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WO2009108081A1 - Penicillium verruculosum filamentous fungus strain producer of a highly active complex of cellulases and accessory enzymes and a method of production of biocatalyst for cellulose and hemicellulose hydrolysis - Google Patents

Penicillium verruculosum filamentous fungus strain producer of a highly active complex of cellulases and accessory enzymes and a method of production of biocatalyst for cellulose and hemicellulose hydrolysis Download PDF

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WO2009108081A1
WO2009108081A1 PCT/RU2008/000559 RU2008000559W WO2009108081A1 WO 2009108081 A1 WO2009108081 A1 WO 2009108081A1 RU 2008000559 W RU2008000559 W RU 2008000559W WO 2009108081 A1 WO2009108081 A1 WO 2009108081A1
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strain
vkm
cellulases
filamentous fungus
cellulose
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PCT/RU2008/000559
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French (fr)
Inventor
Arkady Panteleimonovich Sinitsyn
Oleg Nikolaevich Okunev
Olga Arkadievna Sinitsyna
Vladimir Mikhailovich Chernoglazov
Vladimir Olegovich Popov
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Sinitsyn Arkady Panteleimonovi
Oleg Nikolaevich Okunev
Olga Arkadievna Sinitsyna
Chernoglazov Vladimir Mikhailo
Vladimir Olegovich Popov
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Publication of WO2009108081A1 publication Critical patent/WO2009108081A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium

Definitions

  • This invention relates to biotechnology and can be used in microbiological and food industries, in agriculture and for plant biomass conversion.
  • Complexes of cellulases and hemicellulases enzymes hydrolyzing the main components of plant cell wall can be used for the biodegrading of cellulose and hemicellulose containing fed-stocks, including industrial and agricultural wastes; for the conversion of plant biomass to sugars, bioethanol and other products of microbiological synthesis; for the destruction of plant cell wall; for the hydrolysis of non-starch polysaccharides; as well as in the food and alcohol industries, in brewing and in agriculture as feed additives and for forage silaging.
  • the closest counterpart of the invention by technical essence is the mutant Penicillium funiculosum S2006 BKM F-3887D strain producer of a complex of carbohydrases (cellulases, ⁇ -glucanases, xylanases, pectinases and mannanases) obtained by multistage mutagenesis and selection from the source P. funiculosum VKM F-3661D culture under UV irradiation (RF Patent Application # 2006110721/13, April 4, 2006).
  • carbohydrases cellulases, ⁇ -glucanases, xylanases, pectinases and mannanases
  • the technical task solved by the invention is to broaden the range of highly productive strains producing enzymes of cellulases and hemicellulases complexes having high specific activity and intended for the highly efficient destruction of natural plant biomass and wastes of its industrial and agricultural processing and for the hydrolysis of plant polysaccharides.
  • the technical result that can be achieved by the invention is providing a new highly productive Penicillium genus filamentous fungus strain capable of biosynthesizing a complex of cellulases having high specific activity (cellobiohydrolases, endoglucanases, cellobiases and ⁇ -glucosidases) and accessory enzymes (xylanases and xyloglucanases).
  • the essence of the subject of the invention is the new specially selected P.verruculosum PV2007 filamentous fungus strain producer of a complex of cellulases having a uniquely high specific activity (cellobiohydrolases, endoglucanases, cellobiases and ⁇ -glucosidases), and accessory enzymes (xylanases and xyloglucanases).
  • the P.verruculosum PV2007 filamentous fungus strain is deposited in the Russian Collection of Microorganisms at the G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences, No. VKM F-3972D.
  • the P.verruculosum PV2007 VKM F-3972D strain is obtained by multistage mutagenesis and selection from the initial P.verruculosum 28K VKM F-3764D culture.
  • the spore suspension of P.verruculosum 28K VKM F- 3764D culture is UV irradiated. After irradiation the spores are seeded into Petri dishes containing selective media based on Hetchinson media with 0.5% carboxymethylcellulose (CMC), grown at 28 0 C for 2 days and stained with Congo Red. The highest productivity mutants are selected visually by enlarged clarification zones around the colonies.
  • CMC carboxymethylcellulose
  • the most active mutants selected from the Petri dishes are tested for the productivity of synthesis of cellulases, cellobiases ( ⁇ -glucosidases), xylanases and xyloglucanases by growing in liquid media in shaking flasks.
  • the most active of the flask grown variants are repeatedly UV irradiated and selected in Petri dishes and shaking flasks as described above.
  • Storage conditions the strain can be stored in freeze dried condition for several years and/or on slants with agarized Czapek media or Malt agar at +4°C with necessary reseeding at least once in 3-6 months.
  • the fungus does not grow on glycerin media and on Czapek agar at 5°C. When grown on Czapek agar at 37°C the colonies are 18 mm in diameter, funiculose, moderately dence with brownish-reddish reverse side.
  • Conidiogenesis the penicilli are biverticillae, and the metulae are in closely appressed verticills and smooth (8-10 x 2.5 x 3.0).
  • the phialides are acerose (8-9 x 2.5 x 3.0) and short-necked.
  • the conidia are ellipsoidal, small (2.2 x 1.5 x 2.0) and smooth.
  • Submerged fermentation on soluble substrates produces weakly pelleted branched loose mycelium, initial specific mycelium growth rate being 0.35 h "1 , final specific growth rate being 0.1 h '1 .
  • the strain is mesophilic.
  • the optimum mycelium growth temperature is 32°C (29-34°C), and the optimum cellulase secretion temperature is 28°C (26-29°C).
  • the optimum pH for growth and cellulose secretion is 3.5-5.0.
  • Mycelium growth was also observed at pH 2.5, but cellulases and other carbohydrases formed quite slow.
  • the nystatin resistance is high. Surface grown strain is nystatin resistant to 0.5 ⁇ g/1; 2.5 ⁇ g/1 nystatin suppress the growth. Addition of digitonin (3.5-4.0 ⁇ g/1) or Bengali Pink (30-50 ⁇ g/1) reduces colony size.
  • the strain is prototrophic. It readily assimilates glucose, lactose, glycerin, galactose, xylose, mannose, mannitol, tregalose, sorbose and sorbitol, less readily assimilates arabinose, rhamnose and ribose and unreadily assimilates glucoseamine, desoxyribose, desoxygalactose, desoxyglucose and thioglucose.
  • the strain consumes inorganic and organic nitrogen and assimilates it well in the form of nitrates and ammonia.
  • the strain forms enzyme systems providing for growth on respective complex substrates: cellulose, starch, xylan, laminarin, ⁇ -glucan, lichenin, pectin and galactomannan. It can consume lactic acid at sub-inhibiting concentrations.
  • the carbohydrase biosynthesis catabolite repression is heavily reduced.
  • the degree of carbohydrases biosynthesis catabolite repression is tested as follows. The conidia are seeded to test tubes with the minimum media containing mineral salts, trace amount (0.5 g/1) or yeast extract, amorphous cellulose and the test repressor or antimetabolite (glucose, desoxyglucose, lactose, glycerin etc.).
  • the test tube diameter is 9 mm and the agar column height is 50-60 mm.
  • the test tube is incubated for 4 days at 30°C and then for 20 h at 45°C.
  • the stability of carbohydrases biosynthesis against catabolite repression is judged from the depth of the destruction zone in the amorphous cellulose (the size of the agar column clarification zone in the test tube) in the presence of a repressor or an antimetabolite.
  • the resultant mutant P.verruculosum PV2007 VKM F-3972D strain grown on glucose/potato agar and on sporulating agar (SM-agar) and differs in morphological attributes from the initial P.verruculosum 28K VKM F-3764D strain by the significantly lower sporulation, change in the color of the conidia (from grey-green to dark-grey) and that of the opposite side of the colony when grown on solid media, slower growth on agarized media and higher capability of biosynthesizing cellulases, cellobiases ( ⁇ -glucosidases), xylanases and xyloglucanases in the case of submerged fermentation.
  • This kind of filamentous fungus is not treated as pathogenic in the Provisions for the Registration, Storage, Handling, Distribution and Forwarding of Bacteria, Viruses, Rickettsia, Fungi, Protozoa, Mycoplasma, Bacterial Toxins and Biological Poisons.
  • Submerged fermentation of the P.verruculosum PV2007 VKM F- 3972D strain is carried out under aerobic conditions in fermentation media containing one or multiple carbon source that act as enzyme biosynthesis inductors.
  • the carbon sources may not be direct inductors.
  • the strain Under appropriate conditions using the insoluble substrates, e.g. microcrystalline cellulose (MCC), or soluble ones, e.g. glucose, the strain can secrete a complex of enzymes, such as cellulases (cellobiohydrolases, endoglucanases, cellobiases, ⁇ -glucosidases), xylanases and xyloglucanases, into the fermentation media.
  • Glucose in the fermentation media can be replaced for a cheaper product - such as starch hydrolyzate.
  • Enzyme preparations obtained using the resultant strain can be used as a cultural filtarte, in the form of liquid concentrated preparations produced by ultrafiltration, in dry or granulated forms.
  • the P.verruculosum PV2007 VKM F-3972D fungus culture is grown on Malt or SM agar at 29°C for 7 days and then at room temperature under light for 7 days.
  • the shaking flasks are seeded with 1 ml of suspended spores washed from the agar with water containing 0.1% Twin-80.
  • the strain is grown under aerobic conditions in 750 ml Erlenmeyer shaking flasks containing 100 ml of fermentation media preferably having the following composition, g/1: 50 MCC, 12 wheat bran, 12 yeast extract, 5 (NH 4 ) 2 SO 4 , 5 KH 2 PO 4 , 0.3 MgSO 4 x7H 2 0, 0.3 CaCl 2 x2H 2 O, pH 4.5.
  • the shaking flasks are incubated in the shaker at 32°C and 200 rpm for 120-144 h.
  • the cellobiohydrolase activity is 19-20 U/ml
  • the endoglucanase activity is 320-350 U/ml
  • the ⁇ - glucosidase activity is 20-25 U/ml
  • the cellobiase activity is 10-12 U/ml
  • the xylanase activity is 530-540 U/ml
  • the xyloglucanase activity is 200-220 U/ml.
  • Example 2 P.verruculosum PV2007 VKM F-3972D strain growth in a fermenter.
  • the P.verruculosum PV2007 VKM F-3972D strain is grown in an ANKUM 2M 10 1 fermenter (volume of fermentation media 6.0 1) preferably using a fermentation media of the same composition as for shaker flasks at pH 4.5 and 32°C for 120-144 h. After 36 h of fermentation process and until the end of fermentation process the continuous feeding by glucose to the fermenter at 1 g/l/h by glucose occurred.
  • the cellobiohydrolase activity is 60-65 U/ml
  • the endoglucanase activity is 820- 850 U/ml
  • the ⁇ -glucosidase activity is 60-70 U/ml
  • the cellobiase activity is 25-30 U/ml
  • the xylanase activity is 1550-1600 U/ml
  • the xyloglucanase activity is 610-640 U/ml.
  • the cultural filtrate containing a complex of cellulases and concomitant enzymes is ultrafiltered through hollow fibers (10 kDa cut off), and the UF-concentrate is freeze dried to produce dry enzyme preparation.
  • the cellobiohydrolase activity of the dry enzyme preparation is 900-920U/ml
  • the endoglucanase activity is 14,500-14,900U/ml
  • the ⁇ -glucosidase activity is 1,200-1,250 U/ml
  • the cellobiase activity is 600- 670U/ml
  • the xylanase activity is 23,000-24,000U/ml
  • the xyloglucanase activity is 7,500-8,200 U/ml.
  • Example 3 Comparative analysis activity of P.verruculosum enzyme complex.
  • Activity analysis is carried out by comparing the activity of enzyme preparation obtained as described above and similarly produced preparations of cellulases and accessory enzymes obtained with Penicillium and Trichoderma spp. producer strains. The fermentation preparations and methods used are as described below.
  • Penicillium sp. based preparations PV2007 preparation (obtained with the P.verruculosum PV2007 VKM F-3972D strain as described in Example 2), S-2006 preparation (obtained with the P.funiculosum S2006 VKM F-3887D strain as described in RF Patent Application # 2006110721/13, April 4, 2006); Trichoderma sp.
  • the cellobiohydrolase (avicelase) activity is measured by hydrolyzing MCC (5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) at 50°C for 60 min.
  • the reducing sugar (RS) concentration in the reaction mixture is assayed using the Nelson-Somogy method.
  • the unit cellobiohydrolase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml MCC suspension hydrolysis at 50°C and pH 5.0, releases 1 micromole of RS assayed by the Somogy-Nelson method in glucose equivalent.
  • the endoglucanase (CMCase) activity is measured by hydrolyzing water soluble carboxymethylcellulose Na salt (CMC, 5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) and 5O 0 C for 10 min.
  • the unit endoglucanase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml CMC solution hydrolysis at 50°C and pH 5.0, produces 1 micromole of RS assayed by the Somogy-Nelson method in glucose equivalent (A.P Sinitsin, A.V. Gusakov and V.M. Chernoglazov. Bioconversion of Lignocellulose Materials, Moscow, Moscow State University publishing, pi 44- 156).
  • the ⁇ -glucosidase activity is measured by hydrolyzing 0.5 mg/ml p- nitrophenyl- ⁇ -D-glucoside at pH 5.0 (0.05 M acetate buffer solution) and 40°C for 10 min. The reaction is stopped with 1 M Na 2 CO 3 , followed by measuring of optical density at 400 nm. The unit ⁇ -glucosidase activity is equal to the quantity of enzyme which produces 1 micromole of p-nitrophenol during 1 minute at pH 5.0 and 40°C.
  • the cellobiase activity is measured by hydrolyzing 2.5 mM cellobiose at pH 5.0 (0.1 M acetate buffer solution) and 40°C for 10 min.
  • the unit cellobiase activity is equal to the quantity of enzyme which, during 1 minute of 2.5 mM cellobiose solution hydrolysis at 40°C and pH 5.0, produces 1 micromole of glucose (glucose is determined by the glucose oxidase - peroxidase assay as described in Itogi Nauki I Tekhniki, Biotechnologia Seria, vol. 25, VINITI press, Moscow, 1993, p. 34).
  • the xylanase activity is measured by hydrolyzing birchwood xylan (5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) and 50°C for 10 min.
  • the unit xylanase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml xylan solution hydrolysis at 50 0 C and pH 5.0, produces 1 micromole RS assayed by the Somogy-Nelson method in xylose equivalent (A.P Sinitsin, A.V. Gusakov and V. M. Chernoglazov. Bioconversion of Lignocellulose Materials, Moscow, Moscow State University publishing, pi 44- 156).
  • the xyloglucanase activity is measured by hydrolyzing tamarind xyloglucan (5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) and 50°C for 10 min.
  • the unit xyloglucanase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml xyloglucan solution hydrolysis at 50°C and pH 5.0, produces the 1 micromole of RS assayed be the Somogy-Nelson method in glucose equivalent.
  • the claimed P.verruculosum PV2007 VKM F-3972D strain is capable of producing a complex of highly active cellulases including cellobiohydrolases, endoglucanases, cellobiases ( ⁇ -glucosidases) and accessory enzymes (xylanases and xyloglucanases), thereby providing an active an full enzyme complex and, if necessary, individual enzymes hydrolyzing the main plant cell wall components, biodegrading cellulose and hemicellulose containing substrates, including industrial and agricultural wastes, converting plant biomass to sugars, bioethanol and other microbial synthesis products and producing enzymes for use in the food and alcohol industries, brewing and agriculture as feed additives and for forage silaging.
  • highly active cellulases including cellobiohydrolases, endoglucanases, cellobiases ( ⁇ -glucosidases) and accessory enzymes (xylanases and xyloglucanases)
  • the strain can be grown in fermentation media conventionally used for the industrial fermentation for production of analogous types of enzyme preparations.

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Abstract

This invention relates to biotechnology and can be used in microbiological and food industries, in agriculture and for plant biomass processing.

Description

PENICILLIUM VERRUCULOSUM Filamentous Fungus Strain Producer of a
Highly Active Complex of Cellulases and Accessory Enzymes and a Method of Production of Biocatalyst for Cellulose and Hemicellulose Hydrolysis
This invention relates to biotechnology and can be used in microbiological and food industries, in agriculture and for plant biomass conversion.
Complexes of cellulases and hemicellulases enzymes hydrolyzing the main components of plant cell wall (along with individual cellulolytic and hemicellulolytic enzymes) can be used for the biodegrading of cellulose and hemicellulose containing fed-stocks, including industrial and agricultural wastes; for the conversion of plant biomass to sugars, bioethanol and other products of microbiological synthesis; for the destruction of plant cell wall; for the hydrolysis of non-starch polysaccharides; as well as in the food and alcohol industries, in brewing and in agriculture as feed additives and for forage silaging.
The closest counterpart of the invention by technical essence is the mutant Penicillium funiculosum S2006 BKM F-3887D strain producer of a complex of carbohydrases (cellulases, β-glucanases, xylanases, pectinases and mannanases) obtained by multistage mutagenesis and selection from the source P. funiculosum VKM F-3661D culture under UV irradiation (RF Patent Application # 2006110721/13, April 4, 2006).
The technical task solved by the invention is to broaden the range of highly productive strains producing enzymes of cellulases and hemicellulases complexes having high specific activity and intended for the highly efficient destruction of natural plant biomass and wastes of its industrial and agricultural processing and for the hydrolysis of plant polysaccharides.
The technical result that can be achieved by the invention is providing a new highly productive Penicillium genus filamentous fungus strain capable of biosynthesizing a complex of cellulases having high specific activity (cellobiohydrolases, endoglucanases, cellobiases and β-glucosidases) and accessory enzymes (xylanases and xyloglucanases). The essence of the subject of the invention is the new specially selected P.verruculosum PV2007 filamentous fungus strain producer of a complex of cellulases having a uniquely high specific activity (cellobiohydrolases, endoglucanases, cellobiases and β-glucosidases), and accessory enzymes (xylanases and xyloglucanases).
The P.verruculosum PV2007 filamentous fungus strain is deposited in the Russian Collection of Microorganisms at the G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences, No. VKM F-3972D.
The P.verruculosum PV2007 VKM F-3972D strain is obtained by multistage mutagenesis and selection from the initial P.verruculosum 28K VKM F-3764D culture. The spore suspension of P.verruculosum 28K VKM F- 3764D culture is UV irradiated. After irradiation the spores are seeded into Petri dishes containing selective media based on Hetchinson media with 0.5% carboxymethylcellulose (CMC), grown at 280C for 2 days and stained with Congo Red. The highest productivity mutants are selected visually by enlarged clarification zones around the colonies. The most active mutants selected from the Petri dishes are tested for the productivity of synthesis of cellulases, cellobiases (β-glucosidases), xylanases and xyloglucanases by growing in liquid media in shaking flasks. The most active of the flask grown variants are repeatedly UV irradiated and selected in Petri dishes and shaking flasks as described above. Storage conditions: the strain can be stored in freeze dried condition for several years and/or on slants with agarized Czapek media or Malt agar at +4°C with necessary reseeding at least once in 3-6 months.
Cultural and Morphological Strain Attributes. On Malt agar the colonies grow to 36 mm in diameter on the 7th day of growth at 25°C. The colonies are dense and plane and have convex centers. The mycelium is white, velutinous to lightly floccose or funiculose. The reverse is buff-reddish-orange. Conidiogenesis is light, with a grey-greenish hue. On the 7th day of growth, the colonies grown on Czapek agar with yeast extract are 27-30 mm in diameter, fimiculose, with margins entire, regulare and funiculose surfaces. The mycelium is light and yellowish. The reverse side is buff-orange.
The fungus does not grow on glycerin media and on Czapek agar at 5°C. When grown on Czapek agar at 37°C the colonies are 18 mm in diameter, funiculose, moderately dence with brownish-reddish reverse side.
Conidiogenesis: the penicilli are biverticillae, and the metulae are in closely appressed verticills and smooth (8-10 x 2.5 x 3.0). The phialides are acerose (8-9 x 2.5 x 3.0) and short-necked. The conidia are ellipsoidal, small (2.2 x 1.5 x 2.0) and smooth.
Submerged fermentation on soluble substrates (glucose, fructose, lactose) produces weakly pelleted branched loose mycelium, initial specific mycelium growth rate being 0.35 h"1, final specific growth rate being 0.1 h'1.
Physiological and Biochemical Strain Attributes. The strain is mesophilic. The optimum mycelium growth temperature is 32°C (29-34°C), and the optimum cellulase secretion temperature is 28°C (26-29°C). The optimum pH for growth and cellulose secretion is 3.5-5.0. Mycelium growth was also observed at pH 2.5, but cellulases and other carbohydrases formed quite slow. The nystatin resistance is high. Surface grown strain is nystatin resistant to 0.5 μg/1; 2.5 μg/1 nystatin suppress the growth. Addition of digitonin (3.5-4.0 μg/1) or Bengali Pink (30-50 μg/1) reduces colony size.
The strain is prototrophic. It readily assimilates glucose, lactose, glycerin, galactose, xylose, mannose, mannitol, tregalose, sorbose and sorbitol, less readily assimilates arabinose, rhamnose and ribose and unreadily assimilates glucoseamine, desoxyribose, desoxygalactose, desoxyglucose and thioglucose.
The strain consumes inorganic and organic nitrogen and assimilates it well in the form of nitrates and ammonia.
The strain forms enzyme systems providing for growth on respective complex substrates: cellulose, starch, xylan, laminarin, β-glucan, lichenin, pectin and galactomannan. It can consume lactic acid at sub-inhibiting concentrations. The carbohydrase biosynthesis catabolite repression is heavily reduced. The degree of carbohydrases biosynthesis catabolite repression is tested as follows. The conidia are seeded to test tubes with the minimum media containing mineral salts, trace amount (0.5 g/1) or yeast extract, amorphous cellulose and the test repressor or antimetabolite (glucose, desoxyglucose, lactose, glycerin etc.). The test tube diameter is 9 mm and the agar column height is 50-60 mm. The test tube is incubated for 4 days at 30°C and then for 20 h at 45°C. The stability of carbohydrases biosynthesis against catabolite repression is judged from the depth of the destruction zone in the amorphous cellulose (the size of the agar column clarification zone in the test tube) in the presence of a repressor or an antimetabolite.
The resultant mutant P.verruculosum PV2007 VKM F-3972D strain grown on glucose/potato agar and on sporulating agar (SM-agar) and differs in morphological attributes from the initial P.verruculosum 28K VKM F-3764D strain by the significantly lower sporulation, change in the color of the conidia (from grey-green to dark-grey) and that of the opposite side of the colony when grown on solid media, slower growth on agarized media and higher capability of biosynthesizing cellulases, cellobiases (β-glucosidases), xylanases and xyloglucanases in the case of submerged fermentation.
This kind of filamentous fungus is not treated as pathogenic in the Provisions for the Registration, Storage, Handling, Distribution and Forwarding of Bacteria, Viruses, Rickettsia, Fungi, Protozoa, Mycoplasma, Bacterial Toxins and Biological Poisons.
Submerged fermentation of the P.verruculosum PV2007 VKM F- 3972D strain is carried out under aerobic conditions in fermentation media containing one or multiple carbon source that act as enzyme biosynthesis inductors. As an option, the carbon sources may not be direct inductors. Under appropriate conditions using the insoluble substrates, e.g. microcrystalline cellulose (MCC), or soluble ones, e.g. glucose, the strain can secrete a complex of enzymes, such as cellulases (cellobiohydrolases, endoglucanases, cellobiases, β-glucosidases), xylanases and xyloglucanases, into the fermentation media. Glucose in the fermentation media can be replaced for a cheaper product - such as starch hydrolyzate.
Enzyme preparations obtained using the resultant strain can be used as a cultural filtarte, in the form of liquid concentrated preparations produced by ultrafiltration, in dry or granulated forms.
The practical embodiments of the invention are illustrated by examples that do not limit the scope and essence of the associated claims.
Example 1. P.verruculosum PV2007 VKM F-3972D strain growth in shaking flasks.
To obtain the seeding material (inoculate), the P.verruculosum PV2007 VKM F-3972D fungus culture is grown on Malt or SM agar at 29°C for 7 days and then at room temperature under light for 7 days. The shaking flasks are seeded with 1 ml of suspended spores washed from the agar with water containing 0.1% Twin-80. The strain is grown under aerobic conditions in 750 ml Erlenmeyer shaking flasks containing 100 ml of fermentation media preferably having the following composition, g/1: 50 MCC, 12 wheat bran, 12 yeast extract, 5 (NH4)2SO4, 5 KH2PO4, 0.3 MgSO4x7H20, 0.3 CaCl2x2H2O, pH 4.5. The shaking flasks are incubated in the shaker at 32°C and 200 rpm for 120-144 h. After the end of shaking flasks fermentation, the cellobiohydrolase activity is 19-20 U/ml, the endoglucanase activity is 320-350 U/ml, the β- glucosidase activity is 20-25 U/ml, the cellobiase activity is 10-12 U/ml, the xylanase activity is 530-540 U/ml and the xyloglucanase activity is 200-220 U/ml.
Example 2. P.verruculosum PV2007 VKM F-3972D strain growth in a fermenter.
The P.verruculosum PV2007 VKM F-3972D strain is grown in an ANKUM 2M 10 1 fermenter (volume of fermentation media 6.0 1) preferably using a fermentation media of the same composition as for shaker flasks at pH 4.5 and 32°C for 120-144 h. After 36 h of fermentation process and until the end of fermentation process the continuous feeding by glucose to the fermenter at 1 g/l/h by glucose occurred. After the end of fermentation process, the cellobiohydrolase activity is 60-65 U/ml, the endoglucanase activity is 820- 850 U/ml, the β-glucosidase activity is 60-70 U/ml, the cellobiase activity is 25-30 U/ml, the xylanase activity is 1550-1600 U/ml and the xyloglucanase activity is 610-640 U/ml.
After the end of fermentation process the cultural filtrate containing a complex of cellulases and concomitant enzymes is ultrafiltered through hollow fibers (10 kDa cut off), and the UF-concentrate is freeze dried to produce dry enzyme preparation. The cellobiohydrolase activity of the dry enzyme preparation is 900-920U/ml, the endoglucanase activity is 14,500-14,900U/ml, the β-glucosidase activity is 1,200-1,250 U/ml, the cellobiase activity is 600- 670U/ml, the xylanase activity is 23,000-24,000U/ml and the xyloglucanase activity is 7,500-8,200 U/ml.
Example 3. Comparative analysis activity of P.verruculosum enzyme complex.
Activity analysis is carried out by comparing the activity of enzyme preparation obtained as described above and similarly produced preparations of cellulases and accessory enzymes obtained with Penicillium and Trichoderma spp. producer strains. The fermentation preparations and methods used are as described below.
The following commercial and laboratory enzyme preparations are used. Penicillium sp. based preparations: PV2007 preparation (obtained with the P.verruculosum PV2007 VKM F-3972D strain as described in Example 2), S-2006 preparation (obtained with the P.funiculosum S2006 VKM F-3887D strain as described in RF Patent Application # 2006110721/13, April 4, 2006); Trichoderma sp. based preparations: Celloviridin G20x preparation {T.longibrachiatum) from Promferment Ltd., Russia, BioACE preparation from Dyadic International Ltd., US {T.longibrachiatum), Spezyme CP and Accellerase 1000 preparations from Genencor / Danisco, Celluclast 1.5 L preparation (T.reesei) from Novozymes, Denmark, and Fibrilase HDL 160 preparation (T.reesei) from Iogen, Canada. The specific activities of the fermentation preparations (U/mg protein) are summarized in Table 1. Table 1.
Figure imgf000008_0001
It can be seen from Table 1 that the PV2007 fermentation preparation obtained with the claimed P.verruculosum PV2007 VKM F-3972D strain exhibits the highest specific activity.
Activity determination methods. The cellobiohydrolase (avicelase) activity is measured by hydrolyzing MCC (5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) at 50°C for 60 min. The reducing sugar (RS) concentration in the reaction mixture is assayed using the Nelson-Somogy method. The unit cellobiohydrolase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml MCC suspension hydrolysis at 50°C and pH 5.0, releases 1 micromole of RS assayed by the Somogy-Nelson method in glucose equivalent.
The endoglucanase (CMCase) activity is measured by hydrolyzing water soluble carboxymethylcellulose Na salt (CMC, 5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) and 5O0C for 10 min. The unit endoglucanase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml CMC solution hydrolysis at 50°C and pH 5.0, produces 1 micromole of RS assayed by the Somogy-Nelson method in glucose equivalent (A.P Sinitsin, A.V. Gusakov and V.M. Chernoglazov. Bioconversion of Lignocellulose Materials, Moscow, Moscow State University publishing, pi 44- 156). The β-glucosidase activity is measured by hydrolyzing 0.5 mg/ml p- nitrophenyl-β-D-glucoside at pH 5.0 (0.05 M acetate buffer solution) and 40°C for 10 min. The reaction is stopped with 1 M Na2CO3, followed by measuring of optical density at 400 nm. The unit β-glucosidase activity is equal to the quantity of enzyme which produces 1 micromole of p-nitrophenol during 1 minute at pH 5.0 and 40°C.
The cellobiase activity is measured by hydrolyzing 2.5 mM cellobiose at pH 5.0 (0.1 M acetate buffer solution) and 40°C for 10 min. The unit cellobiase activity is equal to the quantity of enzyme which, during 1 minute of 2.5 mM cellobiose solution hydrolysis at 40°C and pH 5.0, produces 1 micromole of glucose (glucose is determined by the glucose oxidase - peroxidase assay as described in Itogi Nauki I Tekhniki, Biotechnologia Seria, vol. 25, VINITI press, Moscow, 1993, p. 34).
The xylanase activity is measured by hydrolyzing birchwood xylan (5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) and 50°C for 10 min. The unit xylanase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml xylan solution hydrolysis at 500C and pH 5.0, produces 1 micromole RS assayed by the Somogy-Nelson method in xylose equivalent (A.P Sinitsin, A.V. Gusakov and V. M. Chernoglazov. Bioconversion of Lignocellulose Materials, Moscow, Moscow State University publishing, pi 44- 156).
The xyloglucanase activity is measured by hydrolyzing tamarind xyloglucan (5 mg/ml) at pH 5.0 (0.1 M acetate buffer solution) and 50°C for 10 min. The unit xyloglucanase activity is equal to the quantity of enzyme which, during 1 minute of 5 mg/ml xyloglucan solution hydrolysis at 50°C and pH 5.0, produces the 1 micromole of RS assayed be the Somogy-Nelson method in glucose equivalent.
Thus, the claimed P.verruculosum PV2007 VKM F-3972D strain is capable of producing a complex of highly active cellulases including cellobiohydrolases, endoglucanases, cellobiases (β-glucosidases) and accessory enzymes (xylanases and xyloglucanases), thereby providing an active an full enzyme complex and, if necessary, individual enzymes hydrolyzing the main plant cell wall components, biodegrading cellulose and hemicellulose containing substrates, including industrial and agricultural wastes, converting plant biomass to sugars, bioethanol and other microbial synthesis products and producing enzymes for use in the food and alcohol industries, brewing and agriculture as feed additives and for forage silaging.
Achieving high productivity of the strain does not require complex and expensive fermentation media. The strain can be grown in fermentation media conventionally used for the industrial fermentation for production of analogous types of enzyme preparations.

Claims

What is claimed is
1. Penicillium verruculosum PV2007 VKM F-3972D filamentous fungus strain producer of a highly active complex of cellulases and accessory enzymes.
2. Penicillium verruculosum PV2007 VKM F-3972D filamentous fungus strain according to Claim 1 wherein said strain is a producer of a highly active complex of cellobiohydrolases, endoglucanases, cellobiases and β- glucosidases.
3. Penicillium verruculosum PV2007 VKM F-3972D filamentous fungus strain according to Claim 1 wherein said strain is a producer of xylanases and xyloglucanases.
4. A method of obtaining enzyme based biocatalysts for cellulose and hemicellulose hydrolysis wherein said enzyme preparation is obtained by cultivation the Penicillium verruculosum PV2007 VKM F-3972D strain in fermentation media containing cellulose, glucose, mono substituted potassium phosphate, ammonium sulfate, septa-aqueous magnesium sulfate and bi- aqueous calcium chloride in the following concentrations, g/1: 50-60 glucose, 8-12 KH2PO4, 3-7 (NH4)2SO4, 0.2-0.4 MgSO4x7H20, 0.2-0.4 CaCl2x2H2O, pH 4.5-5, the fermentation broth being separated 120-144 h after growth start, followed by biocatalyst separation.
5. A method according to Claim 4 wherein constant glucose content in the fermentation media is maintained by feeding.
PCT/RU2008/000559 2008-02-26 2008-08-19 Penicillium verruculosum filamentous fungus strain producer of a highly active complex of cellulases and accessory enzymes and a method of production of biocatalyst for cellulose and hemicellulose hydrolysis WO2009108081A1 (en)

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