，在温度30℃、转速220rpm的条件下摇床培养48h；将摇床培养48h的菌液3000×g离心15min，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220rpm的条件下诱导培养；诱导培养48h后，3000×g离心5min，取上清用于酶活性检测，从中筛选出高蛋白酶活性的转化子，具体操作请参考毕赤酵母表达操作手册。
4、重组蛋白酶PsAPA的制备
(1)重组工程菌株pPIC9-PsAPA的表达
筛选出酶活较高的转化子，接种于300mL BMGY液体培养基内，在温度30℃，转速220rpm条件下摇床振荡培养48h；摇床振荡培养后5000rpm离心5min，弃上清，再向菌体加入100mL含有0.5％甲醇的BMMY液体培养基，在温度30℃，转速220rpm条件下诱导培养72h。诱导培养期间，间隔24h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72h后12000×g离心10min，收集上清发酵液，检测酶活性并进行SDS-PAGE蛋白电泳分析。
(2)纯化获取重组蛋白酶PsAPA
收集摇瓶表达的重组工程菌株蛋白酶上清液，通过10kDa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10kDa超滤管进一步的浓缩。浓缩能稀释到一定倍数的重组蛋白酶PsAPA，再通过离子交换层析进行纯化，从而得到重组蛋白酶PsAPA。具体地，取重组蛋白酶PsAPA浓缩液2.0mL经预先用20mM Tris-HCl(pH 7.5)平衡过的HiTrap QSepharose XL阴离子柱，然后用0.1mol/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、对酸性蛋白酶PsAPA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重组蛋白酶PsAPA，即酸性蛋白酶PsAPA进行活性分析。具体方法如下：酸性蛋白酶PsAPA与1mL的反应体系反应10min后，加入1mL三氯乙酸(0.4mol/L)终止反应，其中，1mL的反应体系的pH为3.0，温度为30℃，并且含有500μL适当的稀释酶液，500μL底物；终止反应后，将该反应体系12000rpm离心3min，吸500μL上清液加入2.5mL碳酸钠(0.4mol/L)，再加入500μL福林酚试剂，在40℃的温度下显色20min，冷却后在紫外波长680nm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lμmol酪氨酸所需的酶量为1个活性单位(U)。
(1)酸性蛋白酶PsAPA的最适pH及pH稳定性的检测
经过本发明纯化获得的重组蛋白酶PsAPA，即酸性蛋白酶PsAPA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1.0-3.0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。纯化获得的酸性蛋白酶PsAPA在不同pH的缓冲体系中，温度为30℃条件下测定的最适pH结果如图1所示：温度为30℃条件下，酸性蛋白酶PsAPA的最适pH为3.0，在pH为2.5-3.5范围内，该酶能够维持其70％以上的酶活力。
将酶液在不同pH值的缓冲液中于10℃下处理60min，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：酸性蛋白酶PsAPA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)酸性蛋白酶PsAPA最适反应温度以及热稳定性的检测
经过纯化获得的酸性蛋白酶PsAPA在pH为3.0的条件下，测定不同温度(5-40℃)下的酶活性，如图3所示：该酶的最适反应温度为30℃，并且在10℃时依然具有40％以上的酶活力。经过纯化获得的酸性蛋白酶PsAPA分别在30℃、35℃和40℃条件下处理不同时间，再在30℃下进行酶活性测定。结果如图4所示，PsAPA在40℃条件下处理30min能使蛋白完全失活。综上表明，该蛋白酶在低温条件下具有高效的蛋白质水解活性，且具有较低的热灭活温度。这意味着新发明的蛋白酶在食品、医药等领域具有重要的应用价值。
(3)酸性蛋白酶PsAPA的催化特异性
酸性蛋白酶PsAPA的催化特性与天冬氨酸蛋白酶家族的其他蛋白酶基本一致，主要切割底物分子中的疏水性氨基酸残基或者芳香族氨基酸残基间的肽键，例如Leu-Tyr、Phe-Phe、Phe-Tyr等。蛋白酶的活性能够特异性的被Pepstatin A所抑制，Pepstatin A能特异性地结合酸性蛋白酶PsAPA的催化口袋但不被切割，从而抑制了催化残基的活性；因此，该类抑制剂属于底物类似物抑制剂。
6、酸性蛋白酶PsAPA在牛骨胶原蛋白提取中的应用
牛骨胶原蛋白提取过程：
将牛骨经破碎后加入0.1M NaOH溶液(v/w)搅拌8h以去除非胶原成分，然后使用蒸馏水洗涤后沥干，再加入10％正己烷搅拌12h去除脂肪，去除脂肪后使用蒸馏水反复洗涤至溶液成中性然后沥干，获得沥干后的牛骨。用pH为7.4，浓度为0.25M的EDTA-Na2溶液对沥干后的牛骨进行脱除钙盐处理，获得脱钙盐牛骨，将脱钙盐牛骨平均分成两份，其中一份脱钙盐牛骨经加入5倍体积(v/w)的0.5M乙酸溶液和1.6％倍牛骨质量(w/w)的胃蛋白酶，另一份加入5倍体积(v/w)的0.5M乙酸溶液和1.6％倍牛骨质量(w/w)的酸性蛋白酶PsAPA，得到两份混合物，每份混合物均搅拌提取24h，然后双层纱布过滤得到牛骨粗胶原蛋白滤液。向每份滤液中加入NaCl调至NaCl终浓度为0.9M(含0.05M Tris，pH为7.0)，搅拌析出絮状沉淀后经，获得加入NaCl的滤液，将每份加入NaCl的滤液在温度4℃、转速10000g条件下离心20min收集沉淀即为盐析后的牛骨粗胶原蛋白。将每份盐析后的牛骨粗胶原蛋白溶解于0.5M乙酸溶液，依次采用0.1mol/L乙酸和超纯水各透析24h，期间各换液3次，将获得的透析物在-20℃温度下冷冻干燥，即得一份胃蛋白酶辅助提取牛骨胶原蛋白(PSC)以及酸性蛋白酶PsAPA处理后牛骨胶原蛋白(ESC)。然后对PSC和ESC进行提取率计算，并且进行SDS-PAGE蛋白电泳分析，结果分别见图5和图6。
如图5所示：PSC得率仅为8.34±0.27，ESC得率高达11.93±0.53。与胃蛋白酶相比，酸性蛋白酶PsAPA处理显著提高了骨胶原蛋白的提取率。骨胶原蛋白的端肽易发生分子交联降低了其在酸性条件下的溶解度，不利于骨胶原蛋白的提取。酸性蛋白酶PsAPA处理能催化胶原蛋白端肽酶解，提高胶原蛋白在酸性条件下的溶解度，增加胶原蛋白的得率。通过SDS-PAGE蛋白电泳分析如图6所示：酸性蛋白酶PsAPA提取的胶原蛋白条带与胃蛋白酶辅助提取胶原蛋白一致。这表明酸性蛋白酶PsAPA能增加提取液中胶原蛋白浓度，但并不破坏其整体结构。综上表明，酸性蛋白酶PsAPA能显著提高骨胶原蛋白的提取效率，增加骨胶原蛋白得率。
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。
SEQUENCE LISTING
SEQ ID NO.1：
<110> 中国农业科学院农产品加工研究所
<120> 低温酸性蛋白酶PsAPA及其制备方法和应用
<130> 2020
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 374
<212> PRT
<213> Penicillium sp. XT7
<400> 1
Met Ala Ala Ala Ala Pro Thr Gln Ala Ser Lys Phe Ser Leu Glu Gln
1 5 10 15
Val Ser Arg Pro Ala Ser Lys Ser Ser Asn Phe Ala Ser Lys Tyr Ala
20 25 30
Lys Ala Leu Ala Lys Tyr Gly Ala Gln Val Pro Thr Arg Val Gln Ala
35 40 45
Ala Ala Val Ala Ser Gly Val Ala Thr Asn Asn Pro Glu Pro Gln Asp
50 55 60
Val Glu Tyr Leu Thr Pro Val Lys Ile Gly Asp Thr Thr Leu Gln Leu
65 70 75 80
Asp Phe Asp Thr Gly Ser Ala Asp Leu Trp Val Phe Ser Thr Glu Leu
85 90 95
Pro Glu Ser Glu Gln Ser Gly His Ser Val Tyr Asp Thr Ser Ser Gly
100 105 110
Asn Lys Lys Ser Gly Tyr Thr Trp Ser Ile Ser Tyr Gly Asp Gly Ser
115 120 125
Ser Ala Ser Gly Phe Asp Val Tyr Thr Asp Ser Val Thr Val Gly Gly
130 135 140
Ile Ala Val Ser Gly Gln Ala Val Glu Ala Ala Ser Lys Ile Ser Thr
145 150 155 160
Glu Phe Thr Gln Asp Ala Asn Asn Asp Gly Leu Leu Gly Leu Ala Phe
165 170 175
Ser Ser Ile Asn Thr Val Ser Pro Gln Pro Gln Lys Thr Trp Phe Asp
180 185 190
Asn Ala Gln Ser Gln Leu Ala Ser Pro Leu Phe Gly Val Ala Leu Lys
195 200 205
His Asn Ala Pro Gly Val Tyr Asp Phe Gly Phe Ala Asp Ser Ser Lys
210 215 220
Tyr Thr Gly Asp Leu Ala Tyr Thr Asp Val Asp Asn Ser Gln Gly Phe
225 230 235 240
Trp Ser Phe Ser Val Asp Gly Tyr Lys Ala Gly Ser Lys Ser Gly Ala
245 250 255
Gly Phe Asp Gly Ile Ala Asp Thr Gly Thr Thr Leu Leu Leu Leu Asp
260 265 270
Asp Glu Ile Val Ser Ala Tyr Tyr Ser Gln Val Ser Gly Ala Gln Glu
275 280 285
Asp Ala Ser Ala Gly Gly Tyr Val Phe Asp Cys Ser Thr Thr Leu Pro
290 295 300
Asp Phe Ser Val Thr Ile Gly Asp Tyr Thr Ala Thr Val Pro Gly Asp
305 310 315 320
Leu Ile Asn Ala Gly Ser Ser Gly Thr Gly Ser Gly Ser Cys Phe Gly
325 330 335
Gly Ile Ala Ser Asn Ser Gly Ile Gly Phe Ser Ile Phe Gly Asp Ile
340 345 350
Phe Leu Lys Ser Gln Tyr Val Val Phe Asp Ala Ser Gly Pro Arg Leu
355 360 365
Gly Phe Ala Lys Gln Ala
370
SEQ ID NO.2：
<210> 2
<211> 1178
<212> DNA
<213> Penicillium sp. XT7
<400> 2
atggccgctg ctgccccaac ccaagccagc aagttctctc ttgagcaggt ctctcgcccg 60
gcttccaaga gctctaactt tgcttcgaag tatgcaaagg ctcttgccaa gtacggtgcc 120
caagtgccca cccgagttca agctgctgct gtcgctagtg gtgtggctac caacaaccca 180
gagccccagg atgtcgagta cctcactcct gttaagatcg gagacacaac attgcaactg 240
gactttgata ccggttcagc tgatctgtga gtaaagtcat gacttgacat tccgatgagc 300
tagcaaacta acttgtcaat aggtgggttt tctcgactga gcttccagag tcggaacagt 360
caggacactc tgtttatgac accagcagcg gtaacaagaa atctggttat acctggtcca 420
tttcctatgg cgacggtagc agtgccagcg gtgatgtcta cacagactct gtcaccgttg 480
gaggtattgc tgtcagtggt caggctgtcg aagccgcctc gaagatcagc accgagttca 540
cccaggacgc taacaacgac ggtcttctcg gtctggcgtt cagcagcatc aacactgtgt 600
cgccccagcc ccaaaagacc tggttcgata acgcccagtc acagctggcc tcccctctct 660
tcggagttgc tttgaagcac aatgcccctg gtgtttacga tttcggcttt gccgactctt 720
cgaagtatac cggtgatctg gcatacactg atgttgacaa ctcccaggga ttctggagct 780
tctctgttga cggctacaaa gctggtagca agtctggtgc tggattcgat ggtattgccg 840
acaccggtac cactctgctc cttctcgatg acgaaattgt ctcggcatac tactcccaag 900
tctcgggtgc ccaggaggac gccagtgctg gtggctatgt ctttgactgc agcaccactc 960
ttcctgactt cagtgttacc attggtgact acactgccac tgtccccggt gacttaatca 1020
acgctggctc ctctggcact ggctctggtt cttgcttcgg tggaatcgct tccaactcgg 1080
gtattggctt ctccatcttc ggggacatct tcttgaagag ccagtatgtc gttttcgatg 1140
ctagtggacc ccgtcttggc ttcgccaagc aggcttag 1178
SEQ ID NO.3：
<210> 3
<211> 56
<212> DNA
<213> Penicillium sp. XT7
<400> 3
agtaaagtca tgacttgaca ttccgatgag ctagcaaact aacttgtcaa taggtg 56
SEQ ID NO.4：
<210> 4
<211> 1122
<212> DNA
<213> 青霉素XT7
<400> 4
atggccgctg ctgccccaac ccaagccagc aagttctctc ttgagcaggt ctctcgcccg 60
gcttccaaga gctctaactt tgcttcgaag tatgcaaagg ctcttgccaa gtacggtgcc 120
caagtgccca cccgagttca agctgctgct gtcgctagtg gtgtggctac caacaaccca 180
gagccccagg atgtcgagta cctcactcct gttaagatcg gagacacaac attgcaactg 240
gactttgata ccggttcagc tgatctgtgg gttttctcga ctgagcttcc agagtcggaa 300
cagtcaggac actctgttta tgacaccagc agcggtaaca agaaatctgg ttatacctgg 360
tccatttcct atggcgacgg tagcagtgcc agcggtgatg tctacacaga ctctgtcacc 420
gttggaggta ttgctgtcag tggtcaggct gtcgaagccg cctcgaagat cagcaccgag 480
ttcacccagg acgctaacaa cgacggtctt ctcggtctgg cgttcagcag catcaacact 540
gtgtcgcccc agccccaaaa gacctggttc gataacgccc agtcacagct ggcctcccct 600
ctcttcggag ttgctttgaa gcacaatgcc cctggtgttt acgatttcgg ctttgccgac 660
tcttcgaagt ataccggtga tctggcatac actgatgttg acaactccca gggattctgg 720
agcttctctg ttgacggcta caaagctggt agcaagtctg gtgctggatt cgatggtatt 780
gccgacaccg gtaccactct gctccttctc gatgacgaaa ttgtctcggc atactactcc 840
caagtctcgg gtgcccagga ggacgccagt gctggtggct atgtctttga ctgcagcacc 900
actcttcctg acttcagtgt taccattggt gactacactg ccactgtccc cggtgactta 960
atcaacgctg gctcctctgg cactggctct ggttcttgct tcggtggaat cgcttccaac 1020
tcgggtattg gcttctccat cttcggggac atcttcttga agagccagta tgtcgttttc 1080
gatgctagtg gaccccgtct tggcttcgcc aagcaggctt ag 1122
SEQ ID NO.5：
<210> 5
<211> 27
<212> DNA
<213> 人工合成
<400> 5
cggaattcat ggccgctgct gccccaa 27
SEQ ID NO.6：
<210> 6
<211> 34
<212> DNA
<213> 人工合成
<400> 6
ttgcggccgc ctaagcctgc ttggcgaagc caag 34
Claims (6)
1.酸性蛋白酶PsAPA，其特征在于，酸性蛋白酶PsAPA的氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的酸性蛋白酶PsAPA，其特征在于，所述酸性蛋白酶PsAPA的DNA序列如SEQ ID NO.2所示，所述酸性蛋白酶PsAPA的cDNA序列如SEQ ID NO.4所示。
3.如权利要求2所述的酸性蛋白酶PsAPA的制备方法，其特征在于，包括以下步骤：
S1、首先提取Penicillium sp.XT7的总RNA序列，通过反转录获得Penicillium sp.XT7的cDNA序列，以Penicillium sp.XT7的cDNA序列为模板扩增酸性蛋白酶PsAPA的cDNA序列，然后将酸性蛋白酶PsAPA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-PsAPA；
S2、将重组表达载体pPIC9-PsAPA转化到毕赤酵母宿主细胞中，获得重组工程菌株；
S3、将重组工程菌株在30℃条件下培养2-3d，在甲醇诱导下使重组工程菌株表达生产出重组PsAPA的粗酶液；
S4、将粗酶液进行浓缩纯化，从而得到纯化后的重组酸性蛋白酶PsAPA。
4.如权利要求3所述的酸性蛋白酶PsAPA的制备方法，其特征在于，步骤S1中以Penicillium sp.XT7的cDNA为模板扩增酸性蛋白酶PsAPA的cDNA序列的具体包括以下步骤：
S1a、设计酸性蛋白酶PsAPA的特异性引物PsAPA_f核苷酸序列为：CGGAATTCATGGCCGCTGCTGCCCCAA，PsAPA_r核苷酸序列为：TTGCGGCCGCCTAAGCCTGCTTGGCGAAGCCAAG，并将设计好的PsAPA_f和PsAPA_r送至北京华大生物有限公司进行合成；
S1b、以反转录获得的Penicillium sp.XT7的cDNA为模板，以PsAPA_f，PsAPA_r为引物，进行PCR扩增以获取酸性蛋白酶PsAPA的cDNA序列。
5.如权利要求3所述的酸性蛋白酶PsAPA的制备方法，其特征在于，步骤S1中以Penicillium sp.XT7的cDNA序列为模板扩增酸性蛋白酶PsAPA的cDNA序列的条件为：95℃5min；94℃30s，60℃30s，72℃2min，35个循环；72℃10min。
6.如权利要求1所述的酸性蛋白酶PsAPA的应用，其特征在于，所述酸性蛋白酶PsAPA用于骨胶原蛋白提取的应用。
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| DE VRIES,R.P.: "Penicillopepsin [Penicillium subrubescens]，GenBank: OKP11845.1", 《NCBI GENBANK DATABASE》 * |
| 张春晖等: "组合酶水解鸡骨素工艺研究与水解液风味分析", 《晖 核农学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021177752A (ja) * | 2020-05-14 | 2021-11-18 | 中国農業科学院農産品加工研究所 | 低温酸性プロテアーゼＰｓＡＰＡ並びにその調製方法及び応用 |
| JP7008767B2 (ja) | 2020-05-14 | 2022-02-10 | 中国農業科学院農産品加工研究所 | 低温酸性プロテアーゼＰｓＡＰＡ並びにその調製方法及び応用 |
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| Publication number | Publication date |
|---|---|
| JP7008767B2 (ja) | 2022-02-10 |
| JP2021177752A (ja) | 2021-11-18 |
| CN111647584B (zh) | 2021-10-22 |
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