Understanding
the thermostability of cellulases is of high importance
for their application in lignocellulosic biomass degradation,
feedstock, and pulp and paper production. Cellulases have to withstand
high temperatures and harsh conditions in various application areas,
for instance, in bioethanol production. Engineering thermostable
cellulases increases the cellulase lifetime in processes and contributes
to more-sustainable production. Here we report the first KnowVolution
campaign toward improving the thermostability of the endo-β-1,4-glucanase PvCel5A from Penicillium
verruculosum. The C-terminal region of PvCel5A (eighth
α-helix, amino acid residues 280–314) was identified
as a key structural determinant to improve the thermostability
of PvCel5A without affecting its specific activity. The most beneficial
variant, PvCel5A-R17, harbors three substitutions (F16L/Y293F/Q289G);
its half-life at 75 °C improved 5.5-fold (from 32 to 175 min)
and the melting temperature was raised 7.7 °C (from 70.8 °C)
when compared to those of wild-type PvCel5A. Exceptionally, the thermally
improved PvCel5A-R17 variant retained its specific activity at low
temperatures (40 °C). Computational analyses revealed that the
stabilization of the C-terminal region of PvCel5A is responsible for
the improved thermostability. This knowledge will facilitate
shorter times in cellulase engineering and thereby enhance the performance
and sustainability of processes.
Cellobiohydrolase I from Penicillium verruculosum (PvCel7A) has four potential N-glycosylation sites at its catalytic module: Asn45, Asn194, Asn388, and Asn430. In order to investigate how the N-glycosylation influences the activity and other properties of the enzyme, the wild type (wt) PvCel7A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens PCA10 (niaD-) strain, a fungal host for production of heterologous proteins. The rPvCel7A-wt and N45A, N194A, N388A mutants were successfully expressed and purified for characterization, whereas the expression of N430A mutant was not achieved. The MALDI-TOF mass spectrometry fingerprinting of peptides, obtained as a result of digestion of rPvCel7A forms with specific proteases, showed that the N-linked glycans represent variable high-mannose oligosaccharides and the products of their sequential enzymatic trimming, according to the formula (Man)0-13 (GlcNAc)2 , or a single GlcNAc residue. Mutations had no notable effect on pH-optimum of PvCel7A activity and enzyme thermostability. However, the mutations influenced both the enzyme adsorption ability on Avicel and its activity against natural and synthetic substrates. In particular, the N45A mutation led to a significant increase in the rate of Avicel and milled aspen wood hydrolysis, while the substrate digestion rates in the case of N194A and N388A mutants were notably lower relative to rPvCel7A-wt. These data, together with data of 3D structural modeling of the PvCel7A catalytic module, indicate that the N-linked glycans are an important part of the processive catalytic machinery of PvCel7A.
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