100 mV in the redox potential of the M520L and M520F variants of CotA from
Bacillus subtilis, even though the mutated variants suffered from a decrease in
catalytic activity. Targeting substrate specificity of CotA, Gupta et al. [62] obtained
a variant with an increase of more than 100-fold towards 2,2′-amino-bis
(3-ethylbenzothiazoline-6-sulfonic acid (ABTS, a model substrate of laccases and
peroxidases) and with even higher thermostability using a combinatorial approach
of rational design and directed evolution, focusing on the ABTS binding site of the
enzyme. Another CotA from Bacillus pumilus was also evolved using targeted
mutagenesis, resulting in variants with moderate increase in catalytic efficiency
towards ABTS [63]. By removing a methionine-rich helical region of the MCO
CueO from E. coli, resulting in a more accessible substrate binding site, Kataoka
et al. [64] generated an enzyme variant with 30 times increased activity against
ABTS. A recent breakthrough in genetical engineering of laccases came from
MetGen Oy, a commercial enzyme producer, with the product MetZyme
®
LIGNO
™
, a lignin oxidase originated from bacteria that can function at pH 10–11
and at high temperature up to 70 °C [65]. Other types of laccases, including SLAC
and fungal laccases, have also been subjected to protein engineering to increase
catalytic efficiency, substrate scope, expression, thermostability, as well as pH and
salt stability. Many more examples and approaches have been summarized in a
recent comprehensive review [60], as well as a review of laccase-related patents
from 2009 to 2019 [66].
An alternative to broaden the substrate scope of laccases is to improve the
laccase-mediator system (LMS). A LMS of many fungal laccases and
1-hydroxy-benzotriazole (HOBt) has been shown to be able to delignify kraft pulp
[67]. Other LMS consisting of T. versicolor laccase and ABTS or HOBt could
oxidize a phenolic b-O-4 linked lignin dimer (guaiacylglycerol-b-guaiacyl ether,
GBG) [68]. Natural laccase mediators originated from the lignin degradation process
such as vanillin, syringaldehyde, acetosyringone, p-coumaric acid, acetovanillone,
etc., have been studied intensively in LMS. A variety of synthetic substrates, namely
ABTS, HOBt, promazine, phenothiazine, 2,2,6,6-tetramethylpiperidinyloxy or
2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), have also been evaluated in different LMSs with the purpose of developing more affordable, effective, and stable
systems [69, 70]. Recently, Albarrán-Velo et al. synthesized and evaluated nitrogenated derivatives of vanillin and syringaldehyde as mediators in LMS of two
commercial laccases from T. versicolor and Myceliophthora thermophila [71].
A survey of the laccase-related patent landscape showed that 20% of the
patented application is within lignin removal and modification, followed by 15% in
biochemistry and biocatalysis, and 13% in pharma/cosmetics/agrochemicals [66].
Therefore, obtaining new laccases or laccase variants with high redox potentials and
substrate scope through data mining or protein engineering coupled with effective
mediators in new, low-cost LMS is of high interest for industrial applications of
these enzymes.
454
G.-S. Nguyen et al.
Bacillus subtilis, even though the mutated variants suffered from a decrease in
catalytic activity. Targeting substrate specificity of CotA, Gupta et al. [62] obtained
a variant with an increase of more than 100-fold towards 2,2′-amino-bis
(3-ethylbenzothiazoline-6-sulfonic acid (ABTS, a model substrate of laccases and
peroxidases) and with even higher thermostability using a combinatorial approach
of rational design and directed evolution, focusing on the ABTS binding site of the
enzyme. Another CotA from Bacillus pumilus was also evolved using targeted
mutagenesis, resulting in variants with moderate increase in catalytic efficiency
towards ABTS [63]. By removing a methionine-rich helical region of the MCO
CueO from E. coli, resulting in a more accessible substrate binding site, Kataoka
et al. [64] generated an enzyme variant with 30 times increased activity against
ABTS. A recent breakthrough in genetical engineering of laccases came from
MetGen Oy, a commercial enzyme producer, with the product MetZyme
®
LIGNO
™
, a lignin oxidase originated from bacteria that can function at pH 10–11
and at high temperature up to 70 °C [65]. Other types of laccases, including SLAC
and fungal laccases, have also been subjected to protein engineering to increase
catalytic efficiency, substrate scope, expression, thermostability, as well as pH and
salt stability. Many more examples and approaches have been summarized in a
recent comprehensive review [60], as well as a review of laccase-related patents
from 2009 to 2019 [66].
An alternative to broaden the substrate scope of laccases is to improve the
laccase-mediator system (LMS). A LMS of many fungal laccases and
1-hydroxy-benzotriazole (HOBt) has been shown to be able to delignify kraft pulp
[67]. Other LMS consisting of T. versicolor laccase and ABTS or HOBt could
oxidize a phenolic b-O-4 linked lignin dimer (guaiacylglycerol-b-guaiacyl ether,
GBG) [68]. Natural laccase mediators originated from the lignin degradation process
such as vanillin, syringaldehyde, acetosyringone, p-coumaric acid, acetovanillone,
etc., have been studied intensively in LMS. A variety of synthetic substrates, namely
ABTS, HOBt, promazine, phenothiazine, 2,2,6,6-tetramethylpiperidinyloxy or
2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), have also been evaluated in different LMSs with the purpose of developing more affordable, effective, and stable
systems [69, 70]. Recently, Albarrán-Velo et al. synthesized and evaluated nitrogenated derivatives of vanillin and syringaldehyde as mediators in LMS of two
commercial laccases from T. versicolor and Myceliophthora thermophila [71].
A survey of the laccase-related patent landscape showed that 20% of the
patented application is within lignin removal and modification, followed by 15% in
biochemistry and biocatalysis, and 13% in pharma/cosmetics/agrochemicals [66].
Therefore, obtaining new laccases or laccase variants with high redox potentials and
substrate scope through data mining or protein engineering coupled with effective
mediators in new, low-cost LMS is of high interest for industrial applications of
these enzymes.
454
G.-S. Nguyen et al.