Novel (R)-Hydroxynitrile lyase enzyme of Pyrus communis: Purification and characterization of its physicochemical and kinetic properties

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-07-23 DOI:10.1016/j.pep.2025.106785

Asha Kumari , Sheetal , Savitri , Monica Sharma

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引用次数: 0

Abstract

Hydroxynitrile lyases (HNLs) play a vital role in the asymmetric synthesis of drug precursors and plant defence. In this study, an R-specific HNL (PycHNL) was isolated and purified from Pyrus communis (pear) seeds using ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, achieving a 14.31 % yield and 6.9-fold purification. Native PAGE estimated a molecular mass of ∼92 kDa, and SDS-PAGE revealed heterodimeric subunits of 52 and 39 kDa. High-Performance Liquid Chromatography confirmed the presence of flavin adenine dinucleotide (FAD). Peptide sequencing showed no significant similarity with other Rosaceae HNLs but indicated partial identity with serine carboxypeptidases and α/β hydrolase fold proteins from Arabidopsis species. Optimal enzyme activity was observed at pH 5.5 and 30 °C, with stability for up to 6 h. Kinetic analysis revealed a K_m of 11.75 mM, V_max of 227.27 μmol/min/mg, k_cat of 101.46/min, and a half-life of ∼1.9 days. Chiral HPLC analysis demonstrated that PycHNL preferentially synthesized (R)-mandelonitrile with 96.33 % enantiomeric excess and 86.83 % molar conversion, indicating its potential for biocatalytic applications in producing enantiopure nitriles.

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新型梨(R)-羟基腈裂解酶的纯化及其理化和动力学性质的表征。

羟基腈裂解酶（HNLs）在药物前体的不对称合成和植物防御中发挥着重要作用。本研究采用硫酸铵沉淀、凝胶过滤、离子交换层析等方法从梨种子中分离纯化了一种r特异性HNL (PycHNL)，产率为14.31%，纯化率为6.9倍。Native PAGE估计分子质量为~ 92 kDa， SDS-PAGE显示异二聚体亚基为52和39 kDa。高效液相色谱法证实黄素腺嘌呤二核苷酸（FAD）的存在。肽段测序结果显示，与其他蔷薇科hnl没有显著的相似性，但与拟南芥的丝氨酸羧肽酶和α/β水解酶折叠蛋白部分相同。在pH 5.5和30°C条件下，酶活性最佳，稳定性可达6小时。动力学分析表明，其Km为11.75 mM， Vmax为227.27 μmol/min/mg， kcat为101.46/min，半衰期为~ 1.9天。手性高效液相色谱分析表明，PycHNL以96.33%的对映体过量和86.83%的摩尔转化率优先合成(R)-mandelonitrile，表明其在制备对映纯腈方面具有潜在的生物催化应用前景。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.