Abstract
Herein, two genes (LBA0625 and LBA1719) encoding UGPases (UDP-glucose pyrophosphorylase) in Lactobacillus acidophilus (L. acidophilus) were successfully transformed into Escherichia coli BL21 (DE3) to construct recombinant overexpressing strains (E-0625, E-1719) to investigate the biological characteristics of UGPase-0625 and UGPase-1719. The active sites, polysaccharide yield, and anti-freeze-drying stress of L. acidophilus ATCC4356 were also detected. UGPase-0625 and UGPase-1719 belong to the nucleotidyltransferase of stable hydrophilic proteins; contain 300 and 294 amino acids, respectively; and have 20 conserved active sites by prediction. Αlpha-helixes and random coils were the main secondary structures, which constituted the main skeleton of UGPases. The optimal mixture for the high catalytic activity of the two UGPases included 0.5 mM UDP-Glu (uridine diphosphate glucose) and Mg2+ at 37 °C, pH 10.0. By comparing the UGPase activities of the mutant strains with the original recombinant strains, A10, L130, and L263 were determined as the active sites of UGPase-0625 (P < 0.01) and A11, L130, and L263 were determined as the active sites of UGPase-1719 (P < 0.01). In addition, UGPase overexpression could increase the production of polysaccharides and the survival rates of recombinant bacteria after freeze-drying. This is the first study to determine the enzymatic properties, active sites, and structural simulation of UGPases from L. acidophilus, providing in-depth understanding of the biological characteristics of UGPases in lactic acid bacteria.
Key points
• We detected the biological characteristics of UGPases encoded by LBA0625 and LBA1719.
• We identified UGPase-0625 and UGPase-1719 active sites.
• UGPase overexpression elevates polysaccharide levels and post-freeze-drying survival.
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This study received financial support from the National Natural Science Foundation of China (32072195, 41406165, 41641052, 31972093), the Science and Technology Department of Zhejiang Province (2019C02085), and the Education Department of Zhejiang Province (Y202148155, Y202148164, Y202148135).
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NZ and XQZ designed the study and performed the experiments; CYY and QYS prepared the manuscript and analyzed the data; XQZ reviewed and designed the manuscript; DDP, ZW, YXG, and ZDC helped improve the research plan; and CYY and QYS contributed to the revision of the manuscript.
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Zhen, N., Ye, C., Shen, Q. et al. Heterologous expression and biological characteristics of UGPases from Lactobacillus acidophilus. Appl Microbiol Biotechnol 106, 2481–2491 (2022). https://doi.org/10.1007/s00253-022-11856-8
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DOI: https://doi.org/10.1007/s00253-022-11856-8