LC–MS/MS-based identification and biochemical characterization of Dihydrolipoamide dehydrogenase from Bacillus halotolerans
Microchemical Journal, cilt.227, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 227
- Basım Tarihi: 2026
- Doi Numarası: 10.1016/j.microc.2026.118819
- Dergi Adı: Microchemical Journal
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, Chemical Abstracts Core, Chimica, Index Islamicus, Academic Search Ultimate (EBSCO), Engineering Source (EBSCO)
- Anahtar Kelimeler: Bacillus halotolerans, Dihydrolipoamide dehydrogenase, Enzyme characterization LC–MS/MS, Oxidoreductase
- Sivas Cumhuriyet Üniversitesi Adresli: Evet
Özet
Dihydrolipoamide dehydrogenase (DLD; EC 1.8.1.4) is a key flavoprotein oxidoreductase that functions as the E3 component of several multienzyme complexes involved in central energy metabolism. Despite its biochemical importance, information on DLD from halotolerant bacterial sources remains limited. In this study, a Bacillus halotolerans strain isolated from molasse soil was investigated as a novel microbial source of dihydrolipoamide dehydrogenase. The enzyme was partially purified by ammonium sulphate fractionation and characterized by a combination of biochemical assays and nano-liquid chromatography tandem mass spectrometry (LC–MS/MS). SDS–PAGE analysis revealed a dominant protein band within the 24–31 kDa range. LC–MS/MS analysis of the excised band was identified as a dihydrolipoamide dehydrogenase (DLD) homologue. The identified protein exhibited a theoretical molecular weight of 49.7 kDa, which differed from the apparent molecular weight observed by SDS–PAGE. The calculated isoelectric point (pI 5.03) was consistent with those of other bacterial DLD enzymes. The partially purified enzyme exhibited maximum activity at pH 9.0 and 25 °C, with pronounced sensitivity to extreme pH points and elevated temperatures. Kinetic analysis using dihydrolipoamide as substrate revealed a low micromolar apparent Km value (28.2 μM), indicating high substrate affinity. These findings provided preliminary evidence for the presence of a dihydrolipoamide dehydrogenase homologue in Bacillus halotolerans and established a basis for future studies involving further purification and detailed molecular characterization.