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

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

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 (V max )of ClearColi were higher than those of wildtype E. coli (BL21). However, the K M value, which represented the concentration of substrate to reach half the maximum rate (V max ), 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.

ClearColi, N-Acetyl-D-lactosamine (LacNAc), OmpC, Sialyltransferase, Surface display
0141-0229
1-8
Song, Hyun Woo
3c1527ee-0aaf-4093-a3c3-462351ba2ed0
Yoo, Gu
e5ad3031-f4b3-4bcb-96f4-b7b9a80212ae
Bong, Ji Hong
a1ab616a-3da0-46fc-be21-f58286635ddc
Kang, Min Jung
b65a7ea3-da72-4d90-a290-e9bb08669d47
Lee, Seung Seo
ee34fa26-5fb6-48c8-80c2-1f13ec4ccceb
Pyun, Jae Chul
96b800a1-f6d2-43ce-b38f-a8282a371e09
Song, Hyun Woo
3c1527ee-0aaf-4093-a3c3-462351ba2ed0
Yoo, Gu
e5ad3031-f4b3-4bcb-96f4-b7b9a80212ae
Bong, Ji Hong
a1ab616a-3da0-46fc-be21-f58286635ddc
Kang, Min Jung
b65a7ea3-da72-4d90-a290-e9bb08669d47
Lee, Seung Seo
ee34fa26-5fb6-48c8-80c2-1f13ec4ccceb
Pyun, Jae Chul
96b800a1-f6d2-43ce-b38f-a8282a371e09

Song, Hyun Woo, Yoo, Gu, Bong, Ji Hong, Kang, Min Jung, Lee, Seung Seo and Pyun, Jae Chul (2019) Surface display of sialyltransferase on the outer membrane of Escherichia coli and ClearColi. Enzyme and Microbial Technology, 128, 1-8. (doi:10.1016/j.enzmictec.2019.04.017).

Record type: Article

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 (V max )of ClearColi were higher than those of wildtype E. coli (BL21). However, the K M value, which represented the concentration of substrate to reach half the maximum rate (V max ), 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.

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EMT_2019_SSL_JCP - Accepted Manuscript
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More information

Accepted/In Press date: 30 April 2019
e-pub ahead of print date: 1 May 2019
Published date: September 2019
Keywords: ClearColi, N-Acetyl-D-lactosamine (LacNAc), OmpC, Sialyltransferase, Surface display

Identifiers

Local EPrints ID: 431409
URI: http://eprints.soton.ac.uk/id/eprint/431409
ISSN: 0141-0229
PURE UUID: 0b8d42ae-bbd0-4669-a3a6-9f72eeef2fe1
ORCID for Seung Seo Lee: ORCID iD orcid.org/0000-0002-8598-3303

Catalogue record

Date deposited: 31 May 2019 16:30
Last modified: 07 Oct 2020 06:36

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