Based on the recipient strain of Penicillium verruculosum B1-537 (ΔniaD) using the promoter of the cellobiohydrolase I gene, a producer of homologous lytic polysaccharide monooxygenase (PMO) and heterologous β-glucosidase Aspergillus niger (BG), auxiliary enzymes for the “basic” cellulase complex consisting of endoglucanases and cellobiohydrolase, was obtained. The enzyme preparation PMO–BG was obtained, containing 34% PMO and 43% BG, which was used as a source of auxiliary enzymes that increase the effectiveness of basic cellulases P. verruculosum by 20–100% in the bioconversion of various types of cellulose-containing raw materials: microcrystalline cellulose, pretreated with alkali common reed and semi-bleached sulfate hardwood pulp of Arkhangelsk Pulp and Paper Mill JSC.

A recombinant strain producing a complex of extracellular enzymes including chitinase from Myceliophtora thermophila was created based on the fungus Penicillium verruculosum . The activity of the enzyme preparations obtained from the cultural fluid of the producer strain was 0.55, 0.53, and 0.66 U/mg protein with chitin and chitosans with the molecular weight of 200 and 1000 kDa, respectively. The temperature optimum for the recombinant chitinase was 52-65°C; the pH optimum was 4.5-6.2, which corresponded to the published data for this class of the enzymes. The content of heterologous chitinase in the obtained enzyme preparations was 47% of total protein content in the cultural fluid. Enzyme preparations produced by the recombinant P. verruculosum XT403 strain and containing heterologous chitinase were able to degrade the mycelium of micromycetes, including phytopathogenic ones, and were very efficient in the bioconversion of microbiological industry waste.

A possibility to use a concentrate of soybean low molecular substances, the BioAccel preparation, as a component of fermentation media for the cultivation of T. reesei Co-44 strain has been studied. BioAccel is a by-product of the soy protein concentrate production with 3D-stracturing technology. T. reesei Co-44 is a highly active producer of cellulases and hemicellulases with an increased level of xylanase and endoglucanase biosynthesis. The addition of 1-9% BioAccel to the fermentation medium significantly improved the xylanase and endoglucanase activity and the dynamics of their biosynthesis during the T. reesei Co-44 cultivation in flasks. The positive effect of BioAccel grew with the increase in its concentration in the medium. The addition of 5% and 7% of the preparation improved the endoglucanase activity by 1.8 times and the xylanase activity by 40-50%. The maximum increase in the xylanase and endoglucanase activity (twice as compared to the control on average) was obtained with the introduction of 9% BioAccel. Fed-batch fermentation of T. reesei Co-44 in laboratory fermenters according to the industrial scheme showed the promise of using BioAccel as a component of the fermentation medium instead of the expensive yeast extract. concentrate of low molecular soybean compounds, Trichoderma reesei, cultivation, xylanase, endoglucanase This work was financially supported by the Basic Research Program of the Russian Academy of Sciences for 2019-2021 (Project no. 0529-2019-0066).

The effect of polysaccharide monooxygenase (endoglucanase IV) from the fungus Trichoderma reesei on the hydrolysis of polysaccharide substrates by cellulases secreted by the fungus Penicillium verruculosum has been investigated. Supplementation of the enzyme complex from P. verruculosum by endoglucanase IV from T. reesei has been shown to elevate the efficiency of cellulose hydrolysis by 45%.

Изучено влияние полисахаридмонооксигеназы (эндоглюканазы IV) гриба Trichoderma reesei на гидролиз полисахаридных субстратов целлюлазами, секретируемыми грибом Penicillium verruculosum. Показано, что внесение эндоглюканазы IV из T. reesei в комплекс ферментов гриба P. verruculosum позволяет повысить эффективность гидролиза целлюлозы на 45%.

As a result of γ-mutagenesis of Trichoderma longibrachiatum TW1 and the subsequent selection of improved producers, a novel mutant strain, TW1-59-27, capable of efficiently secreting cellulase and xylanase was obtained. In a fed-batch cultivation, the new TW1-59-27 mutant was significantly more active compared with the original TW1 strain. For instance, the activities of cellulase (towards carboxymethylcellulose) and xylanase in the culture broth (CB) increased by 1.8 and two times, respectively, and the protein content increased by 1.47 times. The activity of these enzymes in the dry enzyme preparation derived from the CB of the TW1-59-27 mutant was 1.3–1.8 times higher than that in the preparation derived from the original TW1 strain. It was established that the cellulase from the enzyme preparation of the mutant strain demonstrated the maximum activity at 55–65°C; it occurred in xylanase at 60°C. The pH optima of these enzymes were pH 4.5–5.0 and pH 5.0–6.0, respectively. It was shown that the content of endoglucanases in the enzyme preparation increased from 7% to 13.5%; the effect is largely driven by the elevated secretion of endoglucanase-1. An enzyme preparation with increased endoglucanase-1 content is promising for use as a feed additive in agriculture.

В результате -мутагенеза Trichoderma longibrachiatum TW1 и последующей селекции улучшенных продуцентов получен новый мутантный штамм TW1-59-27, способный эффективно секретировать целлюлазы и ксиланазы. При fed-batch культивировании нового мутанта TW1-59-27 активность ферментов значительно возрастала по сравнению с исходным штаммом TW1. Так, активность целлюлазы (по отношению к карбоксиметилцеллюлозе) и ксиланазы в культуральной жидкости мутантного штамма увеличивалась в 1.8 и 2 раза соответственно, а содержание белка – в 1.47 раз. Активность этих ферментов в сухом ферментном препарате, полученном из КЖ мутанта TW1-59-27, возрастала в 1.3–1.8 раз по сравнению с препаратом из исходного штамма TW1. Условлено что целлюлаза ферментного препарата из мутантного штамма проявляла максимальную активность при 55–65°С, а ксиланаза при 60°С. Оптимум рН для действия этих ферментов – 4.5–5.0 и 5.0–6.0 соответственно. Показано что содержание эндоглюканаз в ферментном препарате увеличивалось с 7 до 13.5% в значительной степени вследствие секреции эндоглюканазы 1. Ферментный препарат с повышенным содержанием эндоглюканазы 1 может быть перспективным для применения в качестве кормовой добавки в сельском хозяйстве.

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.

Commercial and pilot pectate lyase preparations (EC 4.2.2.2) have been compared. They differ in their effect on pectins with different esterification degrees (ED). The activity of the pilot preparation with respect to a substrate with ED = 70% is tenfold lower than with respect to unesterified polygalacturonic acid. For commercial preparations, this activity ratio ranged within 1.5–2. At equal pectate lyase activities, the commercial preparations better remove pectin from crude cotton fabric during its boil off. The laboratory preparation is more efficient for improving the capillarity (wettability) of the fabric owing to the cooperative effect of the pectate lyase, cellulase, and hemicellulase present in the preparation.

The paper describes three Penicillium verruculosum 28K mutants with about threefold enhanced production of five industrially important carbohydrases. The two-stage fermentation process that we developed provided a further two- to threefold increase in the production of carbohydrases. Physiological and biochemical studies showed that the synthesis of all five carbohydrases is inducible. Carboxymethylcellulase, xylanase, and β-glucanase are synthesized under a common regulatory control, as is evident from the concurrent increase in the synthesis of these enzymes in the presence of microcrystalline cellulose. The synthesis of avicelase and β-glucosidase is evidently induced by other cellulose- and hemicellulose-containing compounds present in the fermentation medium and, hence, is regulated independently of the three aforementioned enzymes.

Enzyme preparations were isolated from the culture liquid of five mutant strains of the cellulase producer Penicillium verruculosum. The hydrolytic activities of these preparations against unbleached eucalypt cellulose was compared to that of commercial preparations of Trichoderma reesei (T. longibrachiatum). In the majority of cases, P. verruculosum enzymes provided higher yields of reducing sugars (RSs) and glucose. A correlation was found between the yield of RSs and the avicelase activity of the preparations in the reaction mixture.