Surface display of sialyltransferase on the outer membrane of Escherichia coli and ClearColi

Hyun Woo Song, Gu Yoo, Ji Hong Bong, Min Jung Kang, Seung Seo Lee, Jae Chul Pyun

Research output: Contribution to journalArticle

Abstract

α2,3-Sialyltransferase from Pasteurella multocida (PmST1)is an enzyme that transfers a sialyl group of donor substrates to an acceptor substrate called N-acetyl-D-lactosamine (LacNAc). In this study PmST1 was expressed on the outer membrane of wildtype Escherichia coli (BL21)with lipopolysaccharide (LPS)and ClearColi with no LPS, and then the enzyme activity and expression level of PmST1 were compared. As the first step, the expression levels of PmST1 on the outer membranes of wildtype E. coli (BL21)and ClearColi were compared according to the IPTG induction time, and the absolute amount of surface-displayed PmST1 was calculated using densitometry of SDS-PAGE. As the next step, the influence of LPS on the PmST1 activity was estimated by analyzing Michaelis-Menten plot. The enzyme activity of PmST1 was analyzed by measuring the concentration of CMP, which was a by-product after the transfer of the sialyl group of donor compounds to the acceptor compounds. From a Michaelis–Menten plot, the enzyme activity of the surface-displayed PmST1 and the maximum rate (Vmax)of ClearColi were higher than those of wildtype E. coli (BL21). However, the KM value, which represented the concentration of substrate to reach half the maximum rate (Vmax), was similar for both enzymes. These results represented such a difference in enzyme activity was occurred from the interference of LPS on the mass transport of the donor and acceptor to PmST1 for the sialyl group transfer.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalEnzyme and Microbial Technology
Volume128
DOIs
Publication statusPublished - 2019 Sep

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Sialyltransferases
Enzyme activity
Escherichia coli
Lipopolysaccharides
Display devices
Membranes
Enzymes
Substrates
Cytidine Monophosphate
Isopropyl Thiogalactoside
Byproducts
Pasteurella multocida
Mass transfer
Densitometry
Polyacrylamide Gel Electrophoresis

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Song, Hyun Woo ; Yoo, Gu ; Bong, Ji Hong ; Kang, Min Jung ; Lee, Seung Seo ; Pyun, Jae Chul. / Surface display of sialyltransferase on the outer membrane of Escherichia coli and ClearColi. In: Enzyme and Microbial Technology. 2019 ; Vol. 128. pp. 1-8.
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Surface display of sialyltransferase on the outer membrane of Escherichia coli and ClearColi. / Song, Hyun Woo; Yoo, Gu; Bong, Ji Hong; Kang, Min Jung; Lee, Seung Seo; Pyun, Jae Chul.

In: Enzyme and Microbial Technology, Vol. 128, 09.2019, p. 1-8.

Research output: Contribution to journalArticle

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