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?-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

ABSTRACT: Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable ?-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

SUBMITTER: Krolicka M

PROVIDER: S-EPMC6097783 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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β-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

Krolicka Malgorzata M Hinz Sandra W A SWA Koetsier Martijn J MJ Eggink Gerrit G van den Broek Lambertus A M LAM Boeriu Carmen G CG

Applied microbiology and biotechnology 20180625 17

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable β-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum ...[more]

PMID: 29943052

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Project description:BackgroundLytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity.ResultsHere, we show that three LPMOs from the fungus Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β-(1 → 3, 1 → 4)-glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β-(1 → 3, 1 → 4)-glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds.ConclusionsSeveral of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity toward plant-derived reducing agents in situ.

Project description:BackgroundFilamentous fungi with the ability to use complex carbon sources has been developed as platforms for biochemicals production. Myceliophthora thermophila has been developed as the cell factory to produce lignocellulolytic enzymes and plant biomass-based biofuels and biochemicals in biorefinery. However, low fungal growth rate and cellulose utilization efficiency are significant barriers to the satisfactory yield and productivity of target products, which needs our further exploration and improvement.ResultsIn this study, we comprehensively explored the roles of the putative methyltransferase LaeA in regulating mycelium growth, sugar consumption, and cellulases expression. Deletion of laeA in thermophile fungus Myceliophthora thermophila enhanced mycelium growth and glucose consumption significantly. Further exploration of LaeA regulatory network indicated that multiple growth regulatory factors (GRF) Cre-1, Grf-1, Grf-2, and Grf-3, which act as negative repressors of carbon metabolism, were regulated by LaeA in this fungus. We also determined that phosphoenolpyruvate carboxykinase (PCK) is the core node of the metabolic network related to fungal vegetative growth, of which enhancement partially contributed to the elevated sugar consumption and fungal growth of mutant ΔlaeA. Noteworthily, LaeA participated in regulating the expression of cellulase genes and their transcription regulator. ΔlaeA exhibited 30.6% and 5.5% increases in the peak values of extracellular protein and endo-glucanase activity, respectively, as compared to the WT strain. Furthermore, the global histone methylation assays indicated that LaeA is associated with modulating H3K9 methylation levels. The normal function of LaeA on regulating fungal physiology is dependent on methyltransferase activity.ConclusionsThe research presented in this study clarified the function and elucidated the regulatory network of LaeA in the regulation of fungal growth and cellulase production, which will significantly deepen our understanding about the regulation mechanism of LaeA in filamentous fungi and provides the new strategy for improvement the fermentation properties of industrial fungal strain by metabolic engineering.

Dataset Information

?-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

Publications

β-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

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