P. V. Volkov

N‐linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity

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. Biotechnol. Bioeng. 2016;113: 283–291.

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.

Comparative study of biochemical properties of glucoamylases from the filamentous fungi Penicillium and Aspergillus

Here we report the first isolation to homogeneous forms of two glucoamylases from the fungus Penicillium verruculosum and their study in comparison with known glucoamylases from Aspergillus awamori and Aspergillus niger. Genes that encode glucoamylases from P. verruculosum were cloned and expressed in the fungus Penicillium canescens, and the recombinant glucoamylases were obtained with subsequent study of their molecular weights, isoelectric points, optimal temperature and pH values, and stability. The catalytic activities of the recombinant glucoamylases were determined in relation to soluble potato starch. Changes in molecular mass distribution and content of low molecular weight products during starch hydrolysis by glucoamylases from P. verruculosum, A. awamori, and A. niger were studied. An exo-depolymerization mechanism was established to be the pathway for destruction of starch by the glucoamylases.

Isolation and properties of recombinant inulinases from Aspergillus sp.

The genes inuA and inu1, encoding two inulinases (32nd glycosyl hydrolase family) from filamentous fungi Aspergillus niger and A. awamori, were cloned into Penicillium canescens recombinant strain. Using chromatographic techniques, endoinulinase InuA (56 kDa, pI 3) and exoinulinase Inu1 (60 kDa, pI 4.3) were purified to homogeneity from the enzymatic complexes of P. canescens new transformants. The properties, such as substrate specificity, pH- and T-optima of activity, stability at different temperatures, influence of cations and anions on the catalytic activity, etc., of both recombinant inulinases were studied.

Using an inducible promoter of a gene encoding Penicillium verruculosum glucoamylase for production of enzyme preparations with enhanced cellulase performance

Background Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases. Results Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1. Conclusions The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable to express both heterologous enzymes simultaneously.

Glucoamylases from Penicillium verruculosum and Myceliophthora thermophila: analysis of differences in activity against polymeric substrates based on 3D model structures of the intact enzymes.

Two glucoamylases, a recombinant enzyme from Penicillium verruculosum (PvGla) heterologously expressed in Penicillium canescens RN3-11-7 (niaD-) strain and a native glucoamylase from Myceliophthora thermophila (MtGla), were purified and their properties were studied. MtGla displayed 2-5-fold higher specific activities against soluble starch, amylose and amylopectin than PvGla. MtGla also provided higher glucose yields in extended hydrolysis of the polymeric substrates. Analysis of 3D model structures of the intact PvGla and MtGla, which were built using the 2vn7.pdb crystal structure of the intact Trichoderma reesei glucoamylase (TrGla) as a template, showed that the reason for lower hydrolytic performance of PvGla in comparison to MtGla may be less strong interactions between the enzyme domains as well as a longer (by 17 residues) linker in the first enzyme.

Scythian Trepanations in the Gorny Altai in Hippocratic Times: Modern Expert Appraisal of Ancient Surgical Technologies

OBJECTIVE To report the analysis of 3 cases of ancient trepanation discovered in the craniological collection (153 skulls) of the Pazyryk nomadic culture (500-300 bc) from the Gorny Altai, Russia, and to evaluate the technique, instrumentation, and materials used for cranial surgery as well as the motivation for the trepanations in Scythian times. METHODS A multidisciplinary approach was chosen to study the trepanned skulls. Visual inspection and examination under magnification, multislice computed tomography, high-field magnetic resonance imaging, and coupled plasma mass spectrometry and synchrotron radiation-induced x-ray fluorescence analysis of the bone samples from the site of trephination were used. RESULTS In the Pazyryk culture, trepanation was very likely used to perform the intracranial procedures that were not yet indicated by Hippocrates. No signs of bone infection were detected. Higher copper abundance found at the site of trepanation showed that a bronze knife was the most likely tool used by Scythian surgeons. CONCLUSIONS Our data suggest that the Scythian population of the Altai Mountains had sufficient medical knowledge to perform sophisticated and successful manipulations on the human skulls. Scraping technique with bronze tools was quite effective for prevention of wound infection and resulted in a high survival rate after surgery. In the era of methicillin-resistant Staphylococcus aureus, it may be useful to consider some ancient surgical technologies.

Enzymatic Hydrolysis of Cellulose Using Mixes of Mutant Forms of Cellulases from Penicillium verruculosum

Cellulases are the major components of multienzyme systems applied in processes of bioconversion of renewable lignocellulosic feedstocks to various useful products. The hydrolytic efficiency of enzyme mixes based on recombinant wild-type endoglucanase II, cellobiohydrolases I and II from the Penicillium verruculosum fungus (in the presence of Aspergillus niger β-glucosidase) with mixes of mutant forms of these enzymes in the hydrolysis of cellulosic materials is compared, and the influence of temperature and substrate concentration on the glucose yield is studied. The mutant cellulases represented proteins, in which N-linked glycans were partially removed using site-directed mutagenesis. In the hydrolysis of microcrystalline cellulose and milled aspen wood by mixes of mutant cellulases, the yields of glucose after 24–72 h of an enzymatic reaction were higher by 31–38% and 11–27%, respectively, than those for the compositions based on the wild-type enzymes. The highest product concentrations, using mutant enzyme compositions, are achieved at 50°С when the hydrolysis temperature is varied in the range of 40 to 60°С. Increasing the substrate concentration in the reaction system from 5 to 50 g/L (while maintaining the enzyme dosage at the same level) led to a 2.6–2.8-fold increase in the glucose yield, accompanied by a decrease in the cellulose conversion degree.

Properties of Chimeric Polysaccharide Monooxygenase with an Attached Cellulose Binding Module and Its Use in the Hydrolysis of Cellulose-Containing Materials in the Composition of Cellulase Complexes

The use of recently discovered polysaccharide monooxygenases (PMO) in the composition of cellulase complexes greatly enhances their saccharification ability. Genetic engineering is used in this work to produce a chimeric enzyme based on the Thielavia terrestris PMO with cellulose binding module (CBM) from the Penicillium verruculosum cellobiohydrolase I attached to the PMO С-terminus via a peptide linker. Chimeric PMO exhibits higher (by 24%) activity toward amorphous cellulose and wider substrate specificity than the initial PMO. As a result of the CBM attachment, chimeric PMO acquires the ability to cleave xylan and carboxymethyl cellulose in addition to cellulose and β-glucan, and its activity toward xyloglucan increases by one order of magnitude. Replacing 10% of the highly active cellulase preparation hBGL2 produced by P. verruculosum with the chimeric PMO while retaining the overall dose of the enzymes with regard to their protein concentration increases the yield of sugars during the hydrolysis of microcrystalline cellulose and powdered aspen wood by 24 and 47%, respectively. In addition, the maximum yield of sugars during wood hydrolysis is achieved in 24 h of reaction time, in contrast to hydrolysis with the indicated preparation without the added PMO, which requires 48 h.

Using an Inducible Promoter of the Glucoamylase Gene to Construct New Multienzyme Complexes from Penicillium verruculosum

New recombinant strains of Penicillium verruculosum are created using a new plasmid construct based on an inducible promoter of glucoamylase gene (gla1) that secretes heterologous xylanase E (XylE) from P. canescens. New biocatalysts are produced that contain cellulolytic enzyme preparations (EPs) enriched with XylE. The amount of XylE in the recombinant EPs varies in the optimum range of 11–24% of the total protein while generally preserving the P. verruculosum cellulose complex. The hydrolytic activity of the new EPs with respect to polymeric plant-derived substrates exceeds that of EPs produced using other expression systems and commercial preparations. The new EP glaX-17 surpasses in particular a control EP based on the recipient strain by 13% in the efficiency of hydrolyzing aspen wood and is 20% more efficient than the commercial EP Accelerase Duet. The new EP glaX-17 displays 25% greater efficiency (35–43%) during the hydrolysis of wheat bran than the commercial EP Accelease Duet. The effectiveness is demonstrated of using the new gla1 promoter for the production of EPs (biocatalysts) while preserving the balanced cellulose complex of the strain and optimum yield of heterologous XylE required for the deep hydrolysis of xylan-containing plant biomass.