植物内生真菌木霉一种新型NAD+依赖性木糖醇脱氢酶的表达及生化特性研究。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-02-04 DOI:10.1016/j.pep.2025.106687

Shuping Fei , Wenxiu Hu , Jingwen Shu , Ruirui Zhao , Jiatong Zhao , Mengwei Jiang , Wenwen Wu , Chaoqun Lian , Wanggang Tang

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引用次数: 0

摘要

木糖醇脱氢酶；EC 1.1.1.9)由XYL2基因编码，是真菌木糖代谢途径的关键酶。本研究从植物内生真菌甘姆木霉（Trichoderma gamsii, TgXDH）中分离得到一个XDH，并在大肠杆菌BL21（DE3）中进行了异源表达、纯化和生化表征。序列分析表明，TgXDH全长363个氨基酸，属于含锌中链醇脱氢酶超家族。大小不相容色谱分析和SDS-PAGE显示纯化的重组TgXDH的天然分子质量为~ 155 kDa，由四个相同的分子质量为~ 39 kDa的亚基组成。酶的最适温度为25℃，最适pH为9.5。动力学分析表明，该酶是一种NAD+依赖性酶，具有多元醇底物偏好（基于kcat/Km），其顺序为木糖醇>梨糖醇≈d-山梨醇。NAD+与这三种多元醇的Km值范围为0.23 ~ 0.70 mM。此外，TgXDH与大多数同源物相比，具有较高的底物亲和性。木糖醇、梨糖醇和d-山梨醇的Km值分别为5.23±0.68 mM、8.01±1.22 mM和12.34±1.37 mM。总的来说，这些结果将有助于了解从丝状真菌中提取的新型XDH的生化特性，并为工业生产乙醇提供有前途的XDH。

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Expression and biochemical characterization of a novel NAD+-dependent xylitol dehydrogenase from the plant endophytic fungus Trichoderma gamsii

Xylitol dehydrogenase (XDH; EC 1.1.1.9), encoded by the XYL2 gene, is a key enzyme in the fungal xylose metabolic pathway. In this work, a putative XDH from the plant endophytic fungus Trichoderma gamsii (TgXDH) was hetero-expressed in Escherichia coli BL21(DE3), purified to the homogeneity, and biochemically characterized. Sequence analysis revealed that TgXDH is 363 amino acids long and belongs to the zinc-containing medium-chain alcohol dehydrogenase superfamily. The size-exclusion chromatography analysis and SDS-PAGE showed that the purified recombinant TgXDH had a native molecular mass of ∼155 kDa and was composed of four identical subunits of molecular mass of ∼39 kDa. The optimum temperature and pH of this enzyme were 25 °C and pH 9.5, respectively. Kinetic analysis showed that it is an NAD⁺-dependent enzyme that has a polyol substrate preference (based on k_cat/K_m) in the order xylitol > ribitol ≈ d-sorbitol. The K_m values for NAD⁺ with these three polyols ranged from 0.23 to 0.70 mM. Moreover, TgXDH showed high substrate affinities as compared to most of its homologs. The K_m values for xylitol, ribitol, and d-sorbitol were 5.23 ± 0.68 mM, 8.01 ± 1.22 mM, and 12.34 ± 1.37 mM, respectively. Collectively, the results will contribute to understanding the biochemical properties of a novel XDH from the filamentous fungi and provide a promising XDH for industrial production of ethanol.

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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.