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Transglycosylation by β-mannanase TrMan5A variants and enzyme synergy for synthesis of allyl glycosides from galactomannan

Butler, Samuel J. LU ; Birgersson, Simon LU ; Wiemann, Mathias LU ; Arcos-Hernandez, Monica LU and Stålbrand, Henrik LU (2022) In Process Biochemistry 112. p.154-166
Abstract
Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171K/E205D) of the catalytic module (CM) of the family GH5 Trichoderma reesei β-mannanase (TrMan5A) was expressed in Pichia pastoris and purified. TrMan5A, CM and CM-variants R171K and R171K/E205D had pH optima between pH 4.0–5.3 and showed >80 % remaining activity after incubation at 40 °C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed... (More)
Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171K/E205D) of the catalytic module (CM) of the family GH5 Trichoderma reesei β-mannanase (TrMan5A) was expressed in Pichia pastoris and purified. TrMan5A, CM and CM-variants R171K and R171K/E205D had pH optima between pH 4.0–5.3 and showed >80 % remaining activity after incubation at 40 °C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed by a novel HPLC procedure using an NH2 column. R171K/E205D was superior in reactions with mannotetraose and the acceptor allyl alcohol, it had twice as high propensity for transglycosylation as wild-type TrMan5A. Wild-type TrMan5A produced the highest amounts of allyl β-mannosides (with 1–3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar α-galactosidase to the reaction (to cleave off galactomannan side-groups), gave a 2.1-fold increase of allyl mannosides and simultaneously a significant production of allyl galactopyranoside, increasing overall yield of allyl glycosides 4.4-fold, from 2.2% to 9.8%. The enzymatic synthesis of reactive allyl glycosides opens up for production of novel biomaterials and glycopolymers. (Less)
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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
β-Mannanase, Transglycosylation, Enzymatic synthesis, Enzyme engineering, Synergy, Galactomannan
in
Process Biochemistry
volume
112
pages
13 pages
publisher
Elsevier
external identifiers
  • scopus:85120467967
ISSN
1359-5113
DOI
10.1016/j.procbio.2021.11.028
language
English
LU publication?
yes
id
67a45225-59ae-4092-afef-1108cd35dd7a
date added to LUP
2022-01-04 16:38:25
date last changed
2024-03-23 17:00:31
@article{67a45225-59ae-4092-afef-1108cd35dd7a,
  abstract     = {{Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171K/E205D) of the catalytic module (CM) of the family GH5 <em>Trichoderma reesei</em> β-mannanase (<em>Tr</em>Man5A) was expressed in <em>Pichia pastoris</em> and purified. <em>Tr</em>Man5A, CM and CM-variants R171K and R171K/E205D had pH optima between pH 4.0–5.3 and showed &gt;80 % remaining activity after incubation at 40 °C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed by a novel HPLC procedure using an NH<sub>2</sub> column. R171K/E205D was superior in reactions with mannotetraose and the acceptor allyl alcohol, it had twice as high propensity for transglycosylation as wild-type <em>Tr</em>Man5A. Wild-type <em>Tr</em>Man5A produced the highest amounts of allyl β-mannosides (with 1–3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar α-galactosidase to the reaction (to cleave off galactomannan side-groups), gave a 2.1-fold increase of allyl mannosides and simultaneously a significant production of allyl galactopyranoside, increasing overall yield of allyl glycosides 4.4-fold, from 2.2% to 9.8%. The enzymatic synthesis of reactive allyl glycosides opens up for production of novel biomaterials and glycopolymers.}},
  author       = {{Butler, Samuel J. and Birgersson, Simon and Wiemann, Mathias and Arcos-Hernandez, Monica and Stålbrand, Henrik}},
  issn         = {{1359-5113}},
  keywords     = {{β-Mannanase; Transglycosylation; Enzymatic synthesis; Enzyme engineering; Synergy; Galactomannan}},
  language     = {{eng}},
  pages        = {{154--166}},
  publisher    = {{Elsevier}},
  series       = {{Process Biochemistry}},
  title        = {{Transglycosylation by β-mannanase <i>Tr</i>Man5A variants and enzyme synergy for synthesis of allyl glycosides from galactomannan}},
  url          = {{http://dx.doi.org/10.1016/j.procbio.2021.11.028}},
  doi          = {{10.1016/j.procbio.2021.11.028}},
  volume       = {{112}},
  year         = {{2022}},
}