RU2741078C1 - Penicillium canescens mtcbhi fungus strain producer of thermally stable cellobiohydrolase i and an enzyme preparation based thereon for bioconversion of renewable vegetal raw material into sugars - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to production and production of enzyme preparations (EP) containing highly active and thermostable enzymes. Objective of the invention is to introduce 5 mutations into the cel7A gene encoding the modified form of CBG I, and obtaining dry EP based on the new recombinant strain P. canescens mtCBHI (BKM F-4850D), which is a producer of heterologous mutagenic CBG I P. verruculosum, related to 7-family glycoside-hydrolase (CF 3.2.1.176, molecular weight 55 kDa) for use in biofuel industry for implementation of efficient process of bioconversion of cellulose-containing biomass into industrial sugars.EFFECT: disclosed are the Penicillium canescens mtCBHI fungus strain producer of the thermostable cellobiohydrolase I and an enzyme preparation based thereon for bioconversion of the renewable vegetal raw material into sugars.3 cl, 4 dwg, 2 tbl, 4 ex

Description

The invention relates to the field of biotechnology, namely, to the creation and production of enzyme preparations (FP) containing highly active and thermostable enzymes. The invention can be used in the biofuel industry.

Acceleration of scientific and technological progress, the growth of the world economy stimulate the consumption of traditional energy sources, which will ultimately lead to their shortage. According to the US Department of Energy, the consumption of all types of energy resources in the world (oil products, coal, natural gas, electricity and alternative fuels from renewable energy sources) increased by 40% from 1992 to 2009, while world consumption of oil and oil products increased by almost a third [ A.A. Korotkikh // US biofuel industry in the new century // USA and Canada: economics, politics, culture. 2015. Issue. 5. S 103-118]. In the Russian Federation, the production of heat energy only for the period from 2013 to 2017 increased by 18.14% according to the Ministry of Energy of the Russian Federation [Ministry of Energy of the Russian Federation. Open data // https://minenergo.gov.ru/opendata].

With the further intensification of economic activity and the growth of world energy prices, the replacement of imported oil and other types of mineral fuels with "clean energy" with a predominance of renewable energy sources is one of the most acute global and regional problems.

Plant biomass is the main source of organic matter on Earth. The total reserves of plant biomass are ≈1 trillion tons, and the annual increase in biomass in the world is ≈50 billion τ [Bungay H.R. // Energy: the biomass options. Wiley and Sons, New York. 1981. Bioprocessing of renewable resources to commodity bioproducts, First edition / Edited by V.S. Bisaria and A. Kondo. Wiley and Sons, New York. 2014. Sticklen M. // Curr. Opin. Biotechnol. 2006. Vol. 17. P. 315-319]. Modern technologies make it possible to process plant raw materials into biofuels and other various chemical compounds that are commercially demanded products [From the Sugar Platform to biofuels and biochemical. Final report for the European Commission Directorate-General Energy No. ENER / C2 / 423-2012 / SI2.673791, April 2015].

Cellulose, which is the main component of plant biomass, can be converted into glucose and other fermentable sugars by enzymatic hydrolysis. The sugars obtained in this way are indispensable raw materials for microbiological processes for obtaining fuel (ethanol, butanol, etc.), ethylene, organic and amino acids, polymers, fodder protein and many other useful products.

Deep bioconversion of plant cellulose-containing raw materials into simple sugars is a key stage and is carried out under the action of an enzyme complex. For the efficient conversion of cellulose, enzyme complexes of cellulases obtained using various fungal producer strains (microscopic fungi of the genera Trichoderma, Penicillium, Aspergillus, Humicola, etc.) are used. These enzyme complexes contain, in various ratios, the key enzymes involved in the hydrolysis of cellulose, β-endoglucanases (EG), which degrade cellulose to oligosaccharides, cellobiohydrolases (CBH), which convert cellulose and oligosaccharides into cellobiose, and β-glucosidases cellobiose to glucose [Gusakov A.V., Sinitsyn A.P. Depolymerization of natural biopolymers. Enzymatic hydrolysis of cellulose. Overview. "Chemistry of Biomass: Biofuels and Bioplastics" ed. S.D. Varfolomeeva, ed. Scientific world, Moscow, 2017, p. 65-99, ISBN 978-5-91522-451-2].

The qualitative and quantitative composition of the enzyme complex, as well as the activity and stability of the enzymes included in it, determine the efficiency of hydrolysis of cellulose-containing raw materials. Changing and optimizing the composition of the complex of cellulases makes it possible to obtain enzyme mixtures with increased biocatalytic ability. In addition, the protein engineering of cellulases allows you to increase the catalytic activity and thermal stability of individual enzymes, which opens up new possibilities for increasing the overall activity and stability of the final enzyme mixture [Illanes Α., Cauerhff Α., Wilson L., Castro G.R. // Bioresour. Technol. 2012. Vol. 115. P. 48-57.].

The filamentous fungus Penicillium verruculosum is a promising producer of a hydrolytic complex of carbohydrase enzymes and secretes a cellulase complex of highly active enzymes [Gusakov Α. V, Sinitsyn A.R. Cellulases from Penicillium species for producing fuel from biomass. Review. Biofuels (2012) 3 (4), 463-477], which makes it possible to use it as a basis for obtaining multienzyme complexes for the biodegradation of cellulose-containing materials.

The main component of the Penicillium verruculosum enzyme complex is cellobiohydrolase I (Ce17A), its amount in the complex is almost 50% of the total secreted protein. It has been shown that enzymes of the 7th family of glycoside hydrolases (GH7) play a key role in the process of biomass conversion [Ilmen M., Saloheimo Α., Onnela Μ. L., and Penttila Μ. Ε. (1997)

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl. Environ. Microbiol. 63, 1298-1306], and cellobiohydrolase I is the main limiting rate of biomass degradation by the enzyme [Payne SM, Knott BC, Mayes Η. B., Hans son Η., Himmel Μ. A., Sandgren A., Stahlberg J., and Beckham G T. (2015) Fungal cellulases. Chem. Rev. 115,1308-1448; Suominen P. L., Mantyla A. L., Karhunen T., Hakola S., and Nevalainen H. (1993) High-frequency one-step gene replacement in Trichoderma reesei. 2. Effects of deletions of individual cellulase genes. Mol. Gen. Genet. 241, 523-530]. However, the thermal stability of cellobiohydrolase I of the mesophilic fungus P. verruculosum is limited to 50 ° C, which does not allow its use in processes with elevated temperatures, for example, enzymatic hydrolysis of wood. This property of PPI has a negative effect on the efficiency of industrial enzymatic conversion due to the fundamental dependence of the reaction rate on temperature.

Thus, improving the technical characteristics of Penicillium verruculosum cellobiohydrolase I, in particular, its thermal stability, is an urgent and promising task.

The filamentous fungus Penicillium canescens is a producer of extracellular enzymes. Compared with other fungi, this strain has a number of advantages: it has a higher growth rate and synthesis of extracellular enzymes; the strain is grown on a simple and cheap medium with beet pulp; the fermentation process is easily scalable; received a recipient strain with an auxotrophic sign of selection; the system of expression and transformation of the exogenous DNA strain has been worked out [RF Patent 2001 124 653. Vavilova Ε. Α., Vinetsky Yu.P., Nikolaev I.V., Serebryany V. Α., Chulkin AM, Sinitsyn A.P., Sinitsyna O.A., Okunev O.N., Chernoglazoe V.M. // New transformation system for the expression of fungal genes under the control of regulatory systems of the fungal fungus Penicillium canescnes // Application publication date: 27.07.2003]. This makes it possible to use this mushroom as a basis for obtaining recombinant enzyme-producing strains for practical use in various fields of industry.

One of the modern approaches to increasing thermal stability is enzyme bioengineering using a combination of computer modeling and site-directed mutagenesis. This approach has been successfully applied to increase the thermal stability of P. canescens xylanase A [Denisenko Υ.Α., Gusakov A.V., Rozhkova A.M., Osipov D.O., Zorov I.N., Matys V.Y., Uporov I.V., Sinitsyn A.P. Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability, International Journal of Biological Macromolecules, 2017, v.104, p.665-671]. A similar approach was applied to the modification of CBH I by P. verruculosum.

Thus, the creation of a new strain-producer of thermostable modified CBH I P. verruculosum and the production of EP on its basis, which will be effective in the composition of enzyme mixtures used for biodegradation of plant biomass, is an important and urgent task of modern biotechnology.

The technical problem to be solved by the present invention consists in the introduction of 5 mutations into the cel7A gene encoding the modified form of CBH I, and the production of dry EP based on a new recombinant P. canescens mtCBHI strain (VKM F-4850D), which is a producer of heterologous mutant CBH I P. verruculosum, belonging to the 7th family of glycoside hydrolases (EC 3.2.1.176, molecular weight 55 kDa) for use in the biofuel industry for the efficient process of bioconversion of cellulose-containing biomass into technical sugars.

The technical result of the proposed invention is to reduce the time of enzymatic hydrolysis of cellulose-containing biomass due to the ability of the main active enzyme CBH I to effectively hydrolyze cellulose at elevated temperatures due to an increase in the thermal stability of the enzyme.

The essence of the invention consists in obtaining a recombinant P. canescens fungus strain by transforming it with a genetic construct containing a modified gene encoding a mutant P. verruculosum cellobiohydrolase I, followed by obtaining an enzyme preparation of a thermostable pulp and paper industry on its basis.

The invention is implemented as follows:

- Strain P. canescens mtCBHI (VKM F-4850D) was obtained by transformation of the auxotrophic strain P. canescens RN-3-11 with the expression plasmid pCBHI-mt5, followed by selection on agar medium with 10 mM NaNC> 3 and selection of the most promising clone according to the catalytic criterion activity and thermal stability in relation to MCC.

- The method of obtaining EP provides for submerged cultivation of the producer strain P. canescens mtCBHI (VKM F-4850D) on a medium containing beet pulp, followed by spray drying of the CL and bringing the enzymatic activity of the drug to 0.5 U / mg protein according to MCC.

EFFECT: invention allows to obtain EP with high activity of target cellobiohydrolase I with increased thermal stability. The use of the new FP will increase the efficiency of cellulose hydrolysis and reduce the time of enzymatic treatment.

Cultural, morphological and microscopic features of the P. canescens mtCBHI strain (VKM F-4850D):

It grows on agar media (Czapek's medium with yeast autolysate, Maltz agar, glucose-potato agar, wort agar) at T = 26-30 ° C for 7-10 days, pH 4.5-5.0.

On Czapek's medium with yeast extract, when the fungus is cultivated at 25 ° C on day 7, colonies reach 24-30 mm in diameter, folded, the surface is strongly radially folded, dense, thin, the growth zone grows into agar, has a width of 1.5-2.0 mm. The mycelium is light yellowish, woolly, the center of the colony is convex, conidiogenesis is weak, gray-greenish in color. There is no exudate or soluble pigment. The reverse side is light; in the center of the colony it is pale orange. At a temperature of 37 ° C, colonies with a diameter of 5 mm, mycelium are light, no conidia formation, at a temperature of 5 ° C, no growth.

When growing on Maltz agar, the diameter of the colony is 23-24 mm, the surface is strongly radially folded, dense, thin, the growth zone grows into agar, 1.5-2.0 mm wide. The mycelium is white, woolly, appressed, conidiogenesis is very weak, practically absent. There is no exudate or soluble pigment. The reverse side is light.

When microscopic, the strain has two-tiered, terminal, biverticillate conidiophores, smooth, about 150 microns long, 2-3 microns wide. Metulae are divergent, 10-13 × 2.5-3.0 μm in size, ampulliform phialids, 7-8 × 2.8-3.0 μm in size. Conidia are round, rough, 3.0-3.5 microns in size.

When cultivated in submerged conditions using soluble substrates (glucose, fructose, lactose), a loose branched mycelium with weak pelletization is formed, the specific initial growth rate of the mycelium was 0.35 h ^-1 , at the end of cultivation 0.1 h- ¹ .

Physiological and biochemical characteristics of the strain:

Mesophilene. The optimum temperature for mycelium growth is 32 ° C (29-34 ° C), the optimum for the formation of cellulases is 28 ° C (26-29 ° C). The optimum pH values for growth and secretion of cellulases are 3.5-5.0. Mycelium growth is also observed at pH 2.5, but very weak formation of cellulases and other carbohydrases is observed.

The nystatin resistance is good. With surface cultivation, it is resistant to concentrations up to 0.5 μg / ml, at a concentration of 2.5 μg / ml, growth is inhibited. When digitonin (3.5-4.0 μg / ml) or Bengal rose (30-50 μg / ml) is added to the medium, the colony size decreases.

It is a prototroph. It is able to quickly assimilate glucose, glycerol, galactose, D-mannose, D-mannitol, trehalose, sorbose and sorbitol, and more slowly - D-xylose, L- and D-arabinose, L-rhamnose and ribose. Poorly assimilates: D-glucosamine, deoxyribose, deoxygalactose, 2-deoxy-D-glucose and 5-thio-B-glucose.

Uses inorganic and organic nitrogen, well assimilates nitrate and ammonium forms of nitrogen.

Forms enzyme systems that allow it to grow on the appropriate complex substrates: cellulose, starch, xylan, beta-glucan.

The P. canescens strain mtCBHI (VKM F-4850D) is distinguished by the presence of the production of thermostable heterologous mutant CBH I with a 2.5-fold increased thermal stability with respect to MCC at 65 ° C compared to native CBH I.

In the present invention, the method for determining the activity of cellobiohydrolase I is based on measuring the rate of formation of reducing sugars (BC) by the Shomody-Nelson method during the hydrolysis of a polysaccharide substrate (MCC). A unit of activity is such an amount of enzyme, which leads to the formation of 1 μmol BC per minute at pH 5.0 and 40 ° C [Sinitsyn A. P., Gusakov A. V., Chernoglazoe V. A. Bioconversion of lignocellulosic materials. - M .: MGU, 1995. - 144 s]. The activity of p-NF-P-O-lactoside was determined by the rate of formation of n-nitrophenol during hydrolysis of pNPL; the unit of activity was the amount of enzyme required for the formation of 1 μmol of n-nitrophenol for 1 minute at pH 5.0 and 40 ° C. [Sinitsyn A.P., Chernoglazoe V.M., Gusakov A.V. // Results of Science and Technology, ser. Biotechnology. - M .: VINITI, 1993. T. 25, S. 30-37].

A new FP, mtCBHI-5, obtained on the basis of P. canescens mtCBHI (VKM F-4850D) has improved operational characteristics due to the introduction of 5 mutations into the se17A gene encoding a mutant form of CBH I with high exo-P-1,4 -glucanase activity and increased thermal stability. This is an advantage of this mtCBHI-5 FP.

The new EP mtCBHI-5, obtained on the basis of the QL of the mutant P. canescens mtCBHI (VKM F-4850D), a producer of thermostable highly active CBH I, will have a high profitability in the processes of enzymatic treatment of lignocellulosic biomass due to the possibility of hydrolysis at an elevated temperature.

The possibility of using the invention is illustrated by examples that do not limit the scope and essence of the claims associated with them.

The invention is characterized by the following examples.

Example 1. Protein engineering of CBH I and introduction of point mutations into the seHA gene.

On the basis of bioinformatic analysis of the spatial structure of the protein of P. verruculosum, built on the basis of the spatial structures of crystals of cellobiohydrolase from Trichoderma reesei (Hypocrea jecorina anamorph, PDB ID: 50A5 and 4C4C), an unstructured region of the polypeptide chain was selected (from T301 to N380, in Fig. area outlined in red). The method of network analysis of constraints (CNA, Constraint Network Analysis, http://cpclab.uni-duesseldorf.de/cna) in the structure of the pulp and paper industry in the section 301-380 were identified 5 "weak" points, i.e. 5 positions a / c in which the average stiffness index, Ri, is minimal. These points were T291, V290, E302, V312, G342. Further, by the molecular dynamics method at each “weak” point, the variants of replacing the native amino acid with 19 others were calculated. Thus, taking into account the changes in the stiffness parameter and changes in the stabilization energy, AAG, the most promising replacements were T291P, V290F, E302L, V213W, G342R.

To introduce mutations into the se17A gene, the oligonucleotides shown in Table 1 were used.

The section of the polynucleotide chain containing the five mutations indicated above was synthesized separately at ZAO Evrogen. The structure of the mutated gene region is shown in FIG. 2.

Amplification of PCR products was carried out in a MyCycler thermal cycler (BioRad, USA). As a template, either the genomic DNA of the P. verruculosum strain, previously isolated from a 2-day-old mycelium of the fungus using the DNeasy Plant Mini Kit (QIAGEN, Germany), or the pCBHImt plasmid with a mutated portion of the cbh7A gene, was taken. The full-length gene was obtained In the first step, individual fragments were obtained using the forward oligonucleotides OVERLAP 1-FWD, OVERLAP2-FWD or CBHI B1-UPLIC and reverse OVERLAP 1-REV, OVERLAP2-REV or CBHII_Bl-LOWERLIC. isolated from a 1% agarose gel using the GelExtraction KIT (QIAGEN, Germany) and mixed as templates to obtain the full-length se17A gene by PCR using the edge primers CBHI_B1-UPLIC and CBHII_Bl-LOWERLIC, which contained sites of independent ligation for subsequent ligation gene with a mutation into the pXEG vector.Full-length PCR-πηοπνκτ Gpasmep 1578 bp and The first linearized pXEG vecto were treated with T4 DNA polymerase (Thermo Fisher Scientific Inc., Waltham, MA, USA) in the presence of dATP and dTTP (Thermo Fisher Scientific Inc., Waltham, MA, USA), respectively. The reaction conditions were as follows: 22 ° - 30 min, 80 ° - 30 min. The treated products were ligated into the pXEG vector at a ratio of 50 ng vector with 150 ng insert. Then the mixture was incubated for 1 hour at 22 ° C without the addition of ligase, after which this mixture was transformed into bacterial competent cells of E. coli MachI strain (Invitrogene, Carlsbad, CA, USA) according to the standard transformation protocol [J. Sambrook, D. Russell, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001]. Thus, bacterial colonies were obtained that could grow on a medium with ampicillin, thanks to the bla gene in the resulting plasmid construct.

The resulting clones were screened by PCR. For this, E. coli cells were used as a template, BIOTAQ ™ Red DNA Polymerase (Bioline Reagents Ltd, UK) and terminal oligonucleotides CBHI_B1-UPLIC and CBHI_Bl-LOWERLIC. The reaction was carried out under the following conditions: 1.5 min at 95 ° C (primary denaturation); 30 subsequent cycles - 1 min at 95 ° С (denaturation in a cycle), 2 min at 50 ° С, 1 min at 72 ° С (synthesis); 10 min at 72 ° C, 10 min at 4 ° C. The resulting products were studied using 1% agarose gel electrophoresis in TBE buffer. The cloning efficiency was shown to be 80%. Thus, the pCBHI-mt5 plasmid was obtained, containing the cell A gene with 5 mutations.

Plasimid sequencing showed the presence of the desired mutations in the polynucleotide sequence in the absence of insertions, deletions, or additional mutations in the rest of the cell A.

Example 2. Obtaining a recombinant P. canescens mtCBHI strain (VKM F-4850D) and an mtCBHI-5 enzyme preparation based on it.

Plasmid pCBHI-mt5 obtained in Example 1 was transformed into the recipient strain P. canescens RN-3-11-7 (ΔniaD) together with plasmid pSTA10 in a ratio of 5: 1 (μg of each DNA) according to the standard method [Sambrook, J., and Russell, DW (2001) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, N.Y .; A.Y. Aleksenko, N.A. Makarova, I.V. Nikolaev, A.J. Clutterbuck, Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene, Curr. Genet. 28 (1995) 474-478]. As a result of transformation, more than 40 recombinant strains were obtained. Transformants were cultured in flasks on a standard culture medium of the following composition (g / l): beet pulp - 30, peptone - 50, KH2PO4 - 25, pH 4.5. As a result of the primary screening by the criterion of the presence of a band of 55 kDa, consisting in carrying out electrophoresis of culture liquids secreted by the recombinant strains (data not shown), the P. canescens mtCBHI strain with the highest enzymatic activity on crystalline cellulose (MCC) was selected.

The cultivation of the P. canescens mtCBHI strain was carried out in a 3 L KF-108 fermenter (Prointech, Russia) equipped with a bubbler for air supply to the apparatus and a turbine stirrer on medium 1 of the following composition:

Wednesday 1 (g / l):

Beet pulp - thirty Peptone - 50 KH ₂ PO ₄ - fifteen (NH ₄ ) ₂ SO ₄ - five CaCl ₂ - 0.3 MgO ₄ * 7H ₂ O - 0.3

The cultivation was carried out for 144 h at a pH not lower than 5.2 and 30 ° C.

At the end of the fermentation, the mushroom biomass was removed by centrifugation (4000 rpm for 20 min on an Avanti JXN-26 centrifuge, Vesctap coulter, USA), free of CL cells was concentrated by ultrafiltration (with a cutoff limit of 15 kDa), the ultraconcentrate was dried on a spray dryer (Buchi MiniSpray Dryer B-290, conditions: T _IN = 135 ° C, T _OUT = 55-65 ° C, degree of aspiration 70%, flow rate 0.5 L of CL per hour) to obtain dry FP, which were a beige friable powder readily soluble in an aqueous medium. Thus, dry EP mtCBHI-5 was obtained, the activity of which is shown in Table 2. Enzyme preparations mtCBHI-5 and RN-3-11-7 (an enzyme preparation obtained in a similar way based on the recipient strain) had a basic xylanase activity within the range. The enzyme preparation mtCBHI-5 also exhibited increased activity on microcrystalline cellulose, which indicates additional expression of the introduced cellobiohydrolase I.

Further, mass spectrometric (MS) analysis of samples of target proteins was carried out to confirm amino acid substitutions. Samples of the target protein were used for MS analysis. MS analysis showed the presence of mutations E302L, V213W, G342R. Mutations T291P and V290F did not fall into the zones of protein fragmentation after trypsinolysis; however, the molecular weight of the mutant protein corresponded to the expected 55 kDa, from which it was concluded that there were T291P and V290F mutations, as well as proven E302L, V213W, G342R mutations.

Example 3. Study of thermal stability of P. verruculosum modified CBH I containing mutations T291P, V290F, E302L, V213W, G342R.

To study the thermal stability of modified CBH I, the target enzyme was isolated from EP mtCBHI-5 in a homogeneous form in three stages according to the scheme below using an AKTA purifier chromatograph (GE Health Care, Sweden):

1. Gel permeation chromatography (BioGel P4 column, 0.02 Μ Bis-Tris / HCl buffer; pH 6.80) for desalting the mtCBHI-5 EP solution.

2. Fractionation of desalted FP mtCBHI-5 using anion exchange chromatography (Sourse 15Q column 10 ml, eluent 0.01 Μ Bis-Tris / HCl; pH 6.8) in a NaCl gradient from 0 to 0.4 M.

3. Additional purification of fractions containing mutant CBH I using hydrophobic chromatography (Sourse 15Iso column 1 ml, eluent 50 mM NaAc; pH 5.0) in an ammonium sulfate gradient from 1.4 to 0 M. Thus, the mutant was isolated in a homogeneous form. the form

CBH I from EP mtCBHI-5 obtained on the basis of the recombinant P. canescens mtCBHI strain.

To assess the thermal stability of the mutant CBH I, hydrolysis of MCC (microcrystalline cellulose) was carried out at various temperatures. The experiment was carried out in 2 ml tubes in a thermal shaker at 50, 65, and 75 ° C. The concentration of the substrate in the reaction mixture was 10 g / L (in terms of dry matter), the reaction was carried out in 0.1 Μ acetate buffer, pH 5.0, with stirring at 1000 rpm. The hydrolysis was carried out for two days. To provide additional stirring of the reaction mixture, a small magnetic stirrer (n = 5 mm, c1 = 2 mm) was placed in the test tubes. The homogeneous enzyme was added in terms of protein - 0.5 mg of protein per 1 g of dry matter of the substrate. A homogeneous native enzyme CBH I of P. verruculosum isolated from the P. canescens strain of CBP according to a similar scheme was used as a control.

At certain time intervals, samples were taken from the reaction mixture and the concentration of sugars was determined by HPLC. FIG. 3 is a graph showing the dependence of the concentration of sugars on time at different temperatures.

The mutant form of CBH I was found to be more active and provided a higher yield of sugars at temperatures of 50 °, 65 °, and 75 ° C than the native wild-type CBH I in the same temperature range. For native wild-type CBH I, the yield of sugars was highest at 50 ° C, the yield of sugars at 65 ° C was slightly lower, and at 75 ° C the enzyme was practically inactive. The mutant form of CBH I retained a relatively high activity even at 75 ° C, and the highest yield of sugars was observed at 65 ° C. It can be concluded that at 65 ° C the enzyme was most active.

Thus, the homogeneous mutant CBH I containing 5 amino acid substitutions has increased thermal stability at high temperatures compared to the wild type enzyme. The yield of sugars upon hydrolysis of MCC after 48 hours at 65 ° C under the action of modified CBH I was 2 times higher than that of the wild-type enzyme.

Example 4. Determination of reducing sugars (BC) after hydrolysis of plant substrates.

The experiment was carried out in a cell thermostatted at a given temperature (50 ° C and 65 ° C) placed in a 'TNNOVA 40 Thermo Shaker "(USA). The concentration of the substrate in the reaction mixture was 100 g / L (calculated on dry matter). was carried out in 0.1 Μ acetate buffer with stirring (250 rpm). Hydrolysis was carried out with mixtures of purified enzymes. The mixtures consisted of CBH I (mutant form and native as a control), EG II (endo-1,4-β-glucanase II P .verruculosum) and BG (A. niger β-glucosidase) .The enzymes were dosed according to the protein concentration, in the PP: EGP ratio as 8: 2. The final protein concentration in the reaction mixture was 10 mg / g dry substrate, the volume of the reaction mixture was 20 ml. A. niger β-glucosidase was dosed according to activity at the rate of 40 units of activity per 1 g of dry substrate. Hydrolysis was carried out for 2 days. As a control, instead of mutant PPP, the reaction mixture was supplemented with an appropriate amount of native wild-type PPP isolated from strain P .canesce ns PPI according to the scheme from Example 3. The reaction cell was a plastic vessel with a lid of 50 ml; to provide additional stirring of the reaction mixture, a metal cylinder (d = 7 mm, h = 10 mm) made of stainless steel was placed in the cell. At certain time intervals, samples (0.5 ml each) were taken from the reaction mixture, centrifuged for 3 min at 10000 g, and the concentration of reducing sugars (BC) in the supernatant was measured by the Shomody-Nelson method [Sinitsyn A.P., Gusakov A.V., Chernoglazoe V. .AND. Bioconversion of lignocellulosic materials. - M .: Moscow State University, 1995. - 144 s] and glucose by glucose oxidase method [Polygalina GV, Cherednichenko B.C., Rimareva LV. Determination of enzyme activity. M .: DeLiPrint, 2003. S. 147-169.]. The yield of BC and glucose is shown in FIG. 4.

The hydrolytic capacity of mixtures 2 and 4 containing the native form of CBH I with respect to aspen wood was lower than that of mixtures 1 and 3 containing the mutant form of CBH I. Moreover, the yield of BC and glucose at 65 ° C of mixture 4 was lower than at 50 ° C (mixture 2).

The hydrolytic capacity of mixture 1 at 50 ° C was higher than that of the control mixture under similar conditions. The yield of BC and glucose was 70 and 46 g / l, respectively, which is 13% more than in the control.

As the temperature increased, the hydrolytic capacity of mixture 3 (65 ° C) increased significantly. The yield of BC and glucose was 81 and 54 g / l, respectively, which is 45% more than in the control.

Thus, with an increase in thermal stability in the mutant form of PPP, its hydrolytic capacity significantly increased.

Claims (3)

1. Heterologous cel7 A gene of the fungus Penicillium verruculosum containing mutations in the target coding sequence to provide amino acid substitutions at positions T291P, V290F, E302L, V213W, G342R of cellobiohydrolase I protein with increased thermal stability and increased hydrolytic activity at 65 ° C. 2. Recombinant strain Penicillium canescens mtCBHI (VKM F-4850D), obtained by transforming the cells of the fungus Penicillium canescens RN3-11-7 with a genetic construct with the cel7A gene according to claim 1 and intended for secretion of heterologous thermostable cellobiohydrolase I Penicillium verruculosum. 3. Enzyme preparation mtCBHI-5, obtained on the basis of the recombinant P. canescens strain mtCBHI and characterized by activity on microcrystalline cellulose and n-NF-lactoside - 240 and 124 units per 1 g of the enzyme preparation mtCBHI-5.

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