\u03b2-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin

Malgorzata Krolicka,Carmen G Boeriu,Martijn J Koetsier,Gerrit Eggink,Sandra W A Hinz,Lambertus A M Van Den Broek

doi:10.1007/s00253-018-9166-3

Malgorzata Krolicka, Carmen G Boeriu + Show 4 more

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https://doi.org/10.1007/s00253-018-9166-3

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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 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 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.

Highlights

Large amounts of polysaccharides present in nature are an excellent source of valuable sugars, and chitin is one of them
Hydrolysis products were analyzed by highperformance anion-exchange chromatography (HPAEC) and each reported value was the average of duplicate tests
The deduced 582-amino acid sequence of MthNAG was predicted to have a molecular weight of 62.6 kDa, a theoretical pI of 5.20, an N-terminal 21-amino acid signal peptide, three potential N-linked glycosylation sites, and two potential O-linked glycosylation sites

Summary

Introduction

Large amounts of polysaccharides present in nature are an excellent source of valuable sugars, and chitin is one of them. Chitin is a vastly abundant polysaccharide and is used for the production of chitosan, chitin oligosaccharides, and GlcNAc (Kardas et al 2012). The deacetylated derivative of chitin, has wide application in many fields including medicine, pharmacology, environmental protection, and biobased packaging GlcNAc is considered as an inexpensive and non-toxic treatment for numerous diseases including osteoarthritis, inflammatory bowel disease, viral or bacterial infections, intestinal diseases, and cancer and for proliferation of skin cells during wound healing (Dalirfardouei et al 2016; Salvatore et al 2000; Xu et al 2006; Xu et al 2007a, b; Minami and Okamoto 2007). GlcNAc was proposed as a biological C6 source for bioethanol production through fermentation. It was reported that fermentation with Mucor circinelloides NBRC6746 and Mucor ambiguous NBRC8092 yielded approximately 18.6 and 16.9 g/L ethanol from 50 g/L GlcNAc, respectively (Inokuma et al 2013)

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