20 research outputs found
Converting Bulk Sugars into Functional Fibers: Discovery and Application of a Thermostable β‑1,3-Oligoglucan Phosphorylase
Despite their broad application potential, the widespread
use of
β-1,3-glucans has been hampered by the high cost and heterogeneity
associated with current production methods. To address this challenge,
scalable and economically viable processes are needed for the production
of β-1,3-glucans with tailorable molecular mass distributions.
Glycoside phosphorylases have shown to be promising catalysts for
the bottom-up synthesis of β-1,3-(oligo)glucans since they combine
strict regioselectivity with a cheap donor substrate (i.e., α-glucose
1-phosphate). However, the need for an expensive priming substrate
(e.g., laminaribiose) and the tendency to produce shorter oligosaccharides
still form major bottlenecks. Here, we report the discovery and application
of a thermostable β-1,3-oligoglucan phosphorylase originating
from Anaerolinea thermophila (AtβOGP). This enzyme combines a superior catalytic
efficiency toward glucose as a priming substrate, high thermostability,
and the ability to synthesize high molecular mass β-1,3-glucans
up to DP 75. Coupling of AtβOGP with a thermostable
variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable
β-1,3-(oligo)glucans from sucrose, with a near-complete conversion
of >99 mol %. This cost-efficient process for the conversion of
renewable
bulk sugar into β-1,3-(oligo)glucans should facilitate the widespread
application of these versatile functional fibers across various industries
Indicator diagram applied to the three LPMO regioselectivity types.
<p>Preliminary evaluation of the indicator diagram was done by incubating a member of each LPMO regioselectivity type on PASC: ● = <i>Hj</i>LPMO9A (C1/C4-oxidizer, 1.2–12 μM), ▼ = <i>Pc</i>LPMO9D (C1-oxidizer, 1–10 μM), ○ = <i>Nc</i>LPMO9C (C4-oxidizer, 0.9–2.8 μM).</p
HPAEC-PAD chromatograms of all 3 LPMO representatives.
<p>Activity of <i>Hj</i>LPMO9A (red), <i>Nc</i>LPMO9C (green), <i>Pc</i>LPMO9D (blue) and wildtype <i>P</i>. <i>pastoris</i> CBS7435 broth (black) on 0.5% PASC in the presence of 1 mM ascorbic acid as reducing agent. The six signals (A1-3 and K1-3) that will be evaluated for their use as indicator signal are indicated in the top chromatogram.</p
Indicator diagram demonstrates role of aromatic residues in LPMO regioselectivity.
<p>Slopes, which are a measure of the ratio of C4/C1-oxidation, are listed next to the regression line, together with their standard deviation. ● = <i>Hj</i>LPMO9A wildtype (1.4–14 μM), ○ = Y24A variant (1.2–12 μM), ▼ = Y211 (1.2–12 μM) variant.</p
LPMO regioselectivity.
<p>Oxidation of the C1 position generates a lactone, which is hydrated to a reducing-end aldonic acid. C4-oxidation leads to non-reducing-end 4-ketoaldose formation, which will spontaneously hydrate to gemdiols in aqueous conditions.</p
Aromatic surface residues in the C1/C4-oxidizing <i>Hj</i>LPMO9A.
<p>(A) Homology model of <i>Hj</i>LPMO9A (based on 3ZUD as template) with the aromatic surface residues selected for alanine scanning in pink stick representation. Active site residues are shown as yellow sticks, the copper ion as a blue sphere. (B) 3DM structure based multiple sequence alignment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178446#pone.0178446.ref040" target="_blank">40</a>] of AA9 characterized LPMOs with known regioselectivity. The residues aligned with the Y24, F43, W84 and Y211 aromatic surface residue of <i>Hj</i>LPMO9A are highlighted in pink. Residues in the 3DM core alignment are represented by capitals, the alignment of structurally variable regions are in lower case. The insertion typical for most C1/C4-oxidizing LPMOs is marked in yellow.</p
Effect of mutating aromatic surface residues on <i>Hj</i>LPMO9A regioselectivity.
<p>This effect is determined by comparing the C1/C4-oxidation ratio (slope in the indicator diagram) of the wildtype enzyme (1.4–14 μM) and the variants (1.2–12 μM).</p
Primers used for the creation of mutants of the C1/C4 oxidizing LPMO <i>Hj</i>LPMO9A.
<p>Primers used for the creation of mutants of the C1/C4 oxidizing LPMO <i>Hj</i>LPMO9A.</p
Correlation between release speeds of aldonic acid / 4-ketoaldose peaks and the <i>Hj</i>LPMO9A LPMO load in 500 μL reaction mixture.
<p>Graph A represents the aldonic acid peaks (● = A1, ○ = A2, ▼ = A3); Graph B represents the 4-ketoaldose peaks (■ = K1, □ = K2, ▲ = K3).</p
Biphasic Catalysis with Disaccharide Phosphorylases: Chemoenzymatic Synthesis of α‑d‑Glucosides Using Sucrose Phosphorylase
Thanks
to its broad acceptor specificity, sucrose phosphorylase (SP) has
been exploited for the transfer of glucose to a wide variety of acceptor
molecules. Unfortunately, the low affinity (<i>K</i><sub>m</sub> > 1 M) of SP towards these acceptors typically urges the
addition of cosolvents, which often either fail to dissolve sufficient
substrate or progressively give rise to enzyme inhibition and denaturation.
In this work, a buffer/ethyl acetate ratio of 5:3 was identified to
be the optimal solvent system, allowing the use of SP in biphasic
systems. Careful optimization of the reaction conditions enabled the
synthesis of a range of α-d-glucosides, such as cinnamyl
α-d-glucopyranoside, geranyl α-d-glucopyranoside,
2-<i>O</i>-α-d-glucopyranosyl pyrogallol,
and series of alkyl gallyl 4-<i>O</i>-α-d-glucopyranosides. The usefulness of biphasic catalysis was further
illustrated by comparing the glucosylation of pyrogallol in a cosolvent
and biphasic reaction system. The acceptor yield for the former reached
only 17.4%, whereas roughly 60% of the initial pyrogallol was converted
when using biphasic catalysis