CN112795585B - 一种改进的耐热蜡样芽孢杆菌胶原酶的制备方法 - Google Patents
一种改进的耐热蜡样芽孢杆菌胶原酶的制备方法 Download PDFInfo
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Abstract
本发明涉及一种改进的耐热蜡样芽孢杆菌胶原酶的制备方法，通过引入X到Y个突变对原有蜡样芽孢杆菌胶原酶序列进行改进，所述蜡样芽孢杆菌胶原酶突变体序列为SEQ ID NO7的酶在保持酶活的同时且热稳定性明显提高，因此具备更好的应用前景。
Description
技术领域
本发明涉及基因工程和微生物领域，更具体地说是涉及一种改进的耐热蜡样芽孢胶原酶的制备方法，以及它的一些表征。
背景技术
胶原酶通常被认为是特异性水解天然胶原蛋白和水溶性变性胶原蛋白的酶。其中微生物胶原酶实际上是一种金属内肽酶，其依靠于Zn2+、Ca2+等离子发挥活性。与动物胶原酶相比，微生物胶原酶底物种类更广泛；水解底物更彻底，有的微生物胶原酶可以与胶原蛋白的多个位点发生反应，产生大小均匀的5个残基的短肽，但是动物胶原酶只能与胶原蛋白N端3/4处Gly-Leu或Gly-lle肽键发生反应，产生一个3/4肽段和1/4肽段；微生物胶原酶还更容易获得，因为微生物可以将胶原酶分泌到胞外，通过发酵可以大量生产，而动物胶原酶还需要进行组织培养和提取，工序繁琐且成本很高。
如今，微生物胶原酶在医药领域，食品、皮革等工业有着广泛的应用。其可直接应用于临床治疗，针对掌筋膜挛缩症、青光眼、腰椎间盘突出等疾病有着良好的治疗效果；胶原酶作为制备胶原多肽的主要成分之一，对食品的生产也做出了巨大贡献；胶原酶作为化学加工中的化学试剂替代品在皮革工业中有很多潜在的应用前景。因为它的加入不仅可以生产出质量更好的产品，而且减少了危险和污染化学品的使用。
科学界已经从一些微生物中纯化出了微生物胶原酶，并且已经克隆了它们的相应基因并对其进行了测序，但我国在微生物胶原酶方面的研究与应用还处于刚刚起步阶段，国内市场依旧依赖大量高价进口胶原酶产品，限制了微生物胶原酶的利用。在理想的情况下工业中使用的胶原酶需要活性高、热稳定性好等特点，从而加快反应速率，减少反应成本，因此提高酶的热稳定性成了研究的热点。
发明内容
本发明的目的是利用基因工程技术和微生物技术，设计一种改进的耐热蜡样芽孢杆菌胶原酶，提高胶原酶的热稳定性。
为实现上述目的，本发明采用技术方案的主要原理是：
含有X到Y个突变的蜡样芽孢杆菌胶原酶序列，其特征在于，所述蜡样芽孢杆菌胶原酶突变体序列为SEQ ID NO1、SEQ ID NO2、SEQ ID NO3、SEQ ID NO4、SEQ ID NO5、SEQ IDNO6和SEQ ID NO7，分别包含了8、10、12、15、23、31、39个氨基酸序列突变。
进一步地，根据同源模型分子能量表得到能量最低，理论上结构最稳定的蜡样芽孢杆菌胶原酶突变体序列为：SEQ ID NO7。
进一步地，一种包含蜡样芽孢杆菌胶原酶突变体序列SEQ ID NO7的原核表达系统载体。
进一步地，其所述原核表达系统为大肠杆菌。
进一步地，其所述原核表达系统为大肠杆菌，其表达载体为pET系列载体。
进一步地，其所述原核表达系统为大肠杆菌，表达载体为pET21a载体。
以及利用金属离子亲和层析纯化蜡样芽孢杆菌胶原酶突变体的方法，具体步骤如下：
步骤1：利用Rosetta软件设计好改进后的蜡样芽孢杆菌胶原酶氨基酸序列SEQ IDNO7，通过PCR扩增技术合成蜡样芽孢杆菌胶原酶目的基因，与pET-21a载体组建成为质粒。
步骤2：利用热激法将目的基因转入大肠杆菌感受态细胞中，利用氨苄青霉素对菌落进行筛选。
步骤3：将筛选出的含有目的基因的大肠杆菌通过摇床悬浮培养，并在菌体OD600值达到0.6-0.8时加入IPTG诱导剂诱导其表达。
步骤4：以8000r/min的速度离心收集菌体沉淀并破碎，接着以10000r/min的速度离心得到上清液。
步骤5：用超纯水清洗蛋白纯化镍柱，再将5倍柱体积的缓冲液1清洗纯化柱。随后将破碎过滤后的菌液上样到蛋白纯化镍柱。上样结束后，用5倍柱体积的缓冲液2、缓冲液3先后洗涤蛋白纯化柱，最后用缓冲液4洗涤并收集洗脱下来的蛋白液。其中缓冲液1的组成为0.02M咪唑含0.1M NaCl，0.02M Tris-HCl pH＝8；缓冲液2的组成为0.05M咪唑含0.1M氯化钠，0.02M Tris-HCl pH＝8)；缓冲液3的组成为0.1M咪唑含0.1M氯化钠，0.02M Tris-HClpH＝8；缓冲液4的组成为0.5M咪唑含0.1M氯化钠，0.02M Tris-HCl pH＝8。
与现有技术相比，本发明具有以下优点：
1)本发明选取微生物来源的胶原酶，相比动物来源的胶原酶它水解底物更彻底，更容易获得，而动物胶原酶还需要进行组织培养和提取，工序繁琐且成本很高。
2)本发明采用基因克隆的技术使菌株所产的胶原酶在大肠杆菌的表达系统中进行更为高效的表达。
3)本发明经过基因转化、蛋白的诱导表达、金属离子亲和层析纯化等生物技术得到的胶原酶纯度相对更高。
4)本发明通过引入突变对原有蜡样芽孢杆菌胶原酶序列进行改进，得到了热稳定性较好的胶原酶。
5)本发明纯化出的胶原酶后续可以使用到胶原蛋白的水解中，得到在美容、医药等领域应用很广泛的胶原蛋白多肽，相比酸法碱法水解污染少且安全可靠，而且水解的胶原蛋白还包括了皮革固废物中生皮边角料的胶原蛋白，实现了皮革废弃物的资源化利用。
附图说明
图1为胶原酶的电泳检测结果。
图2为改进前后胶原酶的最适温度对比。
具体实施方式
下面结合具体实施例对本发明作进一步详细说明，但是本发明不局限于以下实施例。
根据同源模型分子能量表得到能量最低，理论上结构最稳定的蜡样芽孢杆菌胶原酶突变体序列为SEQ ID NO7，故本发明各实施例基于序列7实施，含突变序列的蜡样芽孢杆菌胶原酶由PCR扩增技术合成得到。
本发明的实施例中所用培养基及溶液的配置如下：
1L LB液体培养基的配制方法：称取10g NaCl、10g胰蛋白胨、5g酵母浸出粉溶于1L去离子水。
1L LB固体培养基的配制方法：称取10g NaCl、10g胰蛋白胨、5g酵母浸出粉、6.5g琼脂溶于1L去离子水。
下层分离胶12％(10mL)：用移液枪吸取去离子水3.4mL、30％ACr-Bis(29:1)4mL、下层胶缓冲液(4x)2.5mL、10％凝胶聚合催化剂(称0.1g溶于900μL去离子水)0.1mL、TEMED6μL。
上层浓缩胶5％(2mL)：用移液枪吸取去离子水2.33mL，30％ACr-Bis(29:1)0.67mL、上层胶缓冲液(4x)1mL、10％凝胶聚合催化剂(称0.1g溶于900μL水)40μL、TEMED 2μL。
电极缓冲液：称取甘氨酸14.4g，SDS1g，tris 3g，加去离子水定容至1L。
5x上样缓冲液：用移液枪吸取50％甘油10mL，1mol/L Tris-Hcl(pH＝6.8)4mL，β-巯基乙醇1mL，10％ SDS2mL，1％溴酚蓝1.2mL，加去离子水定容至20mL。
染色液：称取考马斯亮蓝R-250 0.25g溶解于91mL甲醇+9mL冰乙酸中。
脱色液：量取甲醇50mL，冰乙酸100mL，加去离子水定容至1L。
实施例1：
将重组质粒溶于超纯水中，使终浓度为100ng/μl，吸取10μl溶解后的质粒加入到感受态大肠杆菌中，同时吸取10μl去离子水加入到另一支感受态大肠杆菌中做空白，共同冰浴30min，接着放入42℃水浴锅水浴90s，然后冰浴3min，最后分别加入1mL液体培养基至感受态大肠杆菌中放入恒温摇床37℃，200r/min恢复1h。各取200μl转化产物均匀涂布在含100μg/mL氨苄青霉素的固体培养基上，37℃倒置平板培养12h，筛选固体平板长出菌落即为含有胶原酶的大肠杆菌菌株。挑取该菌株接种至10mL含100μg/mL的液体培养基中培养，培养条件为37℃，200r/min，培养18h。培养结束后分装至1.5mL无菌离心管内，加入适量80％甘油，保存于-80℃冰箱中。
实施例2：
取一管分装好的菌液，将其加入40mL含100μg/mL氨苄青霉素的液体培养基中，在37℃，180r/min条件下培养12-24h。将培养好的液体继续转入1L含100μg/mL氨苄青霉素的液体培养基中，在37℃下培养至OD600达到0.6-0.8时，加入配好的IPTG溶液，在26℃下诱导10h。诱导后得到的培养液于8000r/min，4℃的条件下离心15min收集菌体，弃上清液，随后用5-10倍体积的缓冲液溶解。将溶解后的菌液超声破碎，超声的功率为400W破碎，超声2s，间歇2s。将超声破碎后的菌液于10000r/min，4℃下离心10min，收集上清液，将上清液用0.45μm的膜过滤，收集滤液于-20℃冰箱中保存。
实施例3：
用超纯水清洗蛋白纯化镍柱，再将5倍柱体积的缓冲液1清洗纯化柱。随后将破碎过滤后的菌液上样到蛋白纯化镍柱。上样结束后，用5倍柱体积的缓冲液2、缓冲液3先后洗涤蛋白纯化柱，最后用缓冲液4洗涤并收集洗脱下来的蛋白液。其中缓冲液1的组成为0.02M咪唑含0.1M NaCl，0.02M Tris-HCl pH＝8；缓冲液2的组成为0.05M咪唑含0.1M氯化钠，0.02M Tris-HCl pH＝8)；缓冲液3的组成为0.1M咪唑含0.1M氯化钠，0.02M Tris-HCl pH＝8；缓冲液4的组成为0.5M咪唑含0.1M氯化钠，0.02M Tris-HCl pH＝8。
实施例4：
本发明采取垂直板电泳槽，将两块长短不一、表面洁净干燥的玻璃板对齐压紧后用夹子夹紧固定在支架上，以免漏胶。
1)下层注入12％分离胶：将分离胶用枪头加至长、短玻璃板之间的缝隙内，大约留出距离玻璃板上方2cm处再用去离子水进行水封，消泡的同时压平胶面避免杂质进入。
2)凝胶：大概30min分离胶聚合后，倒出水，用滤纸条吸干胶表面的水分。
3)上层注入5％浓缩胶：在聚合的分离胶上层注入浓缩胶，立即在浓缩胶溶液中插入干净的梳子，避免带入气泡，室温静置至浓缩胶完全聚合后。
4)标准蛋白样品的制备：用微量进样针吸取标准蛋白质样品Maker 5-10μL至小离心管，准备进样。
5)待测样品的制备：用移液枪吸取待测样品40μL和5X样品缓冲液10μL混合至小离心管，在沸水浴中加热5-10min，取出冷却至室温，准备进样。
6)加样：平稳取出聚合后浓缩胶中的梳子，安装凝胶板到电泳槽内，然后在电泳槽中加满电极缓冲液，注意密封性，用微量进样针按顺序向加样孔内上样，加样量通常为10-15μL。
7)跑胶：电泳接上电泳仪，打开电泳仪电源开关，将电压调至70V，待溴酚蓝染料前沿进入分离胶后，把电压提高到120V，继续电泳直至溴酚蓝移至下端约1-1.5cm，关闭电源停止电泳。
8)染色：将胶慢慢剥离至容器中，加入染色液染色2h。
9)脱色：倒入脱色液，数小时更换一次直至背景接近无色即可。脱色后，可将凝胶浸于水中保存，以备观察。
实施例5：
甘氨酸标准曲线制作
还原茚三酮：取0.5g水合茚三酮用12.5mL沸水溶解。称0.5g抗坏血酸用25mL温水溶解，缓慢加入溶好的水合茚三酮溶液中，边搅拌边加入，静置沉淀，抽滤真空干燥，制备出还原茚三酮，密封保存。显色剂：称取85mg水合茚三酮和15mg还原茚三酮，用10mL乙二醇甲醚溶解。
pH 5.4乙酸缓冲液：量取86mL 2mol/L醋酸钠溶液，加入14mL2mol/L乙酸混合(pH计校正)
方法：取7只试管分成两组，分别加入0、0.1、0.2、0.4、0.6、0.8、1.0mL 0.3mmol/L标准甘氨酸溶液，用水补足到1mL。1mL含0.03-0.3μmol/L的甘氨酸溶液与1mL 2mol/L pH5.4的乙酸缓冲液充分混合，然后加入1mL茚三酮显色液，充分混合，用试管盖盖住，在100℃水浴中加热15min，用自来水冷却，放置15min，加入3mL60％乙醇稀释，用紫外分光光度计于570nm处测量吸光度。以1mL水代替甘氨酸作空白。
实施例6：
在胶原蛋白中分别加入改进前后的胶原酶，在不同反应温度(30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃和70℃)条件下测定其水解液中氨基酸含量，得到酶活以确定胶原酶的最适反应温度。以最适温度下确定的酶活为100％，其余温度下测定的酶活与之相比得到相对酶活。
实施例7：
水解液中甘氨基酸含量的测定：准备1mL酶解溶液，加入1mL2mol/L pH 5.4的乙酸缓冲液和1mL茚三酮显色液，充分混合，在沸水浴中加热15min，冷却后加入3mL 60％乙醇稀释，于570nm测量吸光度。根据所测的吸光值，在标准曲线上即可查得相应氨基酸含量(μmol/L)。
测试部分
按实施例4的步骤得到的结果如图1所示，泳道1代表改进后胶原酶电泳检测结果。结果表明，利用SDS-PAGE电泳检测得到了分子质量约为45kDa的单一蛋白条带，与设计的序列分子大小基本一致,证明蜡样芽孢杆菌胶原酶在大肠杆菌中成功表达纯化。按照实施例6得到改进前后胶原酶的最适温度对比如图2所示。结果表明改进前的胶原酶活力在30℃-50℃之间随着反应温度的升高而逐渐升高，至50℃，酶活达到最大；在55℃仍可以保持90％左右的酶活力。然而当继续升高温度大于55℃之后，胶原酶活力迅速下降并最终失活，可能是因为高温破坏了酶的空间结构，导致酶的活力下降。因此，改进前胶原酶的最适反应温度为50℃。而改进后的胶原酶在30℃-55℃之间随着反应温度的升高而逐渐升高，至55℃，酶活达到最大；在60℃仍可以保持80％左右的酶活力。然而当继续升高温度大于60℃之后，胶原酶活力迅速下降并最终失活。因此，改进前胶原酶的最适反应温度为55℃。证明改进后的胶原酶的热稳定性有了一定的提高。
序列表
<110> 陕西科技大学
<120> 一种改进的耐热蜡样芽孢杆菌胶原酶的制备方法
<141>
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Pro Glu Gln Ser Ile Tyr Ser Leu Glu Glu Leu Phe Arg His Glu Phe
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Tyr Ser Phe Ala Leu Gln Ser Tyr Leu Tyr Thr His Gln Phe Glu Thr
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Phe Asp Lys Ile Gln Asp Leu Ile Arg Ala Asn Asp Val Lys Gly Tyr
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Thr Tyr Thr Phe Asp Asp Gly Ser Ile Val Phe Lys Thr Gly Asp Lys
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Val Ser Glu Glu Lys Ile Lys Arg Leu Tyr Trp Ala Ala Lys Glu Val
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Gly Ile Tyr Ile Glu Glu Thr Gly Thr Phe Phe Thr Tyr Glu Arg Thr
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Pro Glu Gln Ser Ile Tyr Ser Leu Glu Glu Leu Phe Arg His Glu Phe
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Thr His Tyr Leu Gln Gly Arg Tyr Leu Val Pro Gly Leu Phe Gly Arg
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Lys Ser Ile Ile Ser Gly Leu Ser Ser Asp Pro Ala Ser Arg Tyr Thr
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Ala Glu Arg Thr Leu Phe Ala Lys Tyr Gly Ser Trp Asp Phe Tyr Asn
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Tyr Ser Phe Ala Leu Gln Ser Tyr Leu Tyr Thr His Gln Phe Glu Thr
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Phe Asp Lys Ile Gln Asp Leu Ile Arg Ala Asn Asp Val Lys Gly Tyr
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305 310 315 320
Arg Gly Thr Tyr Thr Gly Ser Val Thr Lys Gly Glu Ile Glu Asp Trp
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Val Ser Glu Glu Lys Ile Lys Arg Leu Tyr Trp Ala Ala Lys Glu Val
35 40 45
Lys Ala Gln Phe His Arg Val Ile Gly Asn Asp Lys Pro Leu Glu Pro
50 55 60
Gly Asn Ala Asp Asp Val Leu Thr Ile Val Ile Tyr Asn Ser Pro Asp
65 70 75 80
Glu Tyr Gln Leu Asn Arg Gln Leu Tyr Gly Tyr Glu Thr Asn Asn Gly
85 90 95
Gly Ile Tyr Ile Glu Glu Thr Gly Thr Phe Phe Thr Tyr Glu Arg Thr
100 105 110
Pro Glu Gln Ser Ile Tyr Ser Leu Glu Glu Leu Phe Arg His Glu Phe
115 120 125
Thr His Tyr Leu Gln Gly Arg Tyr Leu Val Pro Gly Leu Phe Gly Arg
130 135 140
Gly Asp Met Tyr Gln Asn Glu Arg Leu Thr Trp Phe Gln Glu Gly Asn
145 150 155 160
Ala Glu Phe Phe Ala Gly Ser Thr Arg Lys Asn Asn Val Val Pro Arg
165 170 175
Lys Ser Ile Ile Ser Gly Leu Ser Ser Asp Pro Ala Ser Arg Tyr Thr
180 185 190
Ala Glu Arg Thr Leu Phe Ala Lys Tyr Gly Ser Trp Asp Phe Tyr Asn
195 200 205
Tyr Ser Phe Ala Leu Gln Ser Tyr Leu Tyr Thr His Gln Phe Glu Thr
210 215 220
Phe Asp Lys Ile Gln Asp Leu Ile Arg Ala Asn Asp Val Lys Gly Tyr
225 230 235 240
Asp Ala Tyr Arg Glu Asn Leu Ser Lys Asp Pro Asn Leu Asn Lys Glu
245 250 255
Tyr Gln Glu Tyr Met Gln Gln Leu Ile Asp Asn Gln Asp Lys Tyr Asn
260 265 270
Val Pro Glu Val Ser Asp Asp Tyr Leu Ala Glu His Ala Pro Lys Ser
275 280 285
Leu Thr Glu Val Lys Lys Glu Ile Ser Lys Thr Leu Pro Met Lys Asp
290 295 300
Thr Lys Met Thr Lys His Lys Ser Gln Phe Phe Asn Thr Phe Thr Leu
305 310 315 320
Arg Gly Thr Tyr Thr Gly Ser Val Thr Lys Gly Glu Ile Glu Asp Trp
325 330 335
Lys Asp Met Ser Lys Arg Ile Asn Glu Ser Leu Glu Gln Leu Ala Gln
340 345 350
Lys Glu Trp Ser Gly Tyr Lys Thr Val Thr Ala Tyr Phe Val Asn Tyr
355 360 365
Arg Val Asn Ser Asn Asn Gln Phe Glu Tyr Asp Val Val Phe His Gly
370 375 380
Ile Ala Lys Asp Asp Gly
385 390
<210> 5
<211> 390
<212> PRT
<213> Artificial Sequence
<400> 5
Asp Asp Glu Lys Ile Arg Glu Glu Gly Lys Glu Gln Tyr Leu Pro Lys
1 5 10 15
Thr Tyr Thr Phe Asp Asp Gly Ser Ile Val Phe Lys Thr Gly Asp Lys
20 25 30
Val Ser Glu Glu Lys Ile Lys Arg Leu Tyr Trp Ala Ala Lys Glu Val
35 40 45
Lys Ala Gln Phe His Arg Val Ile Gly Asn Asp Lys Pro Leu Glu Pro
50 55 60
Gly Asn Ala Asp Asp Val Leu Thr Ile Val Ile Tyr Asn Ser Pro Asp
65 70 75 80
Glu Tyr Gln Leu Asn Arg Gln Leu Tyr Gly Tyr Glu Thr Asn Asn Gly
85 90 95
Gly Ile Tyr Ile Glu Glu Thr Gly Thr Phe Phe Thr Tyr Glu Arg Thr
100 105 110
Pro Glu Gln Ser Ile Tyr Ser Leu Glu Glu Leu Phe Arg His Glu Phe
115 120 125
Thr His Tyr Leu Gln Gly Arg Tyr Leu Val Pro Gly Leu Phe Gly Arg
130 135 140
Gly Asp Met Tyr Gln Asn Glu Arg Leu Thr Trp Phe Gln Glu Gly Asn
145 150 155 160
Ala Glu Phe Phe Ala Gly Ser Thr Arg Lys Asn Asn Val Val Pro Arg
165 170 175
Lys Ser Ile Ile Ser Gly Leu Ser Ser Asp Pro Ala Ser Arg Tyr Thr
180 185 190
Ala Glu Arg Thr Leu His Ala Lys Tyr Gly Ser Trp Asp Phe Tyr Asn
195 200 205
Tyr Ser Phe Ala Leu Gln Ser Tyr Leu Tyr Thr His Asp Phe Glu Thr
210 215 220
Phe Asp Lys Ile Gln Asp Leu Ile Arg Ala Asn Asp Val Lys Gly Tyr
225 230 235 240
Asp Ala Tyr Arg Glu Lys Leu Ser Lys Asp Pro Asn Leu Asn Lys Glu
245 250 255
Tyr Gln Glu Tyr Met Gln Gln Leu Ile Asp Asn Gln Asp Lys Tyr Asn
260 265 270
Val Pro Gln Val Ser Asp Asp Tyr Leu Ala Glu His Ala Pro Lys Ser
275 280 285
Leu Asp Glu Val Lys Lys Glu Ile Ser Lys Thr Leu Pro Met Lys Asp
290 295 300
Thr Lys Met Thr Lys His Lys Ser Gln Phe Phe Asn Thr Phe Thr Leu
305 310 315 320
Arg Gly Thr Tyr Thr Gly Ser Val Thr Lys Gly Arg Ile Glu Asp Trp
325 330 335
Lys Asp Met Ser Lys Arg Ile Asn Glu Ile Leu Glu Gln Leu Ala Gln
340 345 350
Lys Glu Trp Ser Gly Tyr Lys Thr Val Thr Ala Tyr Phe Val Asn Tyr
355 360 365
Arg Val Asn Ser Asn Asn Gln Phe Glu Tyr Asp Val Val Phe His Gly
370 375 380
Ile Ala Lys Asp Asp Gly
385 390
<210> 6
<211> 390
<212> PRT
<213> Artificial Sequence
<400> 6
Asp Asp Glu Lys Ile Arg Glu Glu Gly Lys Glu Gln Tyr Leu Pro Lys
1 5 10 15
Thr Tyr Thr Phe Asp Asp Gly Ser Ile Val Phe Lys Thr Gly Asp Lys
20 25 30
Val Ser Glu Glu Lys Ile Gln Arg Leu Tyr Trp Ala Ala Lys Glu Val
35 40 45
Lys Ala Gln Phe His Arg Val Ile Gly Asn Asp Lys Pro Leu Glu Pro
50 55 60
Gly Asn Ala Asp Asp Val Leu Thr Ile Val Ile Tyr Asn Ser Pro Asp
65 70 75 80
Glu Tyr Gln Leu Asn Arg Gln Leu Tyr Gly Tyr Glu Thr Asn Asn Gly
85 90 95
Gly Ile Tyr Ile Glu Glu Thr Gly Thr Phe Phe Thr Tyr Glu Arg Thr
100 105 110
Pro Glu Gln Ser Ile Tyr Ser Leu Glu Glu Leu Phe Arg His Glu Phe
115 120 125
Thr His Tyr Leu Gln Gly Arg Tyr Leu Val Pro Gly Leu Phe Gly Arg
130 135 140
Gly Asp Met Tyr Gln Asn Glu Arg Leu Thr Trp Phe Gln Glu Gly Asn
145 150 155 160
Ala Glu Phe Phe Ala Gly Ser Thr Arg Thr Asn Asn Val Val Pro Arg
165 170 175
Lys Ser Ile Ile Ser Gly Leu Ser Ser Asp Pro Ala Asn Arg Tyr Thr
180 185 190
Ala Glu Gln Leu Leu His Ala Lys Tyr Gly Ser Trp Asp Phe Tyr Asn
195 200 205
Tyr Ser Phe Ala Leu Gln Ser Tyr Leu Tyr Asn His Asn Phe Glu Thr
210 215 220
Phe Asp Lys Ile Gln Asp Leu Ile Arg Ala Asn Asp Val Lys Gly Tyr
225 230 235 240
Asp Ala Tyr Arg Glu Lys Leu Ser Lys Asp Pro Asn Leu Asn Lys Glu
245 250 255
Tyr Gln Glu Tyr Met Gln Gln Leu Ile Asp Asn Arg Asp Lys Tyr Thr
260 265 270
Val Pro Leu Val Ser Asp Asp Tyr Leu Ala Glu His Ala Pro Lys Ser
275 280 285
Leu Asp Glu Val Lys Lys Glu Ile Glu Lys Thr Leu Pro Met Lys Asn
290 295 300
Thr Lys Met Thr Lys His Lys Ser Gln Phe Phe Asn Thr Phe Thr Leu
305 310 315 320
Arg Gly Thr Tyr Thr Gly Ser Val Thr Lys Gly Arg Ile Glu Asp Trp
325 330 335
Lys Asp Met Ser Lys Arg Val Asn Glu Ile Leu Glu Gln Leu Ala Gln
340 345 350
Lys Glu Trp Ser Gly Tyr Lys Thr Val Thr Ala Tyr Phe Val Asn Tyr
355 360 365
Arg Val Asn Ser Asn Asn Gln Phe Glu Tyr Asp Val Val Phe His Gly
370 375 380
Ile Ala Thr Asp Asp Gly
385 390
<210> 7
<211> 390
<212> PRT
<213> Artificial Sequence
<400> 7
Asp Asp Glu Lys Ile Arg Glu Glu Gly Lys Lys Lys Tyr Leu Pro Lys
1 5 10 15
Thr Tyr Thr Phe Asp Asp Gly Ala Ile Val Phe Lys Thr Gly Asp Lys
20 25 30
Val Ser Glu Glu Lys Ile Gln Arg Leu Tyr Trp Ala Ala Lys Glu Val
35 40 45
Lys Ala Gln Phe His Arg Val Ile Gly Asn Asp Lys Pro Leu Glu Pro
50 55 60
Gly Asn Pro Asp Asp Val Leu Thr Ile Val Ile Tyr Asn Ser Pro Asp
65 70 75 80
Glu Tyr Lys Leu Asn Arg Gln Leu Tyr Gly Tyr Glu Thr Asn Asn Gly
85 90 95
Gly Ile Tyr Ile Glu Glu Thr Gly Thr Phe Phe Thr Tyr Glu Arg Thr
100 105 110
Pro Glu Gln Ser Ile Tyr Ser Leu Glu Glu Leu Phe Arg His Glu Phe
115 120 125
Thr His Tyr Leu Gln Gly Arg Tyr Leu Val Pro Gly Leu Phe Gly Arg
130 135 140
Gly Asp Met Tyr Gln Asn Glu Arg Leu Thr Trp Phe Gln Glu Gly Asn
145 150 155 160
Ala Glu Phe Phe Ala Gly Ser Thr Arg Lys Asn Asn Val Val Pro Arg
165 170 175
Lys Ser Ile Ile Ser Gly Leu Ser Ser Asp Pro Ala Asn Arg Tyr Thr
180 185 190
Ala Glu Gln Leu Leu His Ala Lys Tyr Gly Ser Trp Asp Phe Tyr Asn
195 200 205
Tyr Ser Phe Ala Leu Gln Ser Tyr Leu Tyr Asn His Asn Phe Asp Thr
210 215 220
Phe Asp Lys Ile Gln Asp Leu Ile Arg Ala Asn Asp Val Lys Gly Tyr
225 230 235 240
Asp Ala Tyr Arg Glu Lys Leu Ser Lys Asp Pro Asn Leu Asn Lys Glu
245 250 255
Tyr Gln Glu Tyr Met Gln Gln Leu Ile Asp Asn Arg Asp Asn Tyr Thr
260 265 270
Val Pro Gln Val Ser Asp Asp Tyr Leu Ala Glu His Ala Pro Lys Ser
275 280 285
Leu Asp Glu Val Lys Lys Glu Ile Glu Lys Thr Leu Pro Met Lys Asn
290 295 300
Thr Lys Met Thr Lys His Lys Ser Gln Phe Phe Asn Thr Phe Thr Leu
305 310 315 320
Arg Gly Thr Tyr Thr Gly Ser Val Thr Lys Gly Lys Ser Glu Asp Trp
325 330 335
Lys Asp Met Ser Lys Arg Ile Asn Glu Phe Leu Glu Gln Leu Ala Gln
340 345 350
Lys Glu Trp Ser Gly Tyr Lys Thr Val Thr Ala Tyr Phe Val Asn Tyr
355 360 365
Arg Val Asn Ser Asn Asn Gln Phe Glu Tyr Asp Val Val Phe His Gly
370 375 380
Ile Ala Thr Asp Asp Gly
385 390
Claims (7)
1.一种包含蜡样芽孢杆菌胶原酶突变体的原核表达系统载体，其特征在于，所述蜡样芽孢杆菌胶原酶突变体氨基酸序列为SEQ ID NO7。
2.如权利要求1所述的载体，其特征在于，所述原核表达系统为大肠杆菌，表达载体为pET系列载体。
3.如权利要求2所述的载体，其特征在于，表达载体为pET21a载体。
4.权利要求1-3任一项所述的载体的制备方法，其特征在于，包括如下步骤：
通过PCR扩增技术合成氨基酸序列为SEQ ID NO7的蜡样芽孢杆菌胶原酶基因，将前述基因与pET-21a载体组建成为质粒。
5.一种改进的耐热蜡样芽孢杆菌胶原酶的制备方法，其特征在于，包括如下步骤：
利用热激法将权利要求1-3任一项所述的载体转化到大肠杆菌感受态细胞中，利用氨苄青霉素对菌落进行筛选，通过摇床悬浮培养，在菌体OD600值达到0.6-0.8时加入IPTG诱导剂诱导其表达；离心收集菌体沉淀并破碎，再次离心收集上清液；将破碎过滤后的菌液上清液经蛋白纯化柱洗脱纯化，得到改进的耐热蜡样芽孢杆菌胶原酶。
6.如权利要求5所述的方法，其特征在于，包括如下步骤：
1）将权利要求1-3任一项所述的载体转化到大肠杆菌BL21(DE)3中，利用氨苄青霉素对菌落进行筛选，通过摇床悬浮培养，在菌体OD600值达到0.6-0.8时加入IPTG诱导剂诱导其表达；
2）收集菌体沉淀并破碎得到上清液；
3）用超纯水清洗蛋白纯化镍柱，再将5倍柱体积的缓冲液1清洗纯化柱，随后将破碎离心后的菌液上样到蛋白纯化镍柱；上样结束后，用5倍柱体积的缓冲液2、缓冲液3先后洗涤蛋白纯化柱，最后用缓冲液4洗涤并收集洗脱下来的蛋白液；其中缓冲液1的组成为0.02 M咪唑含0.1 M NaCl、0.02 M Tris-HCl pH=8；缓冲液2的组成为0.05 M咪唑含0.1 MNaCl、0.02 M Tris-HCl pH=8；缓冲液3的组成为0.1 M咪唑含0.1 MNaCl、0.02 M Tris-HCl pH=8；缓冲液4的组成为0.5 M咪唑含0.1 MNaCl、0.02 M Tris-HCl pH=8。
7.权利要求5或6所述方法制备的耐热蜡样芽孢杆菌胶原酶。
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| CN104988127A (zh) * | 2015-05-13 | 2015-10-21 | 天津商业大学 | 在大肠杆菌中表达胶原酶的dna分子、重组胶原酶的方法及应用 |
| CN106957803A (zh) * | 2016-01-08 | 2017-07-18 | 中国科学院天津工业生物技术研究所 | 一株胶原酶生产菌株及其胶原酶基因序列与应用 |
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| CN104988127A (zh) * | 2015-05-13 | 2015-10-21 | 天津商业大学 | 在大肠杆菌中表达胶原酶的dna分子、重组胶原酶的方法及应用 |
| CN106957803A (zh) * | 2016-01-08 | 2017-07-18 | 中国科学院天津工业生物技术研究所 | 一株胶原酶生产菌株及其胶原酶基因序列与应用 |
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