The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on β-Glucan

Abstract

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50°C on β-glucan. Under these conditions specific activity was 239.2±9.1 U mg−1 and the half-life of the enzyme was 84.6±3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2±0.5 mg mL−1 and the Vmax was 0.41±0.02 µmol min−1. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme

“…This might indicate that for the optimum enzymatic activity, ThCel7B must be flexible and that additional enzyme stability might have penalties for its enzymatic activity. A similar difference in pH conditions for the enzyme optimum activity vs. stability has been previously observed for GtCel12A (Miotto et al 2014). Residual enzymatic activity of ThCel7B at highly acidic conditions was quite considerable, since the enzyme maintained 80 % of its initial activity after more than 8 h of incubation at pH 3.0 and even after 72 h it still retained about 20 % of the initial activity (Fig.…”

“…This indicates that the full-length construct is highly glycosylated at the linker peptide region, as it has been observed for other fungal endoglucanases (Sandgren et al 2005). Other recombinant enzymes produced in the A. niger expression system display low pH optimum (see, for example, Miotto et al 2014), which could in theory be attributed to the differences in glycosylation profile of the recombinant proteins produced in such system. However, our data also suggest that CCD has few or no post-translational modifications and it has the same highly acidophilic character of the full-length enzyme (Fig.…”

“…Most of the fungal endoglucanases has optima activity at a temperature around 50 to 55°C (Ding et al 2001;Saha 2004). These biochemical characteristics of ThCel7B might be of biotechnological interest, mainly for second-generation ethanol production, which frequently involves processes run at acidic pH values (Miotto et al 2014).…”

“…1c, d). GtCel12A endoglucanase also has a considerable acidophilic character with pH optimum at 4.5 (Miotto et al 2014). The enzyme was capable to retain about 40 % of its activity at pH 1.0.…”

Enhanced Endoglucanase Production by Bacillus aerius on Mixed Lignocellulosic Substrates

“…This result was attributed to the higher level of hydrolysis of CMC compared with the other substrates. Endoglucanase has high substrate specificity for CMC and lower specificity for crystalline forms of cellulose (Kim 1995;Dobrev and Zhekova 2012;Miotto et al 2014). Hence, endoglucanase, produced by the medium, must have hydrolysed the CMC better, thus producing higher amounts of reducing sugar than the other substrates.…”