Recombinant production, purification, and biochemical characterization of a novel L-lactate dehydrogenase from Bacillus cereus NRC1 and inhibition study of mangiferin

Abstract

Lactate dehydrogenase (LDH, EC 1.1.1.27) is one of the vital glycolytic conditions, especially during anaerobic conditions. It is a significant diagnostic, prognostic, and monitoring biomarker parameter. A 950-bp DNA fragment containing the gene (LDH) encoding LDH was amplified from Bacillus cereus NRC1. The deduced amino acid sequence reveals that B. cereus LDH (Bc-LDH) is highly homologous to the LDHs of Bacillus organisms. All LDH enzymes have a significant degree of conservation in their active site and several additional domains with unidentified functions. The gene for LDH, which catalyzes lactate synthesis, was cloned, sequenced (accession number: LC706200.1), and expressed in Escherichia coli BL21 (DE3). In this investigation, Bc-LDH was purified to homogeneity with a specific activity of 22.7 units/mg protein and a molecular weight of 35 kDa. It works optimally at pH 8.0. The purified enzyme was inhibited by FeCl2, CuCl2, ZnCl2, and NiCl, whereas CoCl2 was found to boost the activity of Bc-LDH. The molecular docking of the 3D model of the Bc-LDH structure with a natural inhibitor, mangiferin, demonstrated excellent LDH inhibition, with a free binding energy of −10.2 kcal/mol. Moreover, mangiferin is a potent Bc-LDH inhibitor that inhibits Bc-LDH competitively and has one binding site with a Ki value of 0.075 mM. The LDH-mangiferin interaction exhibits a low RMSF value (>1.5 Å), indicating a stable contact at the residues. This study will pave the way for more studies to improve the understanding of mangiferin, which could be considered an intriguing candidate for creating novel and improved LDH inhibitors.

Conditional language models enable the efficient design of proficient enzymes

“…Their enzymatic role is crucial in the reversible conversion of pyruvate to lactate, with the interconversion of the coenzymes NAD + and NADH 54,55 . The ability of LDH to mediate this conversion underpins the microbial synthesis of lactic acid, an important precursor for the synthesis of chiral compounds such as drugs and pesticides and, more importantly, in the production of bioplastics such as Poly-L-lactic Acid (PLA) 55,56 . PLA is recognised as a biodegradable, eco-friendly alternative to conventional petroleum-based plastics, aligning with the goal of a more circular economy by reducing waste 57,58 .…”

Overview of cloning in lactic acid bacteria: Expression and its application of probiotic potential in inflammatory bowel diseases

“…Moreover, specific LAB strains exhibit antimicrobial activity against harmful pathogens, making them highly valuable as probiotics, especially those capable of producing enzyme inhibitors. 52,53…”