This study examines the nature of enzymatic degradation of polyethylene terephthalate (PET) films mediated by a novel recombinant LCCICCG 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.

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.

ABSTRACT The gene mutAW encoding Trichoderma harzianum fungus mutanase (MutA, GH71 family, α-1,3-glucanase, EC 3.2.1.59) was cloned and heterologously expressed by the highly productive Penicillium verruculosum fungus. P. verruculosum MutA strain secreted crude enzyme preparations with the recombinant MutA content of 40% of the total secreted protein, and the specific activity increased 150 folds compared to that of enzyme preparation obtained by the host strain. Homogeneous MutA had molecular mass of 70 kDa and displayed maximum of the activity on mutan at pH 5.0 and 50°C, with K m and k cat being 1.0 g/L and 30 s −1 , respectively. At 40-50°C, the MutA was stable for at least 3 h. Glucose was the main product of long-term mutan hydrolysis. HPLC analysis of hydrolysis product of oligo-α-(1→3)-D-glucosides bearing UV-detectable N - trans -cinnamoyl residue in the aglycon clearly indicated that MutA has an endo-processive hydrolytic mode of action. It was demonstrated that MutA can destroy the polysaccharide matrix of both gram-positive and gram-negative pathogenic bacteria biofilms. IMPORTANCE The manuscript describes the properties of a novel recombinant GH71 mutanase Mut A from Trichoderma harzianum . Gene mutAW encoding mutanase was heterologously expressed in the host strain Penicillium verruculosum B1-537 (ΔniaD). The recipient strain has a high secretory ability and allowed to obtain preparations containing the target recombinant enzyme up to 80% of the total protein pool. MutA exhibited a high activity against mutan and negligible or zero activity toward other types of glucans including α-(1→4)-, β-(1→3)-, β-(1→4)-, and β-(1→6)-glucans. By using a series of synthetic oligo-α-(1→3)-D-glucosides, we demonstrated that MutA is an endo-processive enzyme, which hydrolyzes the internal glucosidic bonds and releases glucose from the reducing end sliding into the non-reducing end. MutA recognizes tetrasaccharide as a minimal substrate and hydrolyzes it to trisaccharide and glucose. The effectiveness of the use of MutA for the destruction of clinical isolates of gram-positive and gram-negative bacteria is also described.

Application of new techniques to convert brown algal biomass to glucose suitable for microbiology is implemented by complete enzymatic hydrolysis of cellulose and laminarin. We proposed a scheme for obtaining cellulose-rich fraction (CRF) from the Arctic species Laminaria digitata, Saccharina latissima, Ascophyllum nodosum, Fucus vesiculosus and subsequent saccharification using glycoside hydrolases produced by Penicillium verruculosum and 1,3-β-glucanases from Myceliophtora thermophila. The dependence between the composition, degree of polymerization, and crystallinity of the substrates and its impact on the glucose yield during enzymatic hydrolysis was demonstrated. The enzyme preparations utilized in this study exhibited a conversion degree of up to 72

The review analyzes recent advances, challenges, and practical applications in the field of enzymes within the framework of chemical enzymology and enzyme engineering. The achievements in the fundamental understanding of molecular mechanisms of the catalytic cycle of enzymatic reactions made using quantum mechanics/molecular mechanics methods with supercomputer technologies and bioinformatic approaches are considered. The design of protein biocatalysts with new properties is a fundamentally significant methodology of the bioengineering approach to solving practical problems, which is demonstrated by a number of examples. The increasing role of biocatalysis in medicine and biomedical research is illustrated by addressing the problems of antibiotic synthesis and overcoming antibiotic resistance of bacteria, mechanisms of neurodegenerative diseases and development of drugs to treat Alzheimer's disease, biocatalytic processes of DNA repair and the role of mechanisms of functioning of heme peroxidases in the human body. The use of enzymes to degrade endogenous and exogenous toxicants has been greatly developed in recent decades. The advances and problems of using enzymes in therapy and drug delivery are analyzed. The fundamental role of enzymes in modern analysis and diagnosis is noted. The review considers a new trend in the development of bioanalytical methods using aptamers, multi-analysis systems on biochips, surface-enhanced Raman scattering systems, and bioelectroanalysis. The bibliography includes 460 references.

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.

Abstract The possibility of using the recipient strain Penicillium verruculosum B1-537 (ΔniaD) as a producer of laboratory and industrial enzymes was considered. The advantage of this strain is its ability to secrete a basic cellulase complex consisting of cellobiohydrolases, endoglucanases, and β-glucosidase, which exceeds in its hydrolytic ability the enzyme complex of Hypocrea (Trichoderma) strains. Using the expression system, the basic complex of cellulases of the recipient strain Piptochaetium verruculosum B1-537 (ΔniaD) was supplemented with new (booster) enzymes that are necessary to increase its hydrolytic activity. Enzyme preparations adapted to the processing of various types of renewable plant biomass were obtained.

In this study, CRISPR/Cas9 genome editing was used to knockout the bgl2 gene encoding intracellular β-glucosidase filamentous fungus Penicillium verruculosum. This resulted in a dramatic reduction of secretion of cellulolytic enzymes. The study of P. verruculosum Δbgl2 found that the transcription of the cbh1 gene, which encodes cellobiohydrolase 1, was impaired when induced by cellobiose and cellotriose. However, the transcription of the cbh1 gene remains at level of the host strain when induced by gentiobiose. This implies that gentiobiose is the true inducer of the cellulolytic response in P. verruculosum, in contrast to Neurospora crassa where cellobiose acts as an inducer.

The Penicillium verruculosum filamentous fungus is a highly active producer of cellulolytic complex enzymes, cellobiohydrolases, endoglucanases and β-glucosidases. Using the CRISPR/Cas9 genome editing system, previously adapted to P. verruculosum, a strain with a knockout of the xlnR gene encoding XlnR, one of the main transcription factors of filamentous fungi, has been obtained. The transcription level of cellulolytic genes was determined by quantitative PCR for the P. verruculosum B1-221-151 strain and the new P. verruculosum ΔxlnR strain. The XlnR protein was shown to activate transcription of the cbh1 , egl2 , and bgl1 genes encoding cellobiohydrolase 1, endoglucanase 2, and β-glucosidase, respectively, in the presence of xylose and xylooligosaccharides in the growth medium. It was found that other factors are also involved in the activation of transcription of these genes by cellobiose, cellotriose, sophorose, and gentiobiose, which has a complex effect on the biosynthesis of the cellulolytic complex of enzymes produced by the P. verruculosum fungus.

Methods of the pretreatment of reeds (grinding, hydrothermal treatment, treatment with acid or alkali solutions, organosolv, deep eutectic solvents) and their effect on the subsequent enzymatic hydrolysis by cellulase and hemicellulase complexes have been studied. Substrates with the highest reactivity were obtained by exposing the reeds to a weakly alkaline deep eutectic solvent (DES) and an alkali solution. The depth of enzymatic hydrolysis of these pretreated substrates was 63 and 59

The reactivity during enzymatic hydrolysis of eight industrially produced samples of pulps and semichemical pulps by enzyme preparations of glycosyl hydrolases B151 and F10 produced by a strain of the ascomycete fungus Penicillium verruculosum has been determined. It is shown for the first time that among fibrous pulps available on the market of pulp and paper industry in Russia, the highest level of yield of glucose from the initial wood during biocatalysis using cellulases and hemicellulases is characteristic of semichemical pulps obtained after green liquor cooking of hardwood. A high degree of enzymatic conversion of softwood bleached kraft pulp has been established, which in combination with the possibility of obtaining modified polysaccharide materials from non-hydrolysable residue makes this cellulosic substrate the most promising for the development of biological processes at pulp and paper industries. It is shown that drying of pulp negatively affects the efficiency of cellulose hydrolysis, while mechanical refining improves the performance of the enzymatic saccharification process.

Xylanases are used in several industrial applications, such as feed additives, the bleaching of pulp and paper, and the production of bread, food, and drinks. Xylanases are required to remain active after heat treatment at 80-90 degrees C for 30 s to several minutes due to the conditions of feed pelleting. Also, xylanases need to be active at 60-70 degrees C for several hours while bleaching of pulp and paper or manufacturing of bread, food, and drinks is performed. Xylanases of the glycoside hydrolase family GH10 are good candidates for application in such processes because of their high thermostability and, in particular, as feed additives because of their insensitivity to protein inhibitors in cereal feeds. In the study, the thermostability of GH10 xylanase E from Penicillium canescens was improved to reach a half-inactivation period of 2 min at 80 degrees C compared to 21 s for the wild-type enzyme (WT). Enzymatic activity was increased by 22-48 % at 40-70 degrees C, which improved the action of the enzyme as a feed additive in the gastric system of animals and during bleaching of pulp and paper. Molecular dynamics simulations demonstrated lower flexibility of the tertiary structure of the engineered enzyme at elevated temperatures compared to WT. The residues W113, Q116, W313, and W321 in the (-1) and (-2) subsites for the substrate binding were less flexible. In the simulations, the engineered enzyme had a comparable content of a-helixes, 310helixes, 0-sheets, and 0-bridges as WT, but a lower content of coils and a higher content of 0-turns. (c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Exhaustive enzymatic hydrolysis is performed for semi-bleached sulfate hardwood cellulose (a semi-finished pulp and paper product) at ultra-high concentrations of it in a reaction mixture (up to 300 g/L per dry compound). Russian commercial enzyme preparations are used for hydrolysis. The best seems to be Agroxil Plus, which has high cellulase and endoxylanase activities. A total of 290 g/L of sugars (including 210 g/L of glucose and 30 g/L of xylose) is obtained using Agroxil Plus (20 mg protein/1 g substrate) in combination with an auxiliary β-glucosidase enzyme preparation (2 mg protein/1 g substrate) at an initial semi-bleached cellulose concentration of 300 g/L. The dosage of Agroxil Plus can be halved (10 mg of protein/1 g of substrate with a total concentration of semi-bleached cellulose of 300 g/L) with a high yield of hydrolysis product (270 g/L of sugars, including 200 g/L of glucose and 30 g/L of xylose), due to the fractional addition of a substrate.

Non-edible cellulosic biomass from perennial herbaceous plants is a promising and abundant feedstock for replacing slow-growing woody plants used in biotechnological applications. Herbaceous plant biomass, as other types of plant biomass, requires pretreatment before biochemical conversion. In this study, miscanthus straw was pretreated using different methods and subjected to enzymatic hydrolysis with Penicillium verruculosum enzyme complexes under laboratory conditions. The convertibility after enzymatic hydrolysis varied from 15% to 66%, depending on the pretreatment method. Dilute alkaline pretreatment showed the highest convertibility compared to other methods, reaching up to 66%. The efficiency of dilute acid pretreatment was relatively low compared to other methods. The maximum convertibility was 37% for sulfuric acid pretreatment (the least efficient) and 51% for nitric acid. Convertibility was almost equal with 43% for white liquor and 46% for hot water. The glucose-to-xylose ratio was 4.7:1 for dilute alkaline pretreatment and 11–13:1 for white liquor. Both sulfuric and nitric acid resulted in a low xylose content in the enzymatic hydrolysates. Low-xylose hydrolysates with less than 2% of the glucose amount can be produced by hot water pretreatment. Preparation C, enriched with endoglucanase I from T. reesei and endoglucanase II from P. verruculosum, was found to be the most effective of the different enzyme preparations (EPs) tested.

An Erratum to this paper has been published: https://doi.org/10.1134/S0003683823320030

Thermostability of cellulases can be increased through amino acid substitutions and by protein engineering with predictors of protein thermostability. We have carried out a systematic analysis of the performance of 18 predictors for the engineering of cellulases. The predictors were PoPMuSiC, HoTMuSiC, I-Mutant 2.0, I-Mutant Suite, PremPS, Hotspot, Maestroweb, DynaMut, ENCoM ([Formula: see text] and [Formula: see text], mCSM, SDM, DUET, RosettaDesign, Cupsat (thermal and denaturant approaches), ConSurf, and Voronoia. The highest values of accuracy, F-measure, and MCC were obtained for DynaMut, SDM, RosettaDesign, and PremPS. A combination of the predictors provided an improvement in the performance. F-measure and MCC were improved by 14% and 28%, respectively. Accuracy and sensitivity were also improved by 9% and 20%, respectively, compared to the maximal values of single predictors. The reported values of the performance of the predictors and their combination may aid research in the engineering of thermostable cellulases as well as the further development of thermostability predictors.

Микроскопические грибы широко используются для промышленного производства технических ферментов; важным условием для этого является создание высокоактивных штаммов – продуцентов ферментов. Показана возможность использования для получения таких штаммов двух подходов: индуцированного мутагенеза и методов генетической инженерии. Разработаны схемы индуцированного мутагенеза, обеспечивающие получение высокоактивных продуцентов технических ферментов. В результате гамма-мутагенеза получены мутантные штаммы грибов рода Trichoderma – продуценты целлюлаз, ксиланаз и пектиназы, с различным компонентным составом карбогидразного комплекса, позволяющие получать в культуральной жидкости (КЖ) до 35–40 г/л внеклеточного белка. С помощью комбинированной обработки УФ и гамма-облучением получен стабильный мутантный штамм Aspergillus awamori с увеличенной продукцией глюкоамилазы. Создана система экспрессии Penicillium verruculosum В1-537 (ΔniaD), позволяющая трансформировать реципиентный штамм экспрессионными конструкциями, содержащими целевые гетерологичные или гомологичные гены, функционально связанные с промотором и терминатором сильного индуцибельного промотора гена мажорного секреторного белка целлобиогидролазы I (cbh1) P. verruculosum. Технология создания и отбора активных рекомбинантных штаммов проста и надежна, состав питательной среды и условий культивирования рекомбинантных штаммов – продуцентов различных целевых ферментов стандартизирован. Затраты времени на получение продуцента целевого фермента составляют от 3 до 6 мес. Реципиентный штамм P. verruculosum характеризуется высокой секреторной способностью (до 60 г/л внеклеточного белка в КЖ), а рекомбинантные штаммы-продуценты – высокой продуктивностью целевых ферментов; полученные с их помощью ферментные препараты (ФП) содержат 30‒70 % целевых рекомбинантных ферментов (в некоторых случаях до 80 %) от общего пула белка. Созданы рекомбинантные штаммы – продуценты целлюлаз, β-глюканаз, ксиланаз, фитазы, кислой протеазы, пектин-лиазы, β-глюкозидазы, экзо- и эндоинулиназ. Мутантные и рекомбинантные штаммы – продуценты различных ферментов – используются для промышленного производства на заводе ООО «Агрофермент» различных технических ФП для пищевой промышленности и кормопроизводства. Microscopic fungi are widely used for the industrial production of technical enzymes; an important condition for this is the creation of highly active strains – producers of enzymes. The possibility of using such approaches to obtain highly active strains as induced mutagenesis and genetic engineering methods is shown. Schemes of induced mutagenesis have been developed that ensure the production of highly active producers of technical enzymes. As a result of gamma-mutagenesis, mutant fungal strains of Trichoderma genus were obtained – producers of cellulases, xylanases and pectinase, with different component composition of the carbohydrase complex. The methods used made it possible to obtain up to 35–40 g/l of extracellular protein in the cultural liquid (CL). Using UV and gamma rays combined treatment, a stable mutant Aspergillus awamori strain with increased glucoamylase production was obtained. An expression system for Penicillium verruculosum B1-537 (ΔniaD) has been developed, which makes it possible to transform a recipient strain with expression constructs containing target heterologous or homologous genes functionally linked to the promoter and terminator of the strong inducible promoter of the P. verruculosum major secretory protein cellobiohydrolase I (cbh1) gene. The technology for creating and selection of active recombinant strains is simple and reliable, the composition of the nutrient medium and cultivating conditions for recombinant strains producing various target enzymes are standardized. The time spent on obtaining the target enzyme producer is from 3 to 6 months. The recipient P. verruculosum strain is characterized by a high secretory capacity (up to 60 g/l of extracellular protein in CL), and the recombinant strains are characterized by a high productivity of target enzymes. Enzyme preparations (EP) derived from the recombinant strains contain 30–70 % of the target recombinant enzymes in the total protein pool (in some cases, up to 80 %). Recombinant strains producing cellulases, β-glucanases, xylanases, phytase, acid protease, pectin-lyase, β-glucosidase, exo- and endo-inulinases have been developed. Mutant and recombinant strains – producers of various enzymes for the food industry and feed production are successfully used for industrial production of various technical EPs at the plant LLC «Agroferment».

Enzymes with high thermostability provide bioconversion under elevated temperatures during industrial processes. Additional improvement in the enzyme thermostability allows further increasing the temperature for cost reduction. Aspergillus awamori exo-inulinase is sufficiently thermostable to hydrolyze inulin at temperatures up to 70 degrees C during the industrial process of bioconversion of the polysaccharide inulin into high -fructose syrup. Amino acid substitutions S322P and T521P were designed to improve the thermostability of A. awamori exo-inulinase and increase the temperature of the hydrolysis. The substitutions provided stabilization of alpha-helix and 8 -sheet as detected with molecular dynamics simulations at 70 degrees C. The substitution S322P also provided stabilization of the cleft of the active center, which correlated with the improvement in the thermostability of the enzyme. The variant S322P demonstrated a 1.5 -fold increase in thermostability at 70 degrees C and a 2 degrees C increase in the temperature of inulin hydrolysis.