Method of Lignocellulose Materials Saccharification using enzymes produced by

Penicillium fimiculosum

This invention relates to biotechnology, more specifically, to biotechnological processing and biorefinery of lignocellulose materials, and can be used for the production of fermentable sugars for further ethanol production, as well as feed additives.

Known are many strains producers of carbohydrases capable of decomposition of cellulose containing materials, and methods of their aerobic growth.

Known (RU Patent RF 2057179 C 12 N 9/42, 1996) are Aspergillus niger strains which growth of media containing inducers of cellulolytic enzymes produces a complex of enzymes including cellulase, xylanase, β-glucosidase, β- xylosidase, β-glucanase and laminarinase. The enzyme activities in the growth fluid are as follows: 5.0 U/ml for cellulase, 125.0 U/ml for xylanase, 20.0 U/ml for β-glucosidase, 36.0 U/ml for β-xylosidase, 0.95 U/ml for β-glucanase and 1.25 U/ml for laminarinase.

Although Aspergillus genus fungi form enzyme complexes with containing a wide range of carbohydrases, the activities of most of the individual components composing the complex are insufficiently high and therefore they are useless for practical application.

Cellulose producers are also Trichoderma genus mycelial fungus strains.

Specifically, the Trichoderma reesei MCG 80 mutant strain (US Patent 4472504, C 12 N 9/42, 1984) during continuous fermentation on a 8% cellulose + biotin nutrient medium provides for a cellulase activity of 17.2 U/ml FPA (FPA is the a cellulase complex activity determined using the filter paper assay and

expressed in international units in accordance with the IUPAC recommendation [T. K. Chose., Pure and Appl. Chem., vol.59, 2, p.257-268]).

Known also is the VKM F-3634 D strain of Tr.longibrachiatum mycelial fungus (RU Patent 2195490 C 12 N 9/42, 2002) that produces a complex of carbohydrases comprising cellulases, β-glucanases, xylanases, pectinases and mannanases. The activities of cellulases, β-glucanases, xylanases, pectinases and mannanases are 6.5, 25.0, 20.0, 7.0 and 0.5 U/ml, respectively.

Known is the Tr.reesei BCM 18.2/KK mutant strain (VGNKI-28) (RU Patent 2001949, C 12 N 9/42, 1993) obtained using parasexual processes from the source Tr.reesei IMET 43803 culture, possessing an elevated productivity and providing for a high enzyme (cellulase) yield per unit weight of the used substrate. This strain has enzyme systems providing the growth on media comprising cellulose, starch, chitin, pectin, xylan, laminarin and lichenin. When grown on a sugar beet pulp media, this strain provides for a 3.5-4.2 FPU/ml activity (fermentation time 110 h), when grown in a fermenter on a lactose containing media - 18.2 FPU/ml (81 h) and when grown in a fermenter on cheese whey - 12- 14 FPU/ml (100-110 h).

The disadvantage of the strains described above are the experimentally proven facts confirming that the Trichoderma genus fungi are usually highly productive, primarily, in relation to cellulases, but are far less productive in relation to other carbohydrases (β-glucanases, xylanases, pectinases and mannanases) that are required for the hydrolysis of various polysaccarides comprised, along with cellulose in the renewable biomass. All the Trichoderma strains listed above have low productivities in relation to carbohydrases that are not cellulases (β-glucanases, xylanases, pectinases, mannanases etc.).

Disadvantage of the known method is its complexity and the low catalytic activity of the resultant biologically active catalyst in relation to cellulose containing materials.

Therefore one object of the invention disclosed herein is to provide a Penicillium funiculosum S-2006 mycelial fungus strain that is producer of the carbohydrase complex comprising cellulases, β-glucanases, β-glucosidases (cellobiases), xylanases and xyloglucanases.

Another object of the invention disclosed herein is to provide a method of obtaining a biocatalyst for deriving fermentable sugars from cellulose containing materials.

Still another object of the invention disclosed herein is to provide a method of obtaining fermentable sugars from cellulose containing materials.

During the implementation of the technical solution suggested herein, a newly selected Penicillium funiculosum S-2006 mycelial fungus strain that is producer of the carbohydrase complex comprising cellulases, β-glucanases, β- glucosidases (cellobiases), xylanases and xyloglucanases.

The strain was obtained by multistage mutagenesis and selection from the source Pen.funiculosum BKMF-3661 culture.

Pen.funiculosum strain mutagenesis and selection are achieved by suspending fungus spores in distilled water with the addition of 0.1% Tween-80 and ultraviolet irradiation (a 3 W/cm ² light flux) for 3 min. Following that the reference and the irradiated spores are seeded on Petri dishes with glucose-potato agar and incubated for 48 h at 28°C. The survival rate is calculated thereafter. A 1-5% survival rate is considered sufficient for mutant selection. Each surviving clone is transferred into dishes with a selecting media comprising 0.1% carboximethylcellulose (CMC) or 1% oat xylan (Sigma) and incubated for 48 h at 28 ⁰ C. Improved mutants are selected visually judging by hallo enlightened areas around single colonies. The most active mutants selected from the dishes are tested for the productivity of cellulase, β-glucanase, β-glucosidase (cellobiase), xylanase and xyloglucanase synthesis by growing in a liquid medium in retorts. The most active variants selected by fermentation in shaking flasks are again (multiply) UV irradiated and selected in dishes and shaking flasks as described above.

Storage conditions: the strain can be stored in lyophilized condition for several years and/or in agar slants with a Chapek medium, or in malt agar at +4°C with necessary selection at least once in 3-6 months.

Culture-Morphological Strain Parameters

When grown on Malt agar, the diameter of colonies reaches 36 mm in 7 days at 25°C. The colonies are dense and smooth and have convex centers. The

mycelium is light and fluffy. The reverse side is yellowish-reddish-orange. Conidia genesis is weak and grayish-green. Colonies grown on Chapek agar with yeast extract for 7 days are 27-30 mm in diameter, creased and even edged and have a woolly surface. The mycelium is light and yellowish in color. The reverse side is yellowish-orange. Conidia genesis is weak.

The fungus does not grow on a glycerin containing medium and in Chapek agar at 5 ⁰ C. When grown on Chapek agar at 37°C the colonies are 18 mm in diameter, creased and medium dense, and their reverse sides are brownish- reddish.

Conidia genesis: biverticillate penicilli, depressed smooth metulae (8-10 x 2.5-3.0). Acerosic short-necked phialides (8-9 x 2.5-3.0). Elliptical small 2.2 x 1.5-2.0) smooth conidia.

When grown in submerged fermentation on soluble substrates (glucose, fructose or lactose), the strain forms a loose branching mycelium with weak pelletization, the initial mycelium growth rate being 0.35 h ^"1 and the final mycelium growth rate being 0.1 h ^"1 .

Physiological and Biochemical Strain Parameters

The strain is mesophilic. The optimum mycelium growth temperature is 32 ⁰ C (29-34 ⁰ C), the optimum one for cellulase formation 28 ⁰ C (26-29 ⁰ C). The optimum growth and cellulase secretion pH values are 3.5-5.0. Mycelium also grows at pH 2.5 but at this pH cellulase and other carbohydrase formation is very weak.

The strain has a good resistance to nystatin. In the conditions of solid state fermentation, the strain is stable to nystatin concentrations of up to 0.5 μg/1, and at 2.5 μg/1 concentration the growth is suppressed. Digitonin (3.5-4.0 μg/1) or Bengal rose (30-50 μg/1) addition reduces the size of the colonies.

The strain is prototrophe and is capable of rapidly assimilating glucose, lactose, glycerin, galactose, xylose, D-mannose, D-mannitol, tregalose, sorbose and sorbitol and less rapidly assimilating D-xylose, L- and D-arabinose, L- rhamnose and ribose. Weakly assimilates D-glucosamine, desoxyribose, desoxygalactose, 2-desoxy-D-glucose and 5-thio-D-glucose.

The strain uses inorganic and organic nitrogen and well assimilates nitrate and ammonium forms of nitrogen.

The strain forms enzyme systems providing the growth on appropriate substrates such as cellulose, starch, xylan, laminarin, β-glucan, lichenin, pectin and galactomannan and is capable of utilizing lactic acid at below inhibiting concentrations.

This genus of mycelial fungus is not considered pathogenic in Provisions for the Procedure of Storage, Handling, Dispatching and Transportation of Cultures of Bacteria, Viruses, Rickettsiae, Fungi, Protozoa, Microplasmas, Bacterial Toxins and Biological Poisons.

The suggested Pemfunicidosum strain is grown under aerobic conditions in submerged fermentation on a nutrient medium comprising one or more carbon source that induce biosynthesis of the enzymes. These substrates can also be not enzymes biosynthesis inducing ones. Under appropriate growth conditions based on using soluble substrates, e.g. glucose, the strain is capable of secreting a complex of enzymes, i.e. the cellulase, β-glucanase, β-glucosidase (cellobiase), xylanase and xyloglucanase synthesis by growing in a liquid medium in shaking flasks. Glucose in the growth medium can be replaced for a cheaper product, e.g. starch hydrolysate.

The filter paper activity (FPA) of the cellulases is determined using the conventional method [Ghose T.K. Measurement of Cellulase Activity. Pure Appl. Chem., 1987, v.59, p.257-268] at 50°C and pH 4.8 using chromatographic paper No. 1 manufactured by Whatman, UK, and the dinitrosalicylic assay of reducing sugars. The fermentation activities in relation to carboxymethylcellulose (CMC, produced by Sigma, US), avicel (microcrystalline cellulose of PH 105 produced by Serva, Germany), barley β-glucan (Sigma, US), birch tree xylan (Sigma, US) and tamarind xyloglucan (Megazyme, Australia) are determined from the initial reducing sugars formation rates (in 5-10 minutes of the enzymatic reaction) using the Shomody-Nelson assay of reducing sugars [A.P. Sinitsyn, A. V. Gusakov and LM. Chernoglazov, Bioconversion of Lignocellulose Materials, Handbook, Moscow, Moscow State University, 1995, p. 144-156] from an appropriate

40

6 substrate (5 g/1) at pH 5.0 (0.1 M Na acetate buffer) and 50°C (the activity in relation to avicel is determined at 40 ⁰ C).

The β-glucosidase activity is determined using ra-nitrophenyl-β-glucoside (Sigma, US) as a substrate at pH 5.0 (0.1 MNa acetate buffer) and 40 ⁰ C (reaction time - 10 min). Reaction is terminated by addition Na ₂ CO ₃ . The quantity of the produced «-nitrophenol is calculated using the extinction coefficient (D ₄ O ₀ = 18300 MT ¹ Cm ^"1 ).

The activities unit toward different substrates is expressed as the quantity of the enzyme that causes the hydrolysis of 1 μmole of glycoside bonds or the formation of 1 μmole of the reduce sugars (or «-nitrophenol) in 1 minute under the reaction conditions as specified hereinabove.

The enzyme preparations obtained using the suggested strain can be used in the form of a culture liquid (cultural filtrate), in the form of concentrated liquid preparations obtained by ultrafiltration or vacuum evaporation, or in the form of dry or granulated preparations.

Below the application capacities of the invention will be exemplified with the use of specific embodiments that do not restrict the scope and nature of the claims related thereto.

Example 1. The inoculum is obtained by growing a culture of the claimed Pemfuniculosum S-2006 fungus strain on malt or SM agar at 29 ⁰ C for 7 days and further at room temperature for 5 days. 1 ml of spore suspension washed from the agar with water containing 0.1% of Tween 80 is planted into in 750 ml Erlenmeyer shaking flasks. The strain is grown under aerobic conditions in shaking flasks containing 100 ml of a nutrient medium of the following composition, g/1: 40 sugar beet pulp, 14 malt sprouts, 6 (NELi) ₂ SO ₄ , 2 KH ₂ PO ₄ and 0.6 MgSO ₄ XH ₂ O; pH 5.4. The cultures are incubated in shaking flasks at 3O ⁰ C and 200 rpm for 120 h.

The avicelase, carboxymethylcellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and FPA activities of the fermentation broth after 12O h growth are 15, 130, 140, 10, 380, 100 and 12 U/ml, respectively.

Example 2. The culture is grown in Erlenmeyer shaking flasks as described in Example 1 in a nutrient medium of the following composition, g/1: 40

cellulose, 10 glucose, 6 (NEU) ₂ SO ₄ , 2 KH ₂ PO ₄ and 0.6 MgSO ₄ XH ₂ O; pH 5.4, 3O ⁰ C.

The avicelase, carboxymethylcellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and FPA activities of the fermentation broth after 120 h growth are 25, 260, 275, 15, 510, 140 and 18 U/ml, respectively.

Example 3. The enzyme preparation is obtained by growing the Pen.funiculosum S-2006 fungus strain in an ANCUM-2M fermenter (7.0 1 working volume) on a nutrient medium of the following composition, g/1: 50 cellulose, 10 glucose, 8 KH ₂ PO ₄ , 3 (MIj) ₂ SO ₄ , 0.2 MgSO ₄ x7H ₂ O and 0.2 CaCl ₂ x2H ₂ θ at 28°C and pH maintained in the 4.5-5 range by adding HCl or NH ₄ OH. During the growth, glucose is continuously added to the fermenter at a 2- 3 g/l/h rate. In 144 h, the culture liquid containing a complex of highly active carbohydrases is separated and concentrated by ultrafiltration (on 10 kDa membranes) to obtain liquid biocatalyst.

The avicelase, carboxymethylcellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and FPU activities of the culture filtrate after 144 h growth are 45, 650, 690, 28, 1280, 260 and 42 U/ml, respectively. The avicelase, carboxymethylcellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and FPA activities of the liquid concentrated enzyme preparation are 280, 3900, 4250, 170, 7580, 1570 and 260 U/ml, respectively.

Example 4. The enzyme preparation is obtained by growing the Penjimiculosum S-2006 fungus strain in an ANCUM-2M fermenter (7.0 1 working volume) on a nutrient medium of the following composition, g/1: 60 cellulose, 30 glucose, 12 KH ₂ PO ₄ , 7 CNHU) ₂ SO ₄ , 0.24 MgSO ₄ x7H ₂ O and 0.4 CaCl ₂ x2H ₂ O at 28°C and pH maintained in the 4.5-5 range by adding HCl or IMH ₄ OH. During the growth, glucose (in the form of starch hydrolysate) is permanently added to the fermenter at a 2-3 g/l/h rate. In 144 h, the culture liquid containing a complex of highly active carbohydrases is separated and concentrated by ultrafiltration (on 10 kDa membranes) and subjected to freeze during to obtain dry biocatalyst. The avicelase, carboxymethylcellulase, β- glucanase, β-glucosidase, xylanase, xyloglucanase and FPA activities of the culture liquid after 144 h growth are 58, 820, 870, 38, 1520, 360 and 55 U/ml,

respectively. The avicelase, carboxymethylcellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and FPA activities of the dry enzyme preparation are 870, 13100, 13500, 610, 22900, 5760 and 870 U/ml, respectively.

The efficiency of the lignocellulosic materials saccharification (hydrolysis) method disclosed herein was assessed by comparing the enzyme preparation obtained as described above to counterpart cellulase enzyme preparations.

The following enzyme preparations and lignocellulose materials were used for comparison.

Penicillium genus fungus based enzyme preparations: the S-2006 preparation obtained as described above, the Bl laboratory preparation (P.vetruculosum) developed from Biotechnology Institute, Leipzig, the B400 laboratory preparation {P.verruculosum) from the Microorganism Biochemistry and Physiology Institute, Russian Academy of Sciences, the Cellulase 2000L, Cellulase, HK70 and Beta-glucanase 200L preparations (P.fimiculosum) from Erbslόh GmbH, Germany, the Rovabio Excel preparation (P.fimiculosum) from Adisseo, France; Trichoderma (T. reesei, T. longibrachiatum) genus fungus based enzyme preparations: Celloviridin G20x (T. longibrachiatum) from Promferment Ltd., Russia, the TW-I and TW-307 laboratory preparations (T. longibrachiatum) from the Microorganism Biochemistry and Physiology Institute, Russian Academy of Sciences, the Fibrilase HDL 160 (T.reesei) from Iogen, Canada, the Bio ACE preparation (T. longibrachiatum) from Dyadic International, US, and the Celluclast 1.5L preparation (T.reesei) from Novozymes, Denmark.

The specific activities of the enzyme preparations (in U/mg protein) are shown in Table 1.

Table 1.

Table 1 suggests that the enzyme preparation obtained using the method disclosed herein has the highest hydrolytic activity in relation to cellulose containing materials.

The cellulose containing materials can be wood samples of fir, pine, poplar and maple and wheat straw preliminarily treated with Organosolv organic solvent or using the steam explosion method. Treatment with Organosolv organic solvent (50% ethanol having pH reduced to 2.4 with 10% sulfuric acid) was performed at 195 ⁰ C for 40 min under a 3.2 MPa pressure. For steam explosion, the samples were first impregnated with 4.5% SO ₂ , exposed to 195°C for 4.5 min and then decompressed. Moreover, the cellulose containing materials can be sugar beet pulp, short-fiber cellulose (pulp and paper production waste), parchment pieces and other sources of cellulose containing wastes.

The biocatalyst hydrolytic (saccharification) activity assessment experiment is performed in a reaction cells placed on a shaker thermostated at 50 ⁰ C. The cells are filled with weighed substrate portions (Organosolv or steam exploded hardwood or softwood, preliminarily steam exploded cereal straw, sugar beet pulp, short-fiber cellulose, parchment pieces or other cellulose containing wastes), 6.8 ml of 0.1 M acetate buffer at pH 5.0 and 1 ml of preliminarily diluted enzyme preparation. The reaction mixture is mixed in the shaker at a 250 min ^"1 rotation frequency. The substrate concentration in the reaction mixture is 50 g/1 as recalculated to dry material, and the enzyme preparation is diluted in a way so that the FPA activity is 10 units per 1 g of dry substrate. After 12 h of hydrolysis, 1 ml samples are taken from the reaction mixture, centrifuged for 1 min at 10,000 rpm, and the reducing sugar and glucose concentrations are measured in the supernatant. The enzyme preparation hydrolytic activity criterion is taken as the glucose release per 12 h of insoluble substrate hydrolysis at 5O ⁰ C.

The reducing sugar concentration is determined using the Shomogy- Nelson method [AP. Sinitsyn, AV. GUsakov and I.M. Chernoglazov, Bioconversion of Lignocellulose Materials, Handbook, Moscow, Moscow State University, 1995, p. 144-156]. The glucose concentration is determined using the glucose oxydase - peroxidase method [LV. Berezin, MX. Rabinovich and AP. Sinitsyn, Study of the Capacities of the Kinetic Spectrophotometric Glucose Determination Method, Biochemistry, 1977, Vol. 42, No. 9, p. 1631-1636].

The experimental data suggest that for any source of lignocellulose raw material (softwood or hardwood, cereal straw, sugar beet pulp or pulp and paper

production waste), the largest quantity of fermentable to alcohol glucose was produced after 12 h of hydrolysis by the Penicillium funiculosum S-2006 preparation suggested for the implementation of the method disclosed herein. Its glucose yield was 15-20% higher than those for the commercial and laboratory Penicillium enzyme preparations or commercial and laboratory Trichoderma enzyme preparations.

Thus, the Penicillium funiculosum S-2006 strain suggested herein can produce a complex of highly active carbohydrases including cellulases, β- glucanases, β-glucosidases (cellobiases), xylanases and xyloglucanases, thus allowing the production of the entire enzyme complex and, if necessary, individual enzymes (components) of the lignocellulose raw material saccharification (hydrolysis) enzyme complex.

The technical result achieved by the implementation of the technical solution disclosed herein is an increase in the production yield of fermentable sugars from the cellulose containing material.