Heterologous expression and biological characteristics of UGPases from Lactobacillus acidophilus

Ni Zhen; Congyan Ye; Qiyuan Shen; Xiaoqun Zeng; Zhen Wu; Yuxing Guo; Zhendong Cai; Daodong Pan

doi:10.1007/s00253-022-11856-8

Heterologous expression and biological characteristics of UGPases from Lactobacillus acidophilus

Appl Microbiol Biotechnol. 2022 Apr;106(7):2481-2491. doi: 10.1007/s00253-022-11856-8. Epub 2022 Mar 28.

Authors

Ni Zhen^#^{1

2

3}, Congyan Ye^#^{1

2

3}, Qiyuan Shen^{1

2

3}, Xiaoqun Zeng^{4

5

6}, Zhen Wu^{1

2

3}, Yuxing Guo^{1

7}, Zhendong Cai^{1

2

3}, Daodong Pan^{1

2

3}

Affiliations

¹ State Key Laboratory for Managing Biotic and Chemical Threats To the Quality and Safety of Agro-Products, Ningbo, 315211, China.
² Key Laboratory of Animal Protein Food Processing Technology of Zhejiang Province, Ningbo University, Ningbo, 315800, China.
³ College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315800, China.
⁴ State Key Laboratory for Managing Biotic and Chemical Threats To the Quality and Safety of Agro-Products, Ningbo, 315211, China. zengxiaoqun@nbu.edu.cn.
⁵ Key Laboratory of Animal Protein Food Processing Technology of Zhejiang Province, Ningbo University, Ningbo, 315800, China. zengxiaoqun@nbu.edu.cn.
⁶ College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315800, China. zengxiaoqun@nbu.edu.cn.
⁷ School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, 210097, China.

^# Contributed equally.

PMID: 35344091
DOI: 10.1007/s00253-022-11856-8

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.

Keywords: Active site; Enzymatic properties; Extracellular polysaccharides; Freeze-drying; Lactobacillus acidophilus; UGPase.

MeSH terms

Catalytic Domain
Lactobacillus acidophilus* / genetics
Lactobacillus acidophilus* / metabolism
Protein Structure, Secondary
UTP-Glucose-1-Phosphate Uridylyltransferase* / chemistry
UTP-Glucose-1-Phosphate Uridylyltransferase* / genetics
UTP-Glucose-1-Phosphate Uridylyltransferase* / metabolism
Uridine Diphosphate Glucose / metabolism

Substances

UTP-Glucose-1-Phosphate Uridylyltransferase
Uridine Diphosphate Glucose