CN118240004A - 芽孢杆菌碱性蛋白酶的新型抑制肽混合物及其制备和应用 - Google Patents
芽孢杆菌碱性蛋白酶的新型抑制肽混合物及其制备和应用 Download PDFInfo
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Abstract
本发明涉及芽孢杆菌来源碱性蛋白酶的竞争性抑制剂，发明获得了一种新型抑制肽，该抑制肽及其衍生物对芽孢杆菌来源碱性蛋白酶具有明显的抑制作用，在芽孢杆菌来源碱性蛋白酶的液体酶制剂中添加该抑制肽，蛋白酶活力保存的稳定性明显增强。该抑制剂在环保和安全性上具有明显的优势，有望应用于液体加酶洗涤剂领域，替代传统的硼砂类抑制剂。
Description
技术领域
本发明属于酶工程领域，具体涉及芽孢菌碱性蛋白酶的短肽抑制剂及其生物合成和应用方法。
背景技术
芽孢菌来源的碱性蛋白酶的水解能力强，稳定性好，其最适pH一般为9～11，广泛应用于洗涤剂、食品加工、纺织、医药等领域，在工业蛋白酶制剂中占比最高。因为其在碱性条件下的去污能力很强，并且对蛋白类污垢的效果最佳，所以芽孢菌来源的碱性蛋白酶是洗涤剂添加酶的主要用酶。但同时也因为较强的水解活力，芽孢菌来源的碱性蛋白酶在储存过程中容易水解自身，导致其酶活力快速损失，从而影响该酶的稳定储存和使用效果。因此，需要在加酶洗涤剂中添加适量的蛋白酶抑制剂以保持储存中的稳定性。目前行业中所使用的抑制剂大多都为硼砂类物质，对人类的身体健康不利，对环境也不友好。秉承绿色环保的理念，更加安全健康的酶抑制剂亟待开发。多肽类抑制剂在安全性和环保上较硼砂类物质更有优势，并且抑制能力也跟高，故其成为近年来关于加酶洗涤剂抑制剂的研发热点。
发明内容
本发明的目的在于，寻找芽孢杆菌碱性蛋白酶的多肽类抑制剂，开发其生物合成方法，并应用到液体酶制剂和加酶洗涤剂中。本发明工作发现，序列特征为R1-R2-R3-F的混合抑制肽对芽孢杆菌碱性蛋白酶具有明显的抑制和稳定效果，其中R1、R2、R3分别代表H/P/S/T/G/A/V/I中的任意一种氨基酸，并且，如果N端修饰为酰基化保护基团，C端羧基被醛基化修饰，抑制活性会大大增强，对蛋白酶活力在液体酶制剂或者加酶洗涤剂中保存的稳定性也明显增强。
本发明还开发了所述混合抑制肽的生物合成方法，如下：
(1)合成串联多肽(R1-R2-R3-F)n的DNA序列，n代表4肽R1-R2-R3-F的串联次数，该串联多肽序列中每5-10个R1-R2-R3-F序列间隔中插入一段亲水氨基酸序列；
(2)将串联多肽与糜蛋白酶的DNA序列共同重组到细菌或酵母表达质粒上，优选地，选择pET22b(+)或者pPIC9K作为表达质粒；
(3)将质粒导入工程菌株，发酵表达(R1-R2-R3-F)n序列多肽分子和糜蛋白酶蛋白分子，优选地，选择大肠杆菌或者毕赤酵母作为工程菌株。
(4)制备的(R1-R2-R3-F)n序列多肽分子和糜蛋白酶蛋白分子混合物，孵育水解，并通过膜过滤制备R1-R2-R3-F混合肽；
(5)通过化学合成，将R1-R2-R3-F混合肽的N端修饰为酰基化保护基团，C端羧基被醛基化修饰。
本发明还将所合成的混合抑制肽应用于芽孢杆菌来源碱性蛋白酶的液体酶制剂和加酶洗涤中。
(1)在芽孢杆菌来源碱性蛋白酶液体酶制剂中添加0.2％-2％的R1-R2-R3-F抑制肽混合物，相比未添加抑制剂的空白组，酶活力保留率明显提高，延长了酶活力保存时间。
(2)在芽孢杆菌来源碱性蛋白酶的液体加酶洗涤剂中添加0.002％-0.02％的R1-R2-R3-F抑制肽混合物，相比未添加抑制剂的空白组，酶活力保留率明显提高，延长了酶活力保存时间。
本发明证明所述新型混合肽抑制剂对芽孢杆菌碱性蛋白酶具有抑制和稳定效果的技术方案如下：
1.测定和比较液体酶制剂中添加不同抑制剂对酶活力的稳定效果：
(1)配制含有稳定剂(主要成分为CaCl2和多羟基化合物，如甘油、丙二醇、多糖等，不含有蛋白酶酶活力抑制剂)的芽孢杆菌碱性蛋白酶的酶液。
(2)在含有稳定剂的酶液基础上，分别添加不同抑制剂，实验分为空白组、对照组和实验组。空白组添加空白缓冲液，对照组添加4-甲酰苯硼酸(4-FPBA)，实验组添加R1-R2-R3-F混合肽。将所有酶液密封放置37℃培养箱孵育，于不同时间点取样测定残留的酶活力。
(3)计算不同时间点的酶活保留率，比较不同抑制剂对碱性蛋白酶的稳定效果。
2.测定和比较液体洗涤剂基质中添加不同抑制剂对酶活力的稳定效果：
(1)配制含含有芽孢杆菌碱性蛋白酶的液体洗涤剂。
(2)在液体洗涤剂基质中，分别添加不同的抑制剂，实验分为空白组、对照组和实验组。空白组添加空白缓冲液，对照组添加4-甲酰苯硼酸，实验组添加R1-R2-R3-F混合肽。将所有洗涤剂密封放置37℃培养箱孵育，于不同时间点取样测定残留的酶活力。
(3)计算不同时间点的酶活保留率，比较不同抑制剂对碱性蛋白酶的稳定效果。
本发明的有益效果是：
(1)所发明的混合抑制肽对芽孢来源碱性蛋白酶有显著的抑制和稳定作用，可用于液体酶制剂的复配，提高液体酶的稳定性。
(2)所发明的混合抑制肽，能够提高液体洗涤剂中蛋白酶的稳定性，可替代液体加酶洗涤剂中的硼砂类和4-FPBA类抑制剂，对环境和人体健康都具有更高的安全性。
附图说明
图1，串联多肽发酵水解产物的液相色谱质谱分析
图2，不同抑制剂在稳定剂基质中对枯草杆菌碱性蛋白酶的稳定作用
图3，在洗衣液基质中不同抑制剂在稳定剂中对枯草杆菌碱性蛋白酶的稳定作用
具体实施方式
下面结合实施例对本发明的技术内容做进一步说明，但本发明不只限于这些实施例，不能以下述实施例来限定本发明的保护范围。
实施例1：R1-R2-R3-F混合抑制肽的生物合成和化学修饰
1.通过金唯智生物公司合成人工串联多肽(R1-R2-R3-F)n和牛源糜蛋白酶的DNA序列，并其同时插入到pET22b(+)质粒中。氨基酸序列如下：
人工串联多肽序列：
SIPFPIPFTATFGAPFHVPFRKDTKDFGAGFTAPFSVPFTVPFTDRDRKFPVPFHIPFSATFTAG
FAAPFTRDRKDFSAPFAAHFSASFSAGF
糜蛋白酶蛋白序列：
CGVPAIQPVLSGLSRIVNGEEAVPGSWPWQVSLQDKTGFHFCGGSLINENWVVTAAHCGV
TTSDVVVAGEFDQGSSSEKIQKLKIAKVFKNSKYNSLTINNDITLLKLSTAASFSQTVSAVC
LPSASDDFAAGTTCVTTGWGLTRYTNANTPDRLQQASLPLLSNTNCKKYWGTKIKDAMIC
AGASGVSSCMGDSGGPLVCKKNGAWTLVGIVSWGSSTCSTSTPGVYARVTALVNWVQQT
LAAN
2.将构建的质粒转化导入大肠杆菌BL21(DE3)中
(1)从-80℃取出感受态细胞(100μL)，冰浴融解。
(2)向感受态细胞中加入10μL的连接产物或待转化的质粒2-5μL，轻轻混匀，冰浴放置30min。
(3)42℃，水浴热激30S，冰浴放置2min。
(4)加入37℃预热好的的LB培养基900μL，37℃摇床220r/min振荡培养1小时。
(5)4000r/min离心5min收集菌体，弃去部分上清液后重悬菌体，取适量涂布Amp抗性的LB平板，将平板置于37℃培养箱中过夜培养12-16h。
3.将转化导入质粒后的菌株进行发酵诱导培养
(1)将菌液转接至含Amp抗性的LB液体培养基中(Amp终浓度为100μg/mL)，37℃，220r/min培养至菌液OD600＝0.6-0.8。
(2)加入IPTG(终浓度0.5mmol/L)，16℃，120r/min诱导培养16-20h。
4.发酵菌液的处理
发酵后的菌液以8000r/min离心30min后取沉淀。沉淀进行超声破碎处理。超声破碎参数为：破碎时间3s；间歇时间4s；功率300w；总时间20min；温度4℃；变幅杆Φ10。
5.包涵体的变性与复性
加入8M的尿素使包涵体变性，处理12h左右。后通过透析袋的方法将尿素透析出溶液，得到正常折叠的抑制肽串联多肽分子与糜蛋白酶的混合物。
6.孵育水解获得R1-R2-R3-F抑制肽混合物。
将复性产物，主要是串联多肽与糜蛋白酶的混合物，37℃水解3h，超滤后获得混合抑制肽。
7.将超滤后获得的混合抑制肽进行质谱检测。
使用安捷伦LC-ESI-Q-TOF-MS/MS液相质谱连用仪进行检测，色谱柱型号为xbRIDGE peptide BEH C18(2.1mm x 150mm 300A，3.5μm)，能量：4500v，slope：3.6，offset:4.8，Gas Temp(℃)：325，Gas flow(L/min)：13，流速：0.25ml/min
梯度洗脱条件
质谱检测证明已经成功生产出R1-R2-R3-F抑制肽混合物，结果如图1所示。
8.将制备的混合抑制肽委托第三方公司进行N端的酰基化修饰与羧基端的醛基化修饰。
实施例2：混合抑制肽对芽孢杆菌碱性蛋白酶液体酶制剂的稳定效果
1.使用磷酸缓冲液(pH 6.5)：乙二醇＝3：2溶液配置100mM的4-FPBA、R1-R2-R3-F混合肽。
2.配制含有稳定剂(5mM CaCl2、3％甘油、7％丙二醇)的芽孢杆菌碱性蛋白酶的酶液，在此基础上分别加入4-FPBA、R1-R2-R3-F混合肽，终浓度为16mM。空白组加入空白缓冲液。将所有组别密封放置于37℃培养箱孵育，不同时间点取样测定残留的酶活力。
3.残留酶活力随时间的变化趋势，如图2所示。由图可知，空白组的酶活力下降最快，4-FPBA组的酶活力下降最慢，而R1-R2-R3-F混合肽组的效果明显好于空白组。尽管在16mM添加条件下，R1-R2-R3-F混合肽组的酶活保留效果不及4-FPBA，但在稳定碱性蛋白酶酶活力方面也具有不错的效果。在液体酶中，可以考虑使用本研究发明的新型混合多肽抑制剂。
实施例3：混合抑制肽对液体洗涤剂基质中芽孢杆菌碱性蛋白酶的稳定效果。
1.使用磷酸缓冲液(pH 6.5)：乙二醇＝3：2溶液配置100mM的4-FPBA、R1-R2-R3-F混合肽。从市场购买不加酶的液体洗涤剂。
2.芽孢杆菌碱性蛋白酶的酶液，在此基础上分别加入4-FPBA、R1-R2-R3-F混合肽，后加入到不加酶的液体洗涤剂中，终浓度为16mM。空白组加入空白缓冲液。所有溶液密封放置37℃孵育，于不同时间点取样测定残留的酶活力。
3.残留酶活力随时间的变化趋势如图3所示。由图可知，不含任何抑制剂的空白组的酶活力下降最快，4-FPBA组的酶活力下降最慢，R1-R2-R3-F混合肽组的效果明显好于空白组，并且接近4-FPBA组的效果，说明在液体加酶洗涤剂生产中，可以考虑将4-FPBA替换为本研究发明新型混合多肽抑制剂。
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例所公开的内容。
Claims (4)
1.芽孢杆菌来源碱性蛋白酶的一种抑制肽混合物，其序列特征为R1-R2-R3-F，其中R1、R2、R3分别代表H/P/S/T/G/A/V/I中的任意一种氨基酸，优选地，N端修饰为酰基化保护基团，C端羧基被醛基化修饰。
2.如权利要求1所述抑制肽混合物的生物合成方法：
(1)合成串联多肽(R1-R2-R3-F)n的DNA序列，n代表4肽R1-R2-R3-F的串联次数，该串联多肽序列中每5-10个R1-R2-R3-F序列间隔中插入一段亲水氨基酸序列；
(2)将串联多肽与糜蛋白酶的DNA序列共同重组到细菌或酵母表达质粒上；
(3)将质粒导入工程菌株，发酵表达(R1-R2-R3-F)n串联多肽分子和糜蛋白酶蛋白分子；
(4)制备(R1-R2-R3-F)n串联多肽分子和糜蛋白酶蛋白分子的混合物，孵育水解，并通过膜过滤获得R1-R2-R3-F混合肽；
(5)通过化学合成，将R1-R2-R3-F混合肽的N端修饰为酰基化保护基团，C端羧基被醛基化修饰。
3.如权利要求1所述抑制肽混合物在芽孢杆菌来源碱性蛋白酶液体酶制剂中的应用，其特征是在芽孢杆菌来源碱性蛋白酶液体酶制剂中添加0.2％-2％的R1-R2-R3-F抑制肽混合物。
4.如权利要求1所述抑制肽混合物在芽孢杆菌来源碱性蛋白酶液体加酶洗涤剂中的应用，其特征是在含有芽孢杆菌来源碱性蛋白酶的液体洗涤剂中添加0.002％-0.02％的R1-R2-R3-F抑制肽混合物。
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