Aleksandra M. Rozhkova

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.

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability.

N-Linked glycans are an important component of the processive machinery of cellobiohydrolases

Cellobiohydrolases (CBHs), belonging to glycoside hydrolase families 6 and 7 (GH6 and GH7), are the major components of cellulase systems of filamentous fungi involved in biodegradation of cellulose in nature. Previous studies demonstrated that N-linked glycans in the catalytic domains of GH7 CBHs significantly affect the enzyme activity against cellulosic substrates. The influence of N-linked glycans on the activity and processivity of recombinant GH6 CBH II from Penicillium verruculosum (PvCel6A) was studied using site-directed mutagenesis of the respective Asn residues. Depending on the position of N-glycans on the surface of a protein globule, they affected the enzyme activity against cellulose either negatively or positively. The decrease or increase in the degree of processivity of recombinant forms of PvCel6A generally correlated with activity changes against Avicel. The mechanism of the N-glycan influence seems to be universal for GH6 and GH7 CBHs. The observed effects for CBHs from both families are explained in terms of a mechanistic model that also makes clear our previously published data on the highly active CBH IIb from Myceliophthora thermophila (MtCel6B). This study, together with data of other researchers, strongly suggests that the N-linked glycans in the catalytic domains of GH6 and GH7 CBHs are involved in processive catalytic machinery of these enzymes. Data obtained should be taken into account during development of new and more effective biocatalysts by protein engineering techniques.

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.

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.

Properties and N-glycosylation of recombinant endoglucanase II from Penicillium verruculosum

Cellulases are the main components of enzyme complexes used in biotransformation processes of plant raw materials into valuable commercial products. Endoglucanase II (EG II) from the Penicillium verruculosum fungus was cloned into Penicillium canescens. The homogeneous recombinant EGII form is isolated and its properties are studied in comparison with the native enzyme. The N-glycosylation sites and the structure of the N-linked glycans are been determined using mass spectrometry. The biochemical and catalytic properties, as well as the N-glycosylation type of the obtained recombinant EGII form, appear to be close to the native enzyme. At the two potential N-glycosylation sites (N42 and N194) of both forms of the enzyme, N-linked high mannose glycans (or their enzymatic “trimming” products) according to the general formula (Man)1–9(GlcNAc)2 are detected. No glycosylation is found at the third potential site (N19).

Optimizing the composition of cellulase enzyme complex from Penicillium verruculosum: Enhancing hydrolytic capabilities via genetic engineering

Modern technologies for the enzyme hydrolysis of cellulose-containing raw materials allow the production of sugars from which alcohols (biofuel), organic and amino acids, biopolymers, feed additives, and other value-added products can be obtained via microbiological conversion. Three types of cellulolytic enzymes are required for the bioconversion of cellulose containing materials: endoglucanase, cellobiohydrolase, and ß-glucosidase. The prospects for improving the hydrolytic capabilities of the enzyme complex secreted from Penicillium verruculosum are investigated in this work by means of genetic engineering to add different combinations and ratios of homologous and heterologous cellulases: endoglucanase IV (EGIV) of Trichoderma reesei, endoglucanase II (EGII), and cellobiohydrolase I (CBHI) of P. verruculosum, along with ß-glucosidase (ß-GLU) of Aspergillus niger. The optimum ratio of components is determined and the catalytic activity of enzymatic complexes is increased by as much as 100%.

Trichoderma reesei endoglucanase IV: A new component of biocatalysts based on the cellulase complex of the fungus Penicillium verruculosum for hydrolysis of cellulose-containing biomass

The problem of raising the efficiency of enzyme preparations catalyzing cellulose conversion is among the present-day technological challenges. Here, we report the enhancement of the hydrolytic capacity of cellulase preparations by introducing nonhydrolytic enzymes (polysaccharide monooxygenases) into the cellulolytic complex. An enzyme preparation with an increased hydrolytic capacity has been obtained from the recombinant strain of the fungus Penicillium verruculosum that carries the Trichoderma reesei endoglucanase IV gene. This method allows the efficiency of the cellulase complex to be increased by 20%.