Purification and characterization of two forms of the homologously expressed lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum

Семенова, М. В.; Gusakov, Alexander V.; Telitsin, V. D.; Рожкова, А. М.; Kondratyeva, E. G.; Sinitsyn, A. P.

doi:10.1016/j.bbapap.2019.140297

Cited by 23 publications

(12 citation statements)

References 47 publications

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“…The 2,6-DMP assay has been used in different studies to detect LPMOs peroxidase activity [13–15] to compare different LPMO fractions during purification [16] or to study the thermal stability of an LPMO [17]. However, some users have indicated that the 2,6-DMP assay does not work for certain LPMOs.…”

Section: Introductionmentioning

confidence: 99%

Improved spectrophotometric assay for lytic polysaccharide monooxygenase

Breslmayr

Daly

Požgajčić

et al. 2019

Biotechnol Biofuels

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BackgroundThe availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media.ResultsAn LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4–8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction.ConclusionsThe biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent KM value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.

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Section: Introductionmentioning

confidence: 99%

Improved spectrophotometric assay for lytic polysaccharide monooxygenase

Breslmayr

Daly

Požgajčić

et al. 2019

Biotechnol Biofuels

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“…In any case, besides the doubly oxidized products, the chromatogram showed peaks corresponding to oxidized products only in C1 or C4, which merits further investigation. It should be remarked that, as analyzing the regioselectivity of AA9 LPMOs produced by species from Penicillium or Talaromyces is a difficult task, authors usually do not report the C1 or C4 preference of the enzymes [46,56,57]. This work is expected to lay the foundations for developing further research in the AA9LPMOs produced by these genera, well known for Differences between PASC and CMC degradation can be observed mainly in the size of the peaks eluting at about 60 min (which is clearly more intense in PASC), and in the region of products with a double oxidation, that present many more peaks in CMC degradation.…”

Section: Pasc and Cmc Degradation Analysis By Hpaec-padmentioning

confidence: 99%

Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

Méndez-Líter

Ayuso‐Fernández

Csarman

et al. 2021

IJMS

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The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

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“…The pH curves were plotted using the highest values of absorbance obtained in the analysis for each pH in the scan analysis and showed that AfAA9A and AfAA9B are more active at pH 8 (Figure 2.7). LPMOs with better performance at pH higher than 7 have already been reported both for peroxidase activity in the Breslmayr assay with 2,6-DMP (Semenova et al, 2020) and for activity on cellulose (Breslmayr et al, 2018;Frommhagen et al, 2018b;Hegnar et al, 2019). AfAA9A was more stable at all tested pH, maintaining residual activity above 90% for 24 h, whereas AfAA9B showed a slight decrease in activity after 6 h, losing up to 38 and 22% after 24 h in pH 6 and 7, respectively (Figure 2.7A).…”

Section: Lpmo Functional Characterizationmentioning

confidence: 61%

“…The LPMO from the fungus Penicillium verruculosum (PvLPMO9A), an enzyme with C-terminal extension was studied in comparison with the same protein without this extra peptide. The analysis showed that the two enzymes had a very similar catalytic behavior such as its optimal pH and temperature values, including the collaborative effect with cellulases, except for the thermostability that was superior in full-size enzyme (Semenova et al, 2020). This structural feature opens new perspectives in the LPMOs field.…”

Section: Modular Architecture and C-terminal Extensionsmentioning

confidence: 88%

“…Among the common LPMO domains are the carbohydrate-binding modules (CBMs) that favor the attachment/interaction of enzyme with the substrate and also provide protection against the auto-oxidation process (Courtade et al, 2018;Forsberg et al, 2017). Recently, the presence of additional modules (different from CBMs) and C-terminal extensions, of unknown biological activity, has been reported in different LPMO families (Hemsworth et al, 2014;Lenfant et al, 2017;Semenova et al, 2020). Hemsworth et al described the presence of a conserved domain which is additional to the catalytic domain of a LPMO from AA11 family.…”

Section: Modular Architecture and C-terminal Extensionsmentioning

confidence: 99%

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Novel Aspergillus fumigatus recombinant LPMOs: biochemical characteristics, and their participation in saccharification and photobiocatalysis processes

Mendoza¹

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Purification and characterization of two forms of the homologously expressed lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum

Cited by 23 publications

References 47 publications

Improved spectrophotometric assay for lytic polysaccharide monooxygenase

Improved spectrophotometric assay for lytic polysaccharide monooxygenase

Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

Novel Aspergillus fumigatus recombinant LPMOs: biochemical characteristics, and their participation in saccharification and photobiocatalysis processes

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