Heterologous expression, characterization and evolution prediction of a diaphorase from Geobacillus sp. Y4.1MC1

Abstract

β-hydroxybutyric acid is the most sensitive indicator in ketoacidosis detection, and accounts for nearly 78% of the ketone bodies. Diaphorase is commonly used to detect the β-hydroxybutyric acid in clinical diagnosis. However, the extraction of diaphorase from animal myocardium is complex and low-yield, which is not convenient for large-scale production. In this study, a diaphorase from Geobacillus sp. Y4.1MC1 was e ciently heterologous expressed and puri ed in E. coli a yield of 110 mg/L culture. The optimal temperature and pH of this recombinant diaphorase (rDIA) were 55 °C and 6.5, respectively. It was proved that rDIA was a dual acid-and thermo-stable enzyme, and which showed much more accurate detection of β-hydroxybutyric acid than the commercial enzyme. Additionally, we also investigated the molecular interaction of rDIA with the substrate, and the conformation transition in different pH values by using homology modeling and molecular dynamics (MD) simulation. The results showed that 141-161 domain of rDIA played important role in the structure changes and conformations transmission at different pH values. Moreover, it was predicted that F105W, F105R and M186R mutants were able to improve the binding a nity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were bene t for the stability of rDIA.

Characterization and insight mechanism of an acid-adapted β-Glucosidase from Lactobacillus paracasei and its application in bioconversion of glycosides

“…The cloning, expression and purification of recombinant LpBgla were following and modified from previous reported methods ( Jia et al, 2018 ; Shang et al, 2022 ). The LpBgla gene was amplified using the Lactobacillus paracasei TK1501 genome DNA as template, and the specific primers were LpBgla-F (5′- GGA​ATT​CCA​TAT​GGG​GGT​GGT​AGT​ATC​TAA​CTT​TC-3′) and LpBgla-R (5′- CCC​AAG​CTT​TTA​TCT​AAA​AAT​CAT​TTT​CAC​ATA​CTG​ATG-3′).…”

“…Structural stability analysis of LpBgla at pH 7.5 or 5.5 by MD simulations Form the enzymatic property results, it was indicated that the LpBgla was an acid-adapted β-glucosidase, especially the optimal pH for its activity was 5.5. To investigate the involved molecular mechanism of the acid resistance of LpBgla, the structure and conformation changes of this enzyme at pH 5.5 and 7.5 were analyzed by MD simulations following our previously reported methods (Li Y. et al, 2018;Shang et al, 2022). As shown in Figure 4A, except the last 5 ns, the RMSD values of LpBgla at pH 5.5 were generally lower than that at pH 7.5 during the whole simulation time.…”