Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase

Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase

Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aime...

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Main Authors: Shariza, Jamek, Muschiol, Jan, Holck, Jesper, Zeuner, Birgitte, Busk, Peter K., Mikkelsen, Jørn D., Meyer, Anne S.
Format: Article
Language:English
Published: John Wiley & Sons 2018
Subjects:
TP Chemical technology
Online Access:http://umpir.ump.edu.my/id/eprint/22565/
http://umpir.ump.edu.my/id/eprint/22565/
http://umpir.ump.edu.my/id/eprint/22565/
http://umpir.ump.edu.my/id/eprint/22565/1/Loop%20protein%20engineering%20for%20improved%20transglycosylation%20activity.pdf
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recordtype eprints
spelling ump-225652018-11-29T06:12:10Z http://umpir.ump.edu.my/id/eprint/22565/ Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase Shariza, Jamek Muschiol, Jan Holck, Jesper Zeuner, Birgitte Busk, Peter K. Mikkelsen, Jørn D. Meyer, Anne S. TP Chemical technology Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild‐type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG, had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild‐type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate‐binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG. John Wiley & Sons 2018 Article PeerReviewed pdf en http://umpir.ump.edu.my/id/eprint/22565/1/Loop%20protein%20engineering%20for%20improved%20transglycosylation%20activity.pdf Shariza, Jamek and Muschiol, Jan and Holck, Jesper and Zeuner, Birgitte and Busk, Peter K. and Mikkelsen, Jørn D. and Meyer, Anne S. (2018) Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase. ChemBioChem, 19 (17). pp. 1858-1865. ISSN 1439-4227 https://doi.org/10.1002/cbic.201800181 10.1002/cbic.201800181
repository_type Digital Repository
institution_category Local University
institution Universiti Malaysia Pahang
building UMP Institutional Repository
collection Online Access
language English
topic TP Chemical technology
spellingShingle TP Chemical technology
Shariza, Jamek
Muschiol, Jan
Holck, Jesper
Zeuner, Birgitte
Busk, Peter K.
Mikkelsen, Jørn D.
Meyer, Anne S.
Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase
description Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild‐type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG, had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild‐type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate‐binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG.
format Article
author Shariza, Jamek
Muschiol, Jan
Holck, Jesper
Zeuner, Birgitte
Busk, Peter K.
Mikkelsen, Jørn D.
Meyer, Anne S.
author_facet Shariza, Jamek
Muschiol, Jan
Holck, Jesper
Zeuner, Birgitte
Busk, Peter K.
Mikkelsen, Jørn D.
Meyer, Anne S.
author_sort Shariza, Jamek
title Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase
title_short Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase
title_full Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase
title_fullStr Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase
title_full_unstemmed Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase
title_sort loop protein engineering for improved transglycosylation activity of a β‐n acetylhexosaminidase
publisher John Wiley & Sons
publishDate 2018
url http://umpir.ump.edu.my/id/eprint/22565/
http://umpir.ump.edu.my/id/eprint/22565/
http://umpir.ump.edu.my/id/eprint/22565/
http://umpir.ump.edu.my/id/eprint/22565/1/Loop%20protein%20engineering%20for%20improved%20transglycosylation%20activity.pdf
first_indexed 2023-09-18T22:33:40Z
last_indexed 2023-09-18T22:33:40Z
_version_ 1777416452308992000

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