Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

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Abstract

The ¿-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37°C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 30-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically
Original languageEnglish
Pages (from-to)122-131
JournalBiotechnology and Bioengineering
Volume93
Issue number1
DOIs
Publication statusPublished - 2006

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alpha-Galactosidase
Mutagenesis
Site-Directed Mutagenesis
Enzymes
Mutation
Hexuronic Acids
Melibiose
Galactosidases
Sugar Alcohols
Pentoses
Sequence Alignment
Disaccharides
Substrates
Hydroxyl Radical
Amino acids
Amino Acid Sequence
Catalytic Domain
Amino Acids
Molecules
Acids

Keywords

  • beta-glucosidase
  • pyrococcus-furiosus
  • crystal-structure
  • chain elongation
  • dsm 20083
  • oligosaccharides
  • raffinose
  • amylase
  • hydrolysis
  • resolution

Cite this

@article{5540bf467f3a4a3a9bc4b8f36267fd1f,
title = "Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis",
abstract = "The ¿-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37°C. At high melibiose concentration (600 mM), approximately 64{\%} of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 30-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16{\%}, whereas most of the single mutations showed an increase of 2{\%}-5{\%} compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10{\%}-16{\%}, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4{\%}-7{\%}). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64{\%} to 75{\%} of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically",
keywords = "beta-glucosidase, pyrococcus-furiosus, crystal-structure, chain elongation, dsm 20083, oligosaccharides, raffinose, amylase, hydrolysis, resolution",
author = "S.W.A. Hinz and C.H.L. Doeswijk-Voragen and R. Schipperus and {van den Broek}, L.A.M. and J.P. Vincken and A.G.J. Voragen",
year = "2006",
doi = "10.1002/bit.20713",
language = "English",
volume = "93",
pages = "122--131",
journal = "Biotechnology and Bioengineering",
issn = "0006-3592",
publisher = "Wiley",
number = "1",

}

TY - JOUR

T1 - Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

AU - Hinz, S.W.A.

AU - Doeswijk-Voragen, C.H.L.

AU - Schipperus, R.

AU - van den Broek, L.A.M.

AU - Vincken, J.P.

AU - Voragen, A.G.J.

PY - 2006

Y1 - 2006

N2 - The ¿-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37°C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 30-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically

AB - The ¿-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37°C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 30-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically

KW - beta-glucosidase

KW - pyrococcus-furiosus

KW - crystal-structure

KW - chain elongation

KW - dsm 20083

KW - oligosaccharides

KW - raffinose

KW - amylase

KW - hydrolysis

KW - resolution

U2 - 10.1002/bit.20713

DO - 10.1002/bit.20713

M3 - Article

VL - 93

SP - 122

EP - 131

JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

SN - 0006-3592

IS - 1

ER -