Abstract:Improved understanding of cellulose swelling mechanism is beneficial for increasing the hydrolysis efficiency of cellulosic substrates. Here, we report a family 5 glycoside hydrolase ArCel5 isolated from the cellulose-gelatinizing fungus Arthrobotrys sp. CX1. ArCel5 exhibited low specific hydrolysis activity and high cellulose swelling capability, which suggested that this protein might function as an accessory protein. Homology modeling glycosylation detection revealed that ArCel5 is a multi-domain protein in… Show more
“…The peptides of Mi Xly and the mutants of Ec Xly were obtained by in-gel digestion and used for LC–MS/MS analysis (De Godoy et al 2006 ). The acquired MS data were analyzed using Proteome Discoverer 2.2.1 software as previously described (Yuan et al 2022 ). Fig.…”
Modified xanthan produced by xanthan lyase has broad application prospects in the food industry. However, the catalytic performance of xanthan lyase still needs to be improved through rational design. To address this problem, in this work, the glycosylation and its influences on the catalytic performance of a xanthan lyase (EcXly), which was heterologously expressed in Escherichia coli, were reported. Liquid chromatography coupled to tandem mass spectrometry analysis revealed that the N599 site of EcXly was modified by a single N-glycan chain. Based on sequence alignment and three-dimensional structure prediction, it could be deduced that the N599 site was located in the catalytic domain of EcXly and in close proximity to the catalytic residues. After site-directed mutagenesis of N599 with alanine, aspartic acid and glycine, respectively, the EcXly and its mutants were characterized and compared. The results demonstrated that elimination of the N-glycosylation had diminished the specific activity, pH stability, and substrate affinity of EcXly. Fluorescence spectra further revealed that the glycosylation could significantly affect the overall tertiary structure of EcXly. Therefore, in prokaryotic hosts, the N-glycosylation could influence the catalytic performance of the enzyme by changing its structure. To the best of our knowledge, this is the first report about the post-translational modification of xanthan lyase in prokaryotes. Overall, our work enriched research on the role of glycan chains in the functional performance of proteins expressed in prokaryotes and should be valuable for the rational design of xanthan lyase to produce modified xanthan for industrial application.
Graphical Abstract
“…The peptides of Mi Xly and the mutants of Ec Xly were obtained by in-gel digestion and used for LC–MS/MS analysis (De Godoy et al 2006 ). The acquired MS data were analyzed using Proteome Discoverer 2.2.1 software as previously described (Yuan et al 2022 ). Fig.…”
Modified xanthan produced by xanthan lyase has broad application prospects in the food industry. However, the catalytic performance of xanthan lyase still needs to be improved through rational design. To address this problem, in this work, the glycosylation and its influences on the catalytic performance of a xanthan lyase (EcXly), which was heterologously expressed in Escherichia coli, were reported. Liquid chromatography coupled to tandem mass spectrometry analysis revealed that the N599 site of EcXly was modified by a single N-glycan chain. Based on sequence alignment and three-dimensional structure prediction, it could be deduced that the N599 site was located in the catalytic domain of EcXly and in close proximity to the catalytic residues. After site-directed mutagenesis of N599 with alanine, aspartic acid and glycine, respectively, the EcXly and its mutants were characterized and compared. The results demonstrated that elimination of the N-glycosylation had diminished the specific activity, pH stability, and substrate affinity of EcXly. Fluorescence spectra further revealed that the glycosylation could significantly affect the overall tertiary structure of EcXly. Therefore, in prokaryotic hosts, the N-glycosylation could influence the catalytic performance of the enzyme by changing its structure. To the best of our knowledge, this is the first report about the post-translational modification of xanthan lyase in prokaryotes. Overall, our work enriched research on the role of glycan chains in the functional performance of proteins expressed in prokaryotes and should be valuable for the rational design of xanthan lyase to produce modified xanthan for industrial application.
Graphical Abstract
Plastic pollution is a major global concern to the health and wellbeing of all terrestrial and marine life. However, no sustainable method for waste management is currently viable. This study addresses the optimisation of microbial enzymatic polyethylene oxidation through rational engineering of laccases with carbohydrate-binding module (CBM) domains. An explorative bioinformatic approach was taken for high-throughput screening of candidate laccases and CBM domains, representing an exemplar workflow for future engineering research. Molecular docking simulated polyethylene binding whilst a deep-learning algorithm predicted catalytic activity. Protein properties were examined to interpret the mechanisms behind laccase-polyethylene binding. The incorporation of flexible GGGGS(x3) hinges were found to improve putative polyethylene binding of laccases. Whilst CBM1 family domains were predicted to bind polyethylene, they were suggested to detriment laccase-polyethylene associations. In contrast, CBM2 domains reported improved polyethylene binding and may thus optimise laccase oxidation. Interactions between CBM domains, linkers, and polyethylene hydrocarbons were heavily reliant on hydrophobicity. Preliminary polyethylene oxidation is considered a necessity for consequent microbial uptake and assimilation. However, slow oxidation and depolymerisation rates inhibit the large-scale industrial implementation of bioremediation within waste management systems. The optimised polyethylene oxidation of CBM2-engineered laccases represents a significant advancement towards a sustainable method of complete plastic breakdown. Results of this study offer a rapid, accessible workflow for further research into exoenzyme optimisation whilst elucidating mechanisms behind the laccase-polyethylene interaction.
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