Properties of enzyme preparations and homogeneous enzymes — Endoglucanases EG2 Penicillium verruculosum and LAM Myceliophthora thermophila

This study aimed at evaluating different enzyme combinations in the saccharification of sugarcane bagasse (SCB), soybean husks (SBH) and oil palm empty fruit bunches (EFB) submitted to mild acid and alkaline pretreatments. Enzyme pools were represented by B1 host (crude cellulase/xylanase complexes of Penicillium verruculosum); B1-XylA (Penicillium canescens xylanase A expressed in P. verruculosum B1 host strain); and F10 (Aspergillus niger β-glucosidase expressed in B1 host strain). Enzyme loading was 10 mg protein/g dry substrate and 40 U/g of β-glucosidase (F10) activity. SCB was efficiently hydrolyzed by B1 host after alkaline pretreatment, yielding glucose and reducing sugars at 71 g/L or 65 g/100 g of dry pretreated substrate and 91 g/L or 83 g/100 g, respectively. B1 host performed better also for EFB, regardless of the pretreatment method, but yields were lower (glucose 27–30 g/L, 25–27 g/100 g; reducing sugars 37–42 g/L, 34–38 g/100 g). SBH was efficiently saccharified by the combination of B1 host and B1-XylA, yielding similar concentrations of reducing sugars for both pretreatments (92–96 g/L, 84–87 g/100 g); glucose recovery, however, was higher with alkaline pretreatment (81 g/L, 74 g/100 g). Glucose and reducing sugar yields from initial substrate mass were 42% and 54% for SCB, 36% and 42–47% for SBH and 16–18% and 21–26% for EFB, respectively.

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Construction of Recombinant Producers of Enzyme Preparations for Feed Production with an Expression System Based on Penicillium verruculosum Fungus

Applied Biochemistry and Microbiology ◽

10.1134/s0003683820080104 ◽

2020 ◽

Vol 56 (8) ◽

pp. 875-880 ◽

Cited By ~ 1

Author(s):

A. P. Sinitsyn ◽

O. G. Korotkova ◽

E. A. Rubtsova ◽

O. A. Sinitsyna ◽

E. G. Kondrat’eva ◽

...

Keyword(s):

Expression System ◽

Penicillium Verruculosum ◽

Enzyme Preparations ◽

Feed Production

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Application of complex biocatalysts based on recombinant Penicillium verruculosum enzyme preparations in the hydrolysis of semichemical hardwood pulp

Catalysis in Industry ◽

10.1134/s2070050414040138 ◽

2014 ◽

Vol 6 (4) ◽

pp. 348-354 ◽

Cited By ~ 6

Author(s):

E. V. Novozhilov ◽

A. S. Aksenov ◽

M. L. Demidov ◽

D. G. Chukhchin ◽

G. S. Dotsenko ◽

...

Keyword(s):

Penicillium Verruculosum ◽

Enzyme Preparations ◽

Hardwood Pulp ◽

Hydrolysis Of

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Comparing the efficiency of plant material bioconversion processes using biocatalysts based on Trichoderma and Penicillium verruculosum enzyme preparations

Catalysis in Industry ◽

10.1134/s2070050413010042 ◽

2013 ◽

Vol 5 (1) ◽

pp. 98-104 ◽

Cited By ~ 9

Author(s):

A. V. Chekushina ◽

G. S. Dotsenko ◽

A. P. Sinitsyn

Keyword(s):

Plant Material ◽

Penicillium Verruculosum ◽

Enzyme Preparations

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Production of fruit wines using novel enzyme preparations

OENO One ◽

10.20870/oeno-one.2015.49.3.80 ◽

2015 ◽

Vol 49 (3) ◽

pp. 205 ◽

Cited By ~ 1

Author(s):

Anastasia Volchok ◽

Alexandra Rozhkova ◽

Ivan Zorov ◽

Sergey Shcherbakov ◽

Arkady Sinitsyn

Keyword(s):

Wine Industry ◽

Penicillium Verruculosum ◽

Wine Production ◽

Enzyme Preparations ◽

Fruit Wine ◽

Juice Yield ◽

Negative Effect ◽

New Generation ◽

Enzyme Complexes ◽

Selection Of

Aim: This work describes the activities of new-generation enzymatic preparations in fruit-berry substrates engineered for use in the fruit-wine industry. The enzymes were produced after genetic modification and selection of fungi Penicillium verruculosum, which produce efficient cellulase and pectinase enzymatic complexes. Methods and results: This paper covers the main characteristics of novel multi-enzyme complexes and the results of in-lab fruit-wine production with addition of enzymatic preparations, which could be used on an industrial scale. The juice yield and the content of suspended materials in the enzymatically treated samples were compared. Experiments included the sensory analysis of produced juices and fruit wines.Conclusion: Results show a significant increase in juice yield from the fruit pulp processed with the enzymatic preparations, without any negative effect on the quality and organoleptic attributes of the final product.Significance and impact of the study: The obtained data clearly show that the use of the new-generation enzymatic preparations in the fruit-wine industry is effective.

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Determination of Copy Number of Target Carbohydrase Genes in the Penicillium verruculosum fungus Recombinant Strains

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-5-51-57 ◽

2019 ◽

Vol 35 (5) ◽

pp. 51-57 ◽

Cited By ~ 2

Author(s):

V.Yu. Kislitsin ◽

I.N. Zorov ◽

A.P. Sinitsyn ◽

A.M. Rozhkova

Keyword(s):

Copy Number ◽

Target Gene ◽

Gene Copy Number ◽

Gene Promoter ◽

Gene Copy ◽

Penicillium Verruculosum ◽

Penicillium Canescens ◽

Enzyme Preparations ◽

Recombinant Strains

The protein profiles of the culture liquids of recombinant Penicillium verruculosum strains that secreted an Aspergillus niger β-glucosidase (BGL series) or a Penicillium canescens xylanase A (XYLA series) have been previously analyzed. It was shown that the producers were divided into two groups according to the target protein content: 75-85% (1) and no more than 15% (2). It was established in this work that the copy number of the target gene bgll in the recombinant strains correlated with the β-glucosidase content in the BGL-F10 and BGL-F12 enzyme preparations. However, this proportion was violated in the direction of decreasing the xylanase A relative amount: the target enzyme content of 50% and 17% in the XylA3 and XylA4 preparations corresponded to 20-22 and 6 copies, respectively, of the xylA gene. We believe that this effect is a result of titration of positive transcription factors in the P. verruculosum XylA3 strain containing 20 to 22 copies of the cbhl gene promoter. Penicillium verruculosum, real-time PCR, gene copy number This work was partially supported by the Russian Foundation for Bsic Research (Project number: 18-29-07070).

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Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance

PLoS ONE ◽

10.1371/journal.pone.0170404 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0170404 ◽

Cited By ~ 17

Author(s):

Alexander G. Bulakhov ◽

Pavel V. Volkov ◽

Aleksandra M. Rozhkova ◽

Alexander V. Gusakov ◽

Vitaly A. Nemashkalov ◽

...

Keyword(s):

Inducible Promoter ◽

Penicillium Verruculosum ◽

Gene Encoding ◽

Enzyme Preparations

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Construction of Recombinant Producers of Enzyme Preparations for Fodder Production by Means of Expression System Based on Penicillium verruculosum Fungus

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-4-6-14 ◽

2019 ◽

Vol 35 (4) ◽

pp. 6-14

Author(s):

O.G. Korotkova ◽

E.A. Rubtsova ◽

О.А. Sinitsyna ◽

E.G. Kondrat'eva ◽

A.S. Sereda ◽

...

Keyword(s):

Specific Activity ◽

Expression System ◽

Xylanase Activity ◽

Live Weight ◽

Recipient Strain ◽

Metabolic Energy ◽

Feed Consumption ◽

Penicillium Verruculosum ◽

Enzyme Preparations ◽

Wide Range

Using the expression system based on the recipient strain Penicillium verruculosum 537 (ΔniaD) and promoter of the cellobiohydrolase 1 gene, producing strains were created. That made it possible to obtain new enzyme preparations (EP) for feed production, which are characterized by a high content of exodepolymerases (endo-β-l,4-glucanases and endo-β-l,4-xylanase), degrading non-starch polysaccharides (NSP) of grain cereals used in feed. The content of the endodepolymerases in new EP was 44-68% of the total protein content (compared with 15% in the case of the recipient strain), the content of exodepolymerases (cellobiohydrolases) fell to 8-26% compared to 60% in the case of the EP, obtained using recipient strain. New EP had high specific activity of endodepolymerases, which is 1.8-4.6 times higher than for the EP obtained with the recipient strain; the specific activity of exodepolymerases decreased in 2,0-3,7 times. New EP showed endoglucanase and xylanase activity in a wide range of pH and temperature, including the physiological value of these parameters (pH 3 and 7 and Г=37-38 °C); characterized by a high stability of endodepolymerases under the influence of digestive proteases (pepsin and trypsin), as well as the stability of ndodepolymerases under granulation conditions of animal feed (80 °C). The xylanase of the new EP was not inhibited by the inhibitors of cereal proteins. An experimental batch of new EP was manufactured by the enzyme-producing plant "Agroferment" and tested in broiler and pig feeding. The input of new commercial EP into broiler diets has had a positive impact on the safety of chickens, increasing their live weight and reducing feed cost. The supplementation of commercial EP into pig ration also led to positive results during feeding, contributed to the increase in the average daily growth of pig biomass, reduced feed consumption, protein and metabolic energy per unit of live weight gain, improved digestibility and assimilability of feed nutrients, especially of raw fiber and NSP. Feed enzyme preparation, xylanase, endoglucanase, non-starch polysaccharides, Penicillium verruculosum Funding-The work was done with the financial support of the Ministry of Science and Higher Education (State Task 0104-2019-0009).

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Production of Industrial Enzymes based on the Expression System of the Penicillium verruculosum Fungus

Biotekhnologiya ◽

10.21519/0234-2758-2020-36-6-17-34 ◽

2020 ◽

Vol 36 (6) ◽

pp. 17-34

Author(s):

O.A. Sinitsyna ◽

A.M. Rozhkova

Keyword(s):

Expression System ◽

Recipient Strain ◽

Feed Additives ◽

Penicillium Verruculosum ◽

Industrial Enzymes ◽

Recombinant Enzymes ◽

Enzyme Preparations ◽

Recombinant Strains ◽

Protein Pool ◽

Wide Range

The review provides the data on the creation of highly active recombinant strains producers of a wide range of enzymes for various fields of industry and agriculture using the expression system of the Penicillium verruculosum filamentous fungus: endoglucanases, β-glucanases, xylanases, mannanases, phytases, α-galactosidases, pectin lyases, polygalacturonases, dextranases, exo-inulinases and chitinases. The recipient strain, P. verruculosum B1-537(ΔniaD), has a high secretory capacity (up to 60 g/L of extracellular protein in the culture liquid), and the recombinant producer strains are characterized by high productivity of target enzymes. The obtained enzyme preparations contain, as a rule, 30--70% of the target recombinant enzymes (in some cases, up to 80%) of the total protein pool. The P. verruculosum expression system makes it easy to transform expression constructs containing target heterologous or homologous genes, functionally linked to the promoter and terminator of the strong inducible promoter of the cellobiohydrolase-1 (cbh1) gene, and the genetic transformation of the recipient strain can be carried out simultaneously using several vector constructs. The incorporation of target genes into the chromosome of the recipient strain is an integrative process, and the foreign recombinant proteins encoded by them are not subjected to proteolysis. The technique of primary selection of positive transformants on agar media, as well as secondary selection during their cultivation in shaking flasks and fermenters, is simple and reliable in use, partly owing to standardized parameters: the nutrient composition of the fermentation medium and the culturing conditions of recombinant strains producing various target enzymes. The time spent on creating a producer of the target enzyme, from setting the problem to obtaining a recombinant strain, takes from 3 to 6 months. It is important to emphasize that recombinant P. verruculosum strains are successfully used in industrial processes as producers of various target enzymes. expression system, cloning, industrial enzymes, feed additives, Penicillium verruculosum The work was supported by the Ministry of Science and Higher Education of the Russian Federation (State Assignment 0104-2019-0009).

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