CN107828764A - 一种耐热半胱氨酸蛋白酶及其编码基因与应用 - Google Patents
一种耐热半胱氨酸蛋白酶及其编码基因与应用 Download PDFInfo
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- CN107828764A CN107828764A CN201711325176.4A CN201711325176A CN107828764A CN 107828764 A CN107828764 A CN 107828764A CN 201711325176 A CN201711325176 A CN 201711325176A CN 107828764 A CN107828764 A CN 107828764A
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Abstract
本发明公开一种耐热半胱氨酸蛋白酶及其编码基因与应用。本发明首先公开了耐热半胱氨酸蛋白酶是如下(a)或(b)所示的蛋白质:(a)由序列表SEQ ID NO.1所示的氨基酸序列组成的蛋白质;(b)由序列表SEQ ID NO.1所示的氨基酸序列经过一个或多个氨基酸残基的取代和/或缺失和/或插入得到的具有高温条件下蛋白降解活性的蛋白质。本发明进一步公开了所述耐热半胱氨酸蛋白酶在高温条件下降解蛋白的应用。本发明耐热半胱氨酸蛋白酶在75℃高温条件具有高效的蛋白质降解活性,且在85℃高温溶液中持续30分钟以上,仍保持高效催化活性。
Description
技术领域
本发明涉及酶工程领域。更具体地,涉及一种耐热半胱氨酸蛋白酶及其编码基因与应用。
背景技术
半胱氨酸蛋白酶(EC3.4.22)是一大类重要的蛋白水解酶,其催化活性位点含有亲核的Cys残基。目前得到大规模商业化应用的半胱氨酸蛋白酶主要来源于植物,如木瓜蛋白酶、菠萝蛋白酶等。通过对C1家族半胱氨酸蛋白酶的代表品种——木瓜蛋白酶(papain)的结构研究,其前体含有大小相当的2个结构域,活性位点位于2个结构域形成的裂隙底部。木瓜蛋白酶含有保守氨基酸残基组成的催化三体:Cys25-His159-Asn175,肽链中的Gln残基对酶活也发挥重要作用。木瓜蛋白酶可水解蛋白质和多肽中精氨酸和赖氨酸的羧基端,并能优先水解那些在肽键的N-端具有二个羧基的氨基酸或芳香L-氨基酸的肽键。
以木瓜蛋白酶为代表的半胱氨酸蛋白酶,以其较广的底物选择性,较宽的pH适应范围和良好的稳定性,得到了广泛的应用。例如,在皮革及纺织行业中,皮毛的软化一直是一个重要的步骤,而木瓜蛋白酶可以利用其使胶原蛋白部分降解的机理使得皮毛软化。有研究表明,木瓜蛋白酶以其对角蛋白的水解作用,可以改善毛料织物的染色性能或用于改进蚕丝纤维的质地。此外,还能作为羊毛纤维稳定剂,并且在纺织工业中可以作为毛料织物的防缩剂和整理剂等。
在医药行业中,C1半胱氨酸蛋白酶制剂是重要的外用药,可有效的除去腐烂组织而防止感染,一般被用于清理创口的坏死组织,消除水肿等,也在清除挫伤、烫伤等皮肤各种皮肤表面溃疡方面有着显著的作用。在食品行业中,木瓜蛋白酶被应用于肉类制品改性,通过对肌纤维蛋白的水解作用,改善肉质口感。在啤酒生产中,木瓜蛋白酶是良好的生物澄清剂。
作为C1半胱氨酸蛋白酶的主要商用品种,木瓜蛋白酶已经具有一定的热稳定性,在65℃的中高温条件下,具有较好的催化效率。但研究表明,木瓜蛋白酶持续的高温条件下(85℃以上,30分钟以上时间),活性会显著下降。这种高温条件下的热稳定性,对于酶制剂应用于连续生产,具有重要的意义。例如,在纺织印染行业的煮炼工艺中,引入蛋白酶制剂能够有助于降解纤维表面的丝胶蛋白,改善织物特性。但大规模自动化的煮炼生产作业,要求酶制剂在长时间的高温环境中保持高催化活力,现有的木瓜蛋白酶难以满足该方面的生产需求。
因此,开发一种具有耐受持续高温,并保持较高催化活力的蛋白酶制剂具有重要的应用价值。
发明内容
本发明的一个目的在于提供一种耐热半胱氨酸蛋白酶及其编码基因,该酶可以在高温条件下具有降解蛋白的活性。
本发明的另一个目的在于提供上述耐热半胱氨酸蛋白酶的应用。
为达到上述目的,本发明采用下述技术方案:
本发明提供一种耐热半胱氨酸蛋白酶,被命名为kospC1,来源于厌氧消化污泥微生物菌群(microbial community of anaerobic digestion sludge);所述耐热半胱氨酸蛋白酶是如下(a)或(b)所示的蛋白质:
(a)由序列表SEQ ID NO.1所示的氨基酸序列组成的蛋白质;
(b)由序列表SEQ ID NO.1所示的氨基酸序列经过一个或多个氨基酸残基的取代和/或缺失和/或插入得到的具有高温条件下蛋白降解活性的蛋白质。
其中,所述序列表SEQ ID NO.1所述氨基酸序列由391个氨基酸残基组成。
在本发明中上述耐热半胱氨酸蛋白酶的编码基因也属于本发明的保护范围,所述编码基因如(a)或(b)所示:
(a)如序列表SED ID NO.2所示的核苷酸序列;
(b)编码如序列表SED ID NO.1所示氨基酸序列的核苷酸序列。
其中,序列表SEQ ID NO.2所示的核苷酸序列由1173个碱基组成,其编码序列为自5’端第1到第1173位碱基,编码如序列表SED ID NO.1所示的氨基酸序列的蛋白质。
需要注意的是,含有本发明上述编码基因的表达载体、细胞系、工程菌及宿主菌均属于本发明的保护范围。
本发明还提供了一种表达上述耐热半胱氨酸蛋白酶的方法,是将含有上述耐热半胱氨酸蛋白酶编码基因的重组表达载体导入宿主细胞,表达得到耐热半胱氨酸蛋白酶。
其中,所述宿主可为大肠杆菌、酵母菌、哺乳动物、昆虫、枯草杆菌、芽孢杆菌或乳杆菌等,优选为酵母菌。
所述酵母菌优选为巴氏德毕赤酵母(Pichia pastoris),例如巴氏德毕赤酵母X33。
用于构建所述重组大肠杆菌表达载体及重组酵母菌表达载体的出发载体可为在上述宿主中表达外源基因的表达载体,如可在大肠杆菌中表达的pEB载体,以及在巴氏德毕赤酵母(Pichia pastoris)中表达的pPIC9K、pPIC9、pGAPza等。
上述重组表达载体均可按常规方法构建。
本发明还提供了所述耐热半胱氨酸蛋白酶在高温条件下降解蛋白的应用。
本发明进一步还提供了所述耐热半胱氨酸蛋白酶的编码基因在高温条件下降解蛋白的应用。
优选的,所述蛋白可以为牛血红蛋白、丝胶蛋白、胶原、明胶、酪蛋白、肌纤维蛋白等。
本发明的有益效果如下:
本发明耐热半胱氨酸蛋白酶在75℃高温条件具有高效的蛋白质降解活性,且在85℃高温溶液中持续30分钟以上,仍保持高效催化活性。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出重组质粒pGAPZa-kospC1的表达产物的SDS-PAGE图。
图2示出半胱氨酸蛋白酶与木瓜蛋白酶在不同加热预灭活处理后的活力比较。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
实施例1耐热半胱氨酸蛋白酶基因的获得及其重组表达
1、含耐热半胱氨酸蛋白酶微生物群落总DNA样本的提取
某明胶废水高温厌氧消化(UASB工艺)污泥样品,取样后液氮速冻,于-80℃保存备用。污泥样本经液氮研磨后,参照FAST DNA Spin kit for Soil(Qbiogene,美国)试剂盒提取总DNA。DNA样品经琼脂糖凝胶电泳和紫外分光光度计检测,浓度调至100ng/μL,-20℃保存。
2、耐热半胱氨酸蛋白酶基因的获得
以步骤1中获得的总DNA为模板,利用序列表SED ID NO.3所示的引物1和序列表SED ID NO.4所示的引物2进行PCR反应,扩增耐热半胱氨酸蛋白酶(kospC1)基因的序列。
引物1:5’-CTCGAGGGCTCTTTGATAGATGA-3’(其核苷酸序列如序列表SED ID NO.3所示,划线部分碱基为Xho I位点);
引物2:5’-GCGGCCGCTTAAGTTATGATGGTGTAA-3’(其核苷酸序列如序列表SED IDNO.4所示,划线部分碱基为Not I识别位点)
在PCR反应中,PCR反应条件为:94℃,保持5分钟,接着按以下的温度变化程序循环30次:升温至94℃,保持1分钟,降温至54℃,保持1分钟,升温至68℃,保持2分钟;然后于68℃,保持10分钟,最后于4℃保温10分钟,结束扩增反应。通过琼脂糖电泳分析获得约1.2kb的单一带,PCR产物检测之后经PCR产物纯化试剂盒纯化,并检测浓度。PCR产物经TA克隆,连接pMD18-T载体,转化大肠杆菌DH5α,获得重组质粒pMD18-kospC1测序鉴定。该耐热半胱氨酸蛋白酶基因DNA序列如序列表SEQ ID NO.2所示,其对应的氨基酸序列如序列表SEQ IDNO.1所示。
3、耐热半胱氨酸蛋白酶编码基因序列的重组表达载体的构建
所获得的PCR产物两端具有Xho I和Not I限制性内切酶位点,用Xho I和Not I限制性内切酶对PCR产物和质粒pGAPZAα同时进行双酶切反应。酶切体系50μL:目的片段或质粒20μL,10×Buffer 5μL,Xho I 2μL,Not I 2μL,ddH2O 21μL,酶切条件是37℃反应3h。酶切产物经柱回收后测序验证。目的片段和载体片段用T4DNA连接酶进行体外连接。连接反应体系10μL:目的片段5μL,pGAPZAα载体2μL 10×T4DNA连接缓冲液1μL,T4DNA连接酶(350U/μL)1μL,ddH2O 1μL,16℃连接过夜。连接产物转化大肠杆菌JM109,经卡那霉素抗性筛选,挑取菌落37℃振荡培养6-8h,分别进行PCR鉴定和重组质粒的酶切鉴定。获得的重组表达载体命名为后pGAPZAα-kospC1。对其进行测序,证明克隆连接的DNA序列与序列表SEQ ID NO.2所示序列相同,构建含有耐热半胱氨酸蛋白酶基因序列的重组表达载体pGAPZAα-kospC1正确无误。
4、耐热半胱氨酸蛋白酶在毕赤酵母中的表达
将重组表达载体pGAPZAα-kospC1用BspHI限制性内切酶酶切使之线性化后,采用电击方式,将线性化的载体pGAPZAα-kospC1导入毕赤酵母X33中,经选择性培养基培养,筛选抗博来霉素的高表达菌株。挑取选择性培养基上长出的单菌落接种于5ml YPD液体培养基(酵母膏10g/L、蛋白胨20g/L、葡萄糖20g/L)中,28℃培养12-24小时后,转入500mlBMGY培养基(1%酵母膏、2%蛋白胨、酵母氮源(YNB)1.34%、100mM磷酸缓冲液pH6.0、4×10-5生物素,1%甘油)中继续培养至菌液的OD600=2-3,补加葡萄糖诱导培养,并在此后每隔24小时补加葡萄糖至终浓度为1%,培养至120小时即可停止培养。离心收集上清,取15μL上清用SDS-PAGE电泳进行检测。结果如图1所示:泳道1为蛋白标准分子量marker;泳道2为表达产物,箭头表示目的条带,这表明重组菌株在葡萄糖诱导下表达的蛋白质的分子量约44KDa,与从氨基酸序列推段出的理论分子量(44kd)大小一致。
在毕赤酵母中表达的耐热半胱氨酸蛋白酶可以直接分泌到培养液的上清中,上清液中蛋白成分单一,可直接用于酶活性测定。
实施例2耐热半胱氨酸蛋白酶的活性检测(酪蛋白水解法)
1、原理:
目标底物酪蛋白,在适宜的缓冲条件下,经半胱氨酸蛋白酶水解,释放出游离酪氨酸或含酪氨酸的小肽进入反应溶液中。之后,反应体系内加入三氯乙酸沉淀大分子蛋白质(包括半胱氨酸蛋白酶和未反应的底物酪蛋白),通过福林法测定上清液中酪氨酸含量,可间接反映半胱氨酸蛋白酶的活力水平性的定义:在75℃条件下每分钟水解酪蛋白产生1μg酪氨酸定义为1个蛋白酶活力单位(1U)。
2、实验方法与结果:
2.1试验样品
半胱氨酸蛋白酶kospC1(来源于实施例1);市售木瓜蛋白酶(papain)。测试前,蛋白酶干粉均溶解于缓冲液(1.1mM EDTA,0.067mM巯基乙醇和5.5mM的半胱氨酸盐酸)室温孵育30min;
2.2方法与结果
设置三组预灭活实验,条件即:“未加热”、“30分钟85℃”、“60分钟85℃”,见图2标注;样品预灭活实验完成后,进行酶活性测定:
所有待测样品,均各自设置“实验管”、“空白对照管”反应试管,进行酶活测定,以消除误差。
具体地,“实验管”反应液中添加1ml底物酪蛋白(1%,w/v)和酶溶液1ml,“空白对照管”反应液中添加1ml底物酪蛋白(1%,w/v),“实验管”和“空白对照管”试管,同时放入75℃水浴中,水浴10分钟后,“实验管”,加入2ml三氯乙酸溶液(5%W/V),震荡混匀,终止反应。“空白对照组”试管,依次加入2ml三氯乙酸溶液(5%W/V),再添加1ml酶溶液,震荡混匀,终止反应。
实验管和空白对照管,均经高速离心去除蛋白沉淀后,静置10min,离心后,取上清液1mL于试管中,加入5mL 0.4mol/L Na2CO3溶液及1mL福林酚工作液混匀,40℃保温20min,测680mm处吸光度以比色法测定溶液中酪氨酸含量,记录680nm下吸光度,并以如下公式计算酶活。
酶活
其中:
A680Test:实验管吸光度
A680Blank:空白对照管吸光度
m:酶添加量(g)
t:反应时间(min)
r:标准酪蛋白福林法测定斜率
df:稀释倍数
测试结果如图2所示。未加热预灭活条件下,本发明耐热半胱氨酸蛋白酶kospC1在75℃具有良好的蛋白水解活性,活力可达120万U/g,与木瓜蛋白酶(papain)活性相当。
与之对应,在经受不同加热预灭活处理后(85℃水浴中处理30分钟、60分钟),本发明耐热半胱氨酸蛋白酶kospC1活力仍可保持在90万及70万U/g以上,酶的比活力显著高于经受同样加热处理的木瓜蛋白酶(papain),显示出优良的热稳定性,上述结果如图2所示。
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动,这里无法对所有的实施方式予以穷举,凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
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Claims (9)
1.一种耐热半胱氨酸蛋白酶,其特征在于,所述耐热半胱氨酸蛋白酶是如下(a)或(b)所示的蛋白质:
(a)由序列表SEQ ID NO.1所示的氨基酸序列组成的蛋白质;
(b)由序列表SEQ ID NO.1所示的氨基酸序列经过一个或多个氨基酸残基的取代和/或缺失和/或插入得到的具有高温条件下蛋白降解活性的蛋白质。
2.权利要求1所述耐热半胱氨酸蛋白酶的编码基因。
3.一种如权利要求2所述的编码基因,其特征在于,所述耐热半胱氨酸蛋白酶的编码基因如(a)或(b)所示:
(a)如序列表SED ID NO.2所示的核苷酸序列;
(b)编码如序列表SED ID NO.1所示氨基酸序列的核苷酸序列。
4.含有权利要求2或3所述的编码基因的表达载体、细胞系、工程菌或宿主菌。
5.一种表达权利要求1所述的耐热半胱氨酸蛋白酶的方法,是将含有权利要求2或3所述的编码基因的重组表达载体导入宿主细胞,表达得到耐热半胱氨酸蛋白酶。
6.根据权利要求5所述的方法,其特征在于,用于构建重组表达载体的出发载体为pEB、pPIC9K、pPIC9、pGAPza载体。
7.根据权利要求5所述的方法,其特征在于,所述宿主为大肠杆菌、酵母菌、哺乳动物、昆虫、枯草杆菌、芽孢杆菌或乳杆菌。
8.权利要求1所述耐热半胱氨酸蛋白酶在高温条件下降解蛋白的应用。
9.权利要求2或3所述耐热半胱氨酸蛋白酶的编码基因在高温条件下降解蛋白的应用。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110129303A (zh) * | 2019-05-06 | 2019-08-16 | 武汉轻工大学 | 一种耐高温酸性的果胶酶TsPec及基因与应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007075448A2 (en) * | 2005-12-15 | 2007-07-05 | Rutgers, The State University Of New Jersey | Method for producing plant extracts enriched with protease inhibitors for regulation of appetite and food intake in mammals |
CN101100659A (zh) * | 2007-07-06 | 2008-01-09 | 广西大学 | 一种β-葡萄糖苷酶及其编码基因与应用 |
CN101250514A (zh) * | 2008-04-11 | 2008-08-27 | 东华大学 | 一种化学试剂修饰木瓜蛋白酶的方法 |
CN101395269A (zh) * | 2002-10-10 | 2009-03-25 | 戴弗萨公司 | 蛋白酶、编码这些蛋白酶的核酸及它们的制备和应用方法 |
-
2017
- 2017-12-13 CN CN201711325176.4A patent/CN107828764B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101395269A (zh) * | 2002-10-10 | 2009-03-25 | 戴弗萨公司 | 蛋白酶、编码这些蛋白酶的核酸及它们的制备和应用方法 |
WO2007075448A2 (en) * | 2005-12-15 | 2007-07-05 | Rutgers, The State University Of New Jersey | Method for producing plant extracts enriched with protease inhibitors for regulation of appetite and food intake in mammals |
CN101100659A (zh) * | 2007-07-06 | 2008-01-09 | 广西大学 | 一种β-葡萄糖苷酶及其编码基因与应用 |
CN101250514A (zh) * | 2008-04-11 | 2008-08-27 | 东华大学 | 一种化学试剂修饰木瓜蛋白酶的方法 |
Non-Patent Citations (3)
Title |
---|
GENBANK: "hypothetical protein [Kosmotoga arenicorallina],NCBI Reference Sequence:WP_068347331.1", 《GENBANK》 * |
H.A.SATHISH ET AL.: "Mechanism of solvent induced thermal stabilization of papain", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
乙引等: "木瓜蛋白酶应用开发的性质研究", 《贵州师范大学学报(自然科学版)》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110129303A (zh) * | 2019-05-06 | 2019-08-16 | 武汉轻工大学 | 一种耐高温酸性的果胶酶TsPec及基因与应用 |
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