Ivan N. Zorov

Glucoamylases are highly demanded by the industry for starch saccharification, which necessitates an increase in enzyme activity. To improve the activity of Aspergillus awamori VKPM F-1262 glucoamylase N181Q (AGAM), amino acid substitutions were made in the region of substrate binding. In the case of the variant D237G, there was a 1.3–1.4-fold increase in activity towards soluble starch for the individual enzyme and crude enzyme solution after cultivation in flasks. For the variants G57A and C319T, there was also a 1.1–1.4-fold increase in enzyme activity for the crude flask solution, while the increase in individual enzyme activity was minor. AGAM showed an increase in apparent molecular weight consistent with dimerization in the presence of soluble starch. A high T optimum of 70 °C was observed for AGAM and the variants in the hydrolysis of soluble starch. In further protein engineering, a high T optimum for hydrolysis, which may coincide with dimerization, may be considered as a hypothesis for the selection of thermostable glucoamylases, while an area of binding of oligo- and polysaccharides may be a site for mutagenesis.

This study examines the nature of enzymatic degradation of polyethylene terephthalate (PET) films mediated by a novel recombinant LCC^ICCG PETase enzyme preparation based on P. verruculosum fungus. The investigation was conducted using amorphous PET samples and PET samples with varying degrees of crystallinity as substrates for PETase-catalyzed hydrolysis under different temperature and pH conditions. Mechanical testing revealed that enzymatic treatment reduced the yield stress by 20–25%, tensile strength by approximately twofold, and elongation at break by 5–10 times, while the deformation mechanism remained unchanged. Enzymatic degradation under acidic conditions was ineffective, whereas increasing the pH to 9–10 markedly accelerated PET degradation and the associated deterioration of mechanical properties. Thermal analysis (TGA, DSC) and microscopy (optical and scanning electron microscopy) demonstrated that degradation was localized at the polymer surface, leading to the formation of cavities, cracks, and submicron-sized pores rather than bulk material disintegration. An inverse correlation was observed between PET crystallinity and susceptibility to enzymatic degradation: samples with crystallinity below 13% could be almost completely degraded, whereas samples with crystallinity above 30% exhibited little or no measurable weight loss over the same period. Low-crystallinity PET underwent rapid degradation accompanied by a transient increase in crystallinity, while highly crystalline PET primarily accumulated surface defects that nevertheless caused a substantial loss of mechanical strength. Consequently, the experimental data obtained in this study provide useful information for understanding PET degradation and for future studies on enzymatic PET recycling. The systematization of feedstock characteristics and the elucidated patterns of enzymatic degradation will enable optimization of pretreatment, enzymatic hydrolysis, and monomer recovery process parameters, thereby facilitating the eventual production of secondary raw materials.

Filamentous fungi are widely used in biotechnological processes because they secrete significant amounts of protein, use inexpensive nutrient media, and are predictably scalable in technological processes. Penicillium verruculosum B1-537 (now renamed Talaromyces verruculosus) produces large amounts of secreted protein (up to 70 g/L) and is used for large-scale enzyme production. Although P. verruculosum has an excellent protein expression system under the control of a strong cbh1 promoter, some heterologous enzymes such as Aspergillus awamori glucoamylase (aaGlaA) are still produced in insufficient quantities (15–20% of the total secreted protein), and this limits the application of enzyme preparations derived from P. verruculosum strains in the alcohol industry for the enzymatic treatment of grain starch together with α-amylase. One of the well-known approaches to addressing this is signal peptide replacement to increase protein expression. Therefore, the aim of this study was to investigate the effectiveness of signal peptide replacement. Various signal peptides (SPs), which were previously used for other well-expressed heterologous proteins, such as xylanases, β-glucosidases, and others, were analyzed to determine their effect on aaGlaA secretion. Five plasmids containing signal peptide sequences fused to the glaA gene were constructed and used to transform P. verruculosum. The resulting strains were cultured and screened for protein content and glucoamylase activity. Copy number analysis was performed on the most productive strains. The best one was an SP of homologous glucoamylase in P. verruculosum (pvGlaA). The use of this particular SP increased the secretion of heterologous aaGlaA by 2.5 times when cultivating recombinant strains on cellulose-containing fermentation media for P. verruculosum. Thus, SP replacement is a useful way to increase expression levels in the P. verruculosum expression system. Application of this method in P. verruculosum could address some productivity issues and enable the large-scale production of other industrial and food enzymes.

Sugar beet pulp is a byproduct of white sugar production, and it is quite significant in terms of volume. Every year, tens of millions of tons of beet pulp are produced around the world. However, only a fraction of it is currently used, mainly as animal feed. The composition of beet pulp includes plant polysaccharides, such as cellulose, arabinan, and pectin. Through the process of enzymatic hydrolysis, these polysaccharides are converted into technical C6/C5 sugars, which can be further used as a substrate for the microbial synthesis of various substances, including biofuels, organic acids, and other green chemistry molecules. The current study was designed with a primary objective that focused on the development of a strain that had the potential for enhanced productivity and the capacity to produce enzymes suitable for beet pulp hydrolysis. The pelA and abfA genes, which encode pectin lyase and arabinofuranosidase, respectively, in the fungus Penicillium canescens (VKPM F-178), were cloned and successfully expressed in the recipient strain Penicillium verruculosum B1-537 (VKPM F-3972D). New recombinant strains were created using the expression system of the mycelial fungus P. verruculosum B1-537, which is capable of simultaneously producing pectin lyase and arabinofuranosidase, as well as homologous cellulases. The screening of strains for increased enzymatic activity towards citrus pectin, sugar beet branched arabinan, and microcrystalline cellulose revealed that a B4 clone of P. verruculosum exhibited the greatest potential in sugar beet pulp cake hydrolysis. This clone was selected as the basis for the creation of a new enzyme preparation with enhanced pectin lyase, arabinase, and cellulase activities. The component composition of the enzyme preparation was determined, and the results indicated that the enzyme content comprised approximately 11% pectin lyase, 40% arabinofuranosidase, and 40% cellulases. The primary products of the enzymatic hydrolysis of the unpretreated beet pulp cake were arabinose and glucose. The degree of arabinan and cellulose conversion was observed to be up to 50% and 80%, respectively, after a period of 48 to 72 h of hydrolysis. The new B4 preparation was observed to be highly efficacious in the hydrolysis of beet cake at elevated concentrations of solids (up to 300 g/L) within the reaction mixture. The newly developed strain, as a producer of pectin lyase, arabinofuranosidase, and cellulase complexes, has the potential to be utilized for the bioconversion of sugar beet processing wastes and for the efficient generation of highly concentrated solutions of technical sugars for further implementation in processes of microbial synthesis.

An acidic shift in the pH profile of Clonostachys rosea zearalenone hydrolase (ZHD), the most effective and well-studied zearalenone-specific lactone hydrolase, is required to extend the range of applications for the enzyme as a decontamination agent in the feed and food production industries. Amino acid substitutions were engineered in the active center of the enzyme to decrease the pKa values of the catalytic residues E126 and H242. The T216K substitution provided a shift in the pH optimum by one unit to the acidic region, accompanied by a notable expansion in the pH profile under acidic conditions. The engineered enzyme demonstrated enhanced activity within the pH range of 3–5 and improved the activity within the pH ranging from 6 to 10. The D31N and D31A substitutions also resulted in a two-unit shift in the pH optimum towards acidic conditions, although this was accompanied by a significant reduction in the enzyme activity. The D31S substitution resulted in a shift in the pH profile towards the alkaline region. The alterations in the enzyme properties observed following the T216K substitution were consistent with the conditions required for the ZHD application as decontamination enzymes at acidic pH values (from 3.0 to 6.0).

The filamentous fungus Penicillium verruculosum (anamorph Talaromyces verruculosus) has been shown to be an efficient producer of secreted cellulases, used in biorefinery processes. Understanding the mechanisms of regulation of cellulase gene expression in the fungus P. verruculosum is a current task in industrial biotechnology, since it allows for targeted changes in the composition of the complex secreted by the fungus. Expression of cellulase genes in fungi is regulated mainly at the level of transcription via pathway-specific transcription factors (TF), the majority of which belong to the Zn(II)2Cys6 family of zinc binuclear cluster proteins. Transcriptional regulation of cellulase genes may have a species-specific pattern and involves several transcription factors. In this study, we used a qPCR method and transcriptome analysis to investigate the effect of knockouts and constitutive expression of genes encoding homologues of the regulatory factors XlnR and ClrB from P. verruculosum on the transcription of cbh1, egl2, and bgl1 genes, encoding three key cellulases, cellobiohydrolase, endoglucanase, and β-glucosidase, in the presence of various inducers. We have shown that the transcription factor XlnR of the filamentous fungus P. verruculosum is strictly responsible for the transcription of the main cellulolytic genes (cbh1, egl2, and bgl1) in the presence of xylose and xylobiose, but not in the presence of cellobiose. ClrB/Clr-2, a homologue from P. verruculosum, does not represent the main transcription factor regulating transcription of cellulolytic genes in the presence of selected inducers, unlike in the cases of Aspergillus nidulans, Aspergillus niger, and Penicillium oxalicum; apparently, it has a different function in fungi from the genus Talaromyces. We have also shown that constitutive expression of the transcription factor XlnR resulted in 3.5- and 2-fold increases in the activity of xylanase and β-glucosidase in a B1-XlnR enzyme preparation, respectively. In a practical sense, the obtained result can be used for the production of enzyme preparations based on the P. verruculosum B1-XlnR strain used for the bioconversion of renewable cellulose-containing raw materials into technical sugars.

Aflatoxin B1 (AFB1) produced by some Aspergillus species belongs to the most dangerous contaminants of animal feeds. Development of safe and cost efficient decontamination methods saving feed quality and nutritional value are of paramount importance. The use of recombinant AFB1-detoxifying microbial enzymes represents a promising biotechnological approach meeting the aforementioned requirements. In this study, three AFB1-degrading oxidases (AFOs) from edible basidiomycetes Cantharellus cibarius, Lentinula edodes and Pleurotus eryngii as well as AFO from Armillaria tabescens were expressed in E. coli Rosetta (DE3) and purified by immobilized metal-chelate chromatography. The stabilizing effect of the addition of glycerol and β-mercaptoethanol during protein extraction is shown. The catalytic constants of the recombinant AFOs (rAFOs) and other characteristics, which might be important for their practical application (and optimal temperature and pH, thermolability, regulation of the activity by metal ions and chelating agents, storage stability) were investigated. Among the obtained enzymes, rAFO from P. eryngii (Pe-AFO), which was characterized by the highest specific activity, thermostability and pH stability (especially at acidic pH range), the lowest K_m, and relative resistance to the inhibition by phytate, showed the best AFB1-degrading efficacy. However, Pe-AFO and all other rAFOs significantly decreased the target activity during heating above 45 °C, storage frozen or lyophilization.

Forage grain contamination with aflatoxin B1 (AFB1) is a global problem, so its detoxification with the aim of providing feed safety and cost-efficiency is still a relevant issue. AFB1 degradation by microbial enzymes is considered to be a promising detoxification approach. In this study, we modified an previously developed Pichia pastoris GS115 expression system using a chimeric signal peptide to obtain a new recombinant producer of extracellular AFB1 oxidase (AFO) from Armillaria tabescens (the yield of 0.3 g/L), purified AFO, and selected optimal conditions for AFO-induced AFB1 removal from model solutions. After a 72 h exposure of the AFB1 solution to AFO at pH 6.0 and 30 °C, 80% of the AFB1 was degraded. Treatments with AFO also significantly reduced the AFB1 content in wheat and corn grain inoculated with Aspergillus flavus. In grain samples contaminated with several dozen micrograms of AFB1 per kg, a 48 h exposure to AFO resulted in at least double the reduction in grain contamination compared to the control, while the same treatment of more significantly (~mg/kg) AFB1-polluted samples reduced their contamination by ~40%. These findings prove the potential of the tested AFO for cereal grain decontamination and suggest that additional studies to stabilize AFO and improve its AFB1-degrading efficacy are required.

Sulfite and kraft pulping are two principal methods of industrial delignification of wood. In recent decades, those have been considered as possibilities to pretreat recalcitrant wood lignocellulosics for the enzymatic hydrolysis of polysaccharides and the subsequent fermentation of obtained sugars to valuable bioproducts. Current work compares chemistry and technological features of two different cooking processes in the preparation of polysaccharide substrates for deep saccharification with P. verruculosum glycosyl hydrolases. Bleached kraft and sulfite pulps were subjected to hydrolysis with enzyme mixture of high xylanase, cellobiohydrolase, and β-glucosidase activities at a dosage of 10 FPU/g of dry pulp and fiber concentration of 2.5, 5, and 10%. HPLC was used to analyze soluble sugars after hydrolysis and additional acid inversion of oligomers to monosaccharides. Kraft pulp demonstrated higher pulp conversion after 48 h (74–99%), which mostly resulted from deep xylan hydrolysis. Sulfite-pulp hydrolysates, obtained in similar conditions due to higher hexose concentration (more than 50 g/L), had higher fermentability for industrial strains producing alcohols, microbial protein, or organic acids. Along with saccharification, enzymatic modification of non-hydrolyzed residues occurred, which led to decreased degree of polymerization and composition changes in two industrial pulps. As a result, crystallinity of kraft pulp increased by 1.3%, which opens possibilities for obtaining new types of cellulosic products in the pulp and paper industry. The high adaptability and controllability of enzymatic and fermentation processes creates prospects for the modernization of existing factories.