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

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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Abstract

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.