Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability.

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15⁻21% increase in specific activity against carboxymethylcellulose and ß-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52⁻58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15⁻22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.

Enhancement of the enzymatic cellulose saccharification by Penicillium verruculosum multienzyme cocktails containing homologously overexpressed lytic polysaccharide monooxygenase.

The gene lpmo1 encoding Penicillium verruculosum lytic polysaccharide monooxygenase (PvLPMO9A) was sequenced and homologously overexpressed in P. verruculosum B1-537 (ΔniaD) auxotrophic strain under the control of the cbh1 gene promoter in combination with either the cbh1 signal sequence (sCBH1-X series of samples) or the native lpmo1 signal sequence (sLPMO1-X series). Three enzyme samples of the sCBH1-X series were characterized by a lower overall content of cellobiohydrolases (CBHs: 26-45%) but slightly higher content of endoglucanases (EGs: 17-23%) relative to the reference B1-537 preparation (60% of CBHs and 14% of EGs), while the PvLPMO9A content in them made up 9-21% of the total secreted protein. The PvLPMO9A content in four enzyme preparations of the sLPMO1-X series was much higher (30-57%), however the portion of CBHs in most of them (except for sLPMO1-8) decreased even to a greater extent (to 21-42%) than in the samples of the sCBH1-X series. Two enzyme preparations (sCBH1-8 and sLPMO1-8), in which the content of cellulases was substantially retained and the portion of PvLPMO9A was 9-30%, demonstrated the increased yields of reducing sugars in 48-h saccharification of Avicel and milled aspen wood: 19-31 and 11-26%, respectively, compared to the reference cellulase cocktail.

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.

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 ß -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases. RESULTS: Genes bglI, encoding ß-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.

Effect of N-linked glycosylation on the activity and other properties of recombinant endoglucanase IIa (Cel5A) from Penicillium verruculosum.

Endoglucanase IIa from Penicillium verruculosum (PvCel5A) has three potential N-glycosylation sites: Asn19, Asn42 and Asn194. In order to study the role of N-glycosylation, the wild type (wt) PvCel5A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens. All forms of the rPvCel5A were successfully expressed and purified for characterization. The MALDI-TOF mass spectrometry peptide fingerprinting showed that N-glycans linked to Asn42 and Asn194 represent variable oligosaccharides, according to the formula (Man)1-9(GlcNAc)2. No evidence for Asn19 glycosylation was found. Mutations had no notable effect on the enzyme thermostability; however, the N-linked glycans stabilized the enzyme against proteolytic attack. For N42A and N194A mutants, a slight shift of pH-optimum to pH 5.0 was observed (from pH-optimum of 4.5 for the native enzyme, rPvCel5A-wt and N19A mutant). The N19A mutation led to a notable decrease in the specific activity against carboxymethylcellulose and barley ß-glucan (by 26% and 12% relative to the rPvCel5A-wt), while the N42A and N194A mutants displayed 12-13% and 32-35% increase in the activities. Similar effects of the mutations were observed in prolonged hydrolysis of ß-glucan and milled aspen wood by rPvCel5A forms in the presence of purified ß-glucosidase.