Taxonomic identification of the thermotolerant and fast-growing fungus Lichtheimia ramosa H71D and biochemical characterization of the thermophilic xylanase LrXynA

María Teresa Alvarez-Zúñiga,Jorge E Campos,Alejandro Santiago-Hernández,María Eugenia Hidalgo-Lara,Sergio Trejo-Estrada,Johan Rodríguez-Mendoza,Patricia Pavón-Orozco

doi:10.1186/s13568-017-0494-y

Abstract

The zygomycete fungus Lichtheimia ramosa H71D, isolated from sugarcane bagasse compost, was identified by applying phylogenetic analysis based on the DNA sequence of the Internal Transcribed Spacer (ITS), and subsequent secondary structure analysis of ITS2. L. ramosa H71D was able to grow over a wide range of temperatures (25–45 °C), manifesting optimal growth at 37 °C. A 64 kDa xylanase (named LrXynA) was purified from the culture supernatant of L. ramosa H71D grown on 2% carboxymethylcellulose (CMC), as the only carbon source. LrXynA displayed optimal activity at pH 6 and temperature of 65 °C. The enzyme retained more than 50% of its maximal activity over a broad range of pH values (4.5–7.5). Enzyme half-life (t½) times at 55, 65 and 75 °C were 80, 25, and 8 min, respectively. LrXynA showed higher affinity (kM of 2.87 mg/mL) and catalytic efficiency (kcat/kM of 0.651 mg s/mL) towards Beechwood xylan in comparison to other substrates such as Birchwood xylan, Oat-spelt xylan, CMC, Avicel and Solka floc. The predominant final products from LrXynA-mediated hydrolysis of Beechwood xylan were xylobiose and xylotriose, suggesting that the enzyme is an endo-β-1,4 xylanase. Scanning electron microscopy (SEM) imaging of sugar cane bagasse (SCB) treated with LrXynA, alone or in combination with commercial cellulases, showed a positive effect on the hydrolysis of SCB. To our knowledge, this is the first report focusing on the biochemical and functional characterization of an endo-β-1,4 xylanase from the thermotolerant and fast-growing fungus Lichtheimia ramosa.

Highlights

Xylan is in order to cellulose, in terms of the major structural components of plant cell walls, and is the second most abundant renewable polysaccharide in nature (Collins et al 2002)
In the context of fungi, Ascomycetes and Basidiomycetes have been widely studied; very few studies focus on Zygomycetes and their enzymes. This is why our study focused on the biochemical and functional characterization of one of the enzymes involved in the xylanolytic activity produced by the thermotolerant Zygomycete, L. ramosa H71D
It has been reported that the ITS2 secondary structure analysis can improve the phylogenetic resolution obtained from the primary sequence (Keller et al 2008), and the combination and simultaneous analysis of sequence and structural ITS2 RNA data supplemented with indel coding binaries yielded robust phylogenetic hypotheses as measured by bootstrap values for ancestral haplotypes (Poczai et al 2015)

Summary

Introduction

Xylan is in order to cellulose, in terms of the major structural components of plant cell walls, and is the second most abundant renewable polysaccharide in nature (Collins et al 2002). Xylanases and the microorganisms that produce them are interesting because they have extensive biotechnological applications. These enzymes are currently used in waste management in order to degrade xylan for the production of renewable fuels and chemicals. Likewise, they are used in food, agro-fiber, and paper and pulp industries, where xylanases help to reduce environmental impact (Collins et al 2002). In terms of biotechnological application, thermostable enzymes have several generic advantages as the high specific activity, that is often associated with this kind of enzymes, reduces the required amount of enzyme and prolongs hydrolysis time due to greater stability than that exhibited by mesophilic enzymes (Viikari et al 2007)

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