三(2-羧乙基)膦盐酸盐提高麦芽低聚糖生成酶的活力和热稳定性
首发时间:2020-01-09
摘要:来源于多糖降解菌的直链麦芽低聚糖生成酶(Sd-MFA酶)能够将淀粉水解为直链麦芽低聚糖,且水解产物中麦芽五糖的含量约占总产物的35%,使该酶在麦芽五糖的制备和分离中具有天然优势,但是其水解活力仅为275.3 U/mg,在45 C时的半衰期也只有4.2 min,限制了其工业化应用。因此本研究旨在通过三(2-羧乙基)膦盐酸盐(TCEP)的添加提高Sd-MFA酶的活力和热稳定性。实验结果表明,TCEP可以显著增强Sd-MFA酶的活力和热稳定性,在TCEP的终浓度为20 mM时,Sd-MFA酶的活力最高,达到547.4 U/mg,比对照组提高了1倍,并且能将Sd-MFA酶45 C下的半衰期延长5倍。通过模拟Sd-MFA酶的三维结构,结合圆二色谱和荧光光谱等手段,分析了TCEP影响Sd-MFA酶的机理。对Sd-MFA酶的三维结构分析表明,Sd-MFA酶含有14个半胱氨酸,其中6个位于酶的表面,暴露于表面的半胱氨酸可能由于溶剂或空气中存在的氧气而发生氧化,而TCEP可以使半胱氨酸处于还原状态而不被氧化,进而有助于提高Sd-MFA酶的稳定性。荧光光谱和圆二色谱结果表明,在热处理过程中,Sd-MFA酶的荧光强度增强,同时α-螺旋含量降低,β-折叠含量增加,但是TCEP会减弱它们的变化程度,因而可以保护酶的三级结构和二级结构。本研究提供了一种增强Sd-MFA酶的活力和热稳定性的方法,提高了该酶在工业上的生产应用前景,也可为改善其它酶的使用性能提供参考。
关键词: 直链麦芽低聚糖 直链麦芽低聚糖生成酶 活力 热稳定性 三(2-羧乙基)膦盐酸盐
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Tris (2-carboxyethyl) phosphine addition enhances the thermal stability of a maltooligosaccharide-forming amylase from Saccharophagus degradans
Abstract:Maltooligosaccharide-forming amylases from Saccharophagus degradans (Sd-MFAse) can hydrolyze starch into maltooligosaccharide, and maltopentose accounts for 35% of the total product, which gives Sd-MFAse great advantages in the preparation and separation of maltopentose. But its activity and half-life at 45 C is only 275.3 U/mg and 4.2 min, respectively, which limits its industrial application. Therefore, this study aims to improve the activity and thermal stability of Sd-MFAse. The results showed that TCEP could activate and stabilize this Sd-MFAse. The most significant increase (about 100%) in Sd-MFAse was achieved by adding 20 mM TCEP, besides, 20 mM TCEP prolonged the half-life of Sd-MFAse at 45 C by 5-fold, compared to a control. The mechanism of this effect was illuminated by protein modelling, far-UV circular dichroism and intrinsic fluorescence spectra. The results indicated that there were 14 cysteine in the structure of Sd-MFAse. Six of these cysteines were exposed to the solvent and are therefore easily oxidized. TCEP could protect cysteines from oxidation. Fluorescence spectroscopy and circular dichroism analysis indicated that TCEP helped protect the tertiary and secondary structure of Sd-MFAse. This study provides a effective method to enhance the activity and thermal stability of Sd-MFAse, which improves the industrial production and application prospect of the enzyme,and can also provide a reference for improving the performance of other enzymes.
Keywords: maltooligosaccharide maltooligosaccharide-forming amylase activity thermal stability Tris (2-carboxyethyl) phosphine hydrochloride
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三(2-羧乙基)膦盐酸盐提高麦芽低聚糖生成酶的活力和热稳定性
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