Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.
Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.
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DOI：
10.1186/s40409-015-0025-8
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Arantes EC
中科院分区：
文献类型：
--
作者：
Bordon KC;Wiezel GA;Cabral H;Arantes EC
Crotalus durissus terrificus venom（CdtV）是巴西研究最多的蛇毒之一。尽管存在几个众所周知的蛋白质，其L-氨基酸氧化酶（LAAO）尚未被研究过。本研究旨在分离、表征和评价来自CdtV的LAAO--bordonein-L的酶稳定性。通过阳离子交换、凝胶过滤和亲和层析分离酶，然后通过反相快速蛋白质液相色谱来确认其纯度。随后，通过Edman降解测定其N-末端氨基酸序列。酶的活性和稳定性进行了评估，通过微孔板比色测定和分子量估计通过SDS-PAGE使用高碘酸希夫染色和质谱法测定。N端前39个氨基酸残基与其他蛇毒L-氨基酸氧化酶具有高度的同源性。Bordonein-L是一种同源二聚体糖蛋白，通过凝胶过滤评价约101 kDa。其单体通过SDS-PAGE估计约为53 kDa，通过MALDI-TOF质谱测定约为58，702 Da。该酶在pH 7.0时表现出最大活性，在4 °C下储存5天后其活性损失约50%。在2.8%甘露醇或8.5%蔗糖中保存时，Bordonein-L的活性高于对照。这项研究是开拓性的，其分离，表征和酶稳定性评价的LAAO从CdtV，命名为bordonein-L。这些结果很重要，因为它们增加了关于LAAO稳定性的知识，旨在延长其保质期。因为在长时间储存后保持酶活性对于使其生物技术用途以及其功能研究是必不可少的。
Crotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV. The enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry. The first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-L’s activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose. This research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies.