Heterologous Expression of Thermogutta terrifontis Endo-Xanthanase in Penicillium verruculosum, Isolation and Primary Characterization of the Enzyme.

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, ß-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of ß-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Designing enzyme cocktails from Penicillium and Aspergillus species for the enhanced saccharification of agro-industrial wastes.

The aim of this study was to develop optimized enzyme cocktails, containing native and recombinant purified enzymes from five fungal species, for the saccharification of alkali- and acid-pretreated sugarcane bagasse (SCB), soybean hulls (SBH) and oil palm empty fruit bunches (EFB). Basic cellulases were represented by cellobiohydrolase I (CBH) and endo-glucanase II (EG) from Penicillium verruculosum and ß-glucosidase (BG) from Aspergillus niger. Auxiliary enzymes were represented by endo-xylanase A (Xyl), pectin lyase (PNL) and arabinoxylanhydrolase (AXH) from Penicillium canescens, ß-xylosidase (BX) from Aspergillus japonicus, endo-arabinase (ABN) from A. niger and arabinofuranosidase (Abf) from Aspergillus foetidus. Enzyme loads were 5 mg protein/g dry substrate (basic cellulases) and 1 mg/g (each auxiliary enzyme). The best choice for SCB and EFB saccharification was alkaline pretreatment and addition of Xyl + BX, AXH + BX or ABN + BX + Abf to basic cellulases. For SBH, acid pretreatment and basic cellulases combined with ABN + BX + Abf or Xyl + BX performed better than other enzyme preparations.

Cloning, purification and study of recombinant GH3 family ß-glucosidase from Penicillium verruculosum.

A novel bgl1 gene, encoding GH3 family ß-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 °C was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis.

Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species.

The dexA gene encoding Penicillium funiculosum dextranase (GenBank accession MH581385) belonging to family 49 of glycoside hydrolases (GH49) was cloned and heterologously expressed in two recipient strains, P. canescens RN3-11-7 and P. verruculosum B1-537. Crude enzyme preparations with the recombinant dextranase content of 8-36% of the total secreted protein were obtained on the basis of new Penicillium strains. Both recombinant forms of the dextranase were isolated in a homogeneous state using chromatographic techniques. The purified enzymes displayed very similar properties, that is, pI 4.55, activity optima at pH 4.5-5.0 and 55-60 °C and a melting temperature of 60.7-60.9 °C. They were characterized by similar specific activities (1020-1340 U/mg) against dextrans with a mean molecular mass of 20, 70 and 500 kDa, as well as similar kinetic parameters in the hydrolysis of 70 kDa dextran (Km = 1.10-1.11 g/L, kcat = 640-680 s-1). However, the recombinant dextranases expressed in P. canescens and P. verruculosum had different molecular masses according to the data of SDS-PAGE (∼63 and ∼60 kDa, respectively); this was the result of different N-glycosylation patterns as MALDI-TOF mass spectrometry analysis showed. The main products of dextran hydrolysis at its initial phase were isomaltooligosaccharides, while after the prolonged time (24 h) the reaction system contained isomaltose and glucose as the major products and minor amounts of other oligosaccharides.

Homologous cloning, purification and characterization of highly active cellobiohydrolase I (Cel7A) from Penicillium canescens.

Penicillium canescens is a filamentous fungus that normally does not secrete notable levels of cellulase activity. Cellobiohydrolase I of P. canescens (PcCel7A) was homologously cloned into a host strain RN3-11-7 (niaD-) and then expressed under the control of a strong xylA promoter. Using three steps of chromatography, PcCel7A was purified. The enzyme displayed maximum activity at pH 4.0-4.5. PcCel7A was stable at 50°C and pH 4.5 at least for 3h, while at 60°C it lost 45% of activity after 30min of incubation. When equalized by protein concentration, PcCel7A demonstrated a higher performance in prolonged hydrolysis of Avicel and milled aspen wood than CBH I (Cel7A) from Trichoderma reesei, the most industrially utilized cellulase at this moment. The high catalytic efficiency of the PcCel7A makes it a potential candidate for industrial applications.

N-glycosylation patterns in two α-l-arabinofuranosidases from Penicillium canescens belonging to the glycoside hydrolase families 51 and 54.

Using MALDI-TOF mass spectrometry (MS) peptide fingerprinting procedure followed by the analysis of MS data with the GlycoMod tool from the ExPASy proteomic site, N-glycosylation of two GH51 and GH54 family α-l-arabinofuranosidases (Abf51A and Abf54A) from Penicillium canescens was studied. Variable N-linked glycans were identified at five out of eight potential N-glycosylation sites in the Abf51A and one out of three potential N-glycosylation sites in the Abf54A. The discriminated glycans represented high-mannose oligosaccharides (Man)x(GlcNAc)2 with a number of Man residues up to 7 or the products of sequential enzymatic trimming of a high-mannose glycan with α-mannosidases and ß-N-acetylhexosaminidases. The Abf54A peptide, containing the Asn254 glycosylation site, and one peptide from the Abf51A, containing the Asn163 glycosylation site, were found to exist not only in glycosylated, but also in a native non-modified form.

Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase.

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.