CN112898375B - 一种芽孢杆菌来源碱性蛋白酶的新型抑制肽 - Google Patents

一种芽孢杆菌来源碱性蛋白酶的新型抑制肽 Download PDF

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CN112898375B
CN112898375B CN202110191324.8A CN202110191324A CN112898375B CN 112898375 B CN112898375 B CN 112898375B CN 202110191324 A CN202110191324 A CN 202110191324A CN 112898375 B CN112898375 B CN 112898375B
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alkaline protease
bacillus
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aapf
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路福平
王洪彬
李雪
李奕鸣
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Tianjin University of Science and Technology
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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

一种芽孢杆菌来源碱性蛋白酶的新型抑制肽
技术领域
本发明属于酶工程领域，具体涉及芽孢杆菌来源碱性蛋白酶的抑制肽。
背景技术
芽孢菌来源的碱性蛋白酶，因为水解能力强、生产效率高等优势，在工业蛋白酶制剂中占比最高，广泛应用于洗涤、食品和纺织等行业。蛋白酶制剂分为固体制剂和液体制剂，其中液体制剂的应用逐渐增多，尤其是在液体加酶洗涤剂行业。随着中国洗涤用品工业的不断发展，其产业结构发生巨大变化，液体洗涤剂所占市场比重也逐年攀升。芽孢杆菌来源的微生物产碱性蛋白酶是主要的液体洗涤剂添加用酶，其在碱性条件下去污能力较高，对血渍、汗渍、油渍等蛋白类污垢效果最佳。由于蛋白酶水解蛋白质的特性，蛋白酶在液体体系中会水解自身和其他种类酶制剂，因此芽孢菌来源的碱性蛋白酶在其液体制剂或者液体洗涤剂的稳定体系产品中，需要添加其竞争性抑制剂抑制其活力，以保持其在储存期间的稳定性。目前液体加酶洗涤剂中行业中广泛使用的蛋白酶抑制剂为硼砂类物质，其对人体健康和环境并不友好，具有潜在的生殖毒性。随着环保理念逐渐深入人心，开发绿色、环保和安全性高的碱性蛋白酶抑制剂具有十分重要的意义。多肽类抑制剂，在环保和安全性上具有更高的优势，在液体酶和液体加酶洗涤剂领域有望成为现有抑制剂的最佳替代。
发明内容
本发明的目的在于，为芽孢杆菌来源的碱性蛋白酶寻找新型的多肽类抑制剂。本发明工作研究发现，序列特征为R1-A-A-P-F-R2的短肽对芽孢杆菌来源的碱性蛋白酶具有明显的抑制和稳定效果，其中其中A为丙氨酸；P为脯氨酸；F为苯丙氨酸；R1代表N端没有或者含有保护基团，R2代表C端没有或者含有基团修饰。优选地，N端保护基团为琥珀酰基(Suc)、乙酰基(Ac)等酰基保护基团，C端羧基被醛基化修饰(-H)时，对酶活力的抑制和稳定效果较好。通过添加所发明的抑制肽配制含有芽孢杆菌来源碱性蛋白酶的液体酶组合制剂，该制剂中蛋白酶活力保存的稳定性明显增强。
本发明证明所述新型多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.测定和比较不同抑制剂对酶活力的抑制率：
(1)配制芽孢杆菌碱性蛋白酶的酶液。
(2)在酶液基础上，分别进行不同添加，实验分为空白组、对照组、实验组。空白组添加缓冲液，对照组添加4-甲酰苯硼酸(4-FPBA)的酶液，实验组分别添加多肽AAPF、Suc-AAPF、AAPF-H、Suc-AAPF-H。4-FPBA为目前芽孢菌来源碱性蛋白酶在用的竞争性抑制剂。
(3)根据国标GB/T23527-2009测定碱性蛋白酶活力，计算酶活力抑制率，比较不同抑制剂的抑制性能。
2.测定和比较不同抑制剂对酶活力的稳定性能：
(1)配制含有稳定剂(主要成分为CaCl2、甘油、丙二醇等，不含有酶活力抑制剂)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，分别进行不同添加，实验分为空白组、对照组、实验组。空白组添加缓冲液，对照组添加4-甲酰苯硼酸(4-FPBA)，实验组分别添加AAPF、Suc-AAPF、AAPF-H、Suc-AAPF-H。将所有溶液密封放置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性能。
本发明的有益效果是：
(1)所发明的抑制肽对芽孢来源的碱性蛋白酶有一定的抑制和稳定作用，可用于液体酶制剂的复配，延长液体酶的稳定性。
(2)所发明的抑制肽可替代液体加酶洗涤剂中的硼砂类抑制剂，对环境和人体健康都具有更高的安全性。
附图说明
图1，不同抑制剂对枯草杆菌碱性蛋白酶的抑制效果
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
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：抑制肽对芽孢杆菌碱性蛋白酶的抑制效果
1.使用硼酸缓冲液(pH 10.5)配制100mM 4-FPBA、Suc-AAPF、Suc-AAPF-H的母液。
2.使用硼酸缓冲液(pH 10.5)稀释克劳氏芽孢杆菌来源的碱性蛋白酶的酶液，然后分别加入2mM的4-FPBA、抑制肽AAPF和Suc-AAPF-H，空白组不添加抑制剂。根据国标GBT23527-2009测定碱性蛋白酶酶活，再计算酶活抑制率。
3.酶活抑制率测定结果如图1所示，添加浓度2mM条件下，Suc-AAPF对碱性蛋白酶的酶活抑制率为42％，而Suc-AAPF-H的抑制率为46％，达到阳性对照4-FPBA抑制率(55％)的84％。Suc-AAPF-H、Suc-AAPF均对芽孢杆菌来源的碱性蛋白酶有抑制活性，且醛基化修饰后抑制活性也有所增强。
实施例2：抑制肽对芽孢杆菌碱性蛋白酶酶活保留率的影响
1.使用硼酸缓冲液(pH 10.5)配置100mM的4-FPBA、Suc-AAPF、Suc-AAPF-H母液。
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、Suc-AAPF、Suc-AAPF-H，使其终浓度为2mM。空白组加入缓冲液。将所有溶液密封放置37℃培养箱孵育，于不同时间点取样测定残留的酶活力。
3.残留酶活力随时间的变化趋势，如图2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，而Suc-AAPF-H组的效果明显好于空白组，也好于Suc-AAPF组，说明醛基化修饰可以增强该抑制肽对酶活力的稳定作用。尽管2mM添加条件下，Suc-AAPF-H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具有明显的作用，在实际应用中可以通过提升使用浓度来增强稳定效果。

Claims (1)

1.一种含有芽孢杆菌来源碱性蛋白酶的液体酶组合制剂，其特征是含有芽孢杆菌来源碱性蛋白酶的一种抑制肽，其序列为R1-A-A-P-F-R2，其中 A为丙氨酸，P为脯氨酸，F为苯丙氨酸，R1代表琥珀酰基化保护基团，R2代表C端羧基被醛基化修饰。
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