RU2293115C1 - Strain of fungus penicillium canescens as producer of secreted endo-(1-4)-beta-xylanase - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, microbiology, biochemistry, enzymes.

SUBSTANCE: invention proposes the fungus strain Penicillium canescens VKPM F-912 no containing genes encoding endoglucanases that is a producer of endo-(1-4)-beta-xylanase. On media with beet pulp this strain is able to accumulate up to 88 U of activity of secreted endo-(1-4)-beta-xylanase and 0.13 U of cellulase activity, not above, in 1 ml of cultural fluid for 120-144 h of the fermentation process.

EFFECT: valuable biochemical properties of strain.

1 tbl, 2 dwg, 2 ex

Description

The invention relates to the field of microbiological industry and is a producer strain of the secreted enzyme endo (1-4) beta-xylanase Penicillium canescens (hereinafter P.canescens), constructed by transformation and genetic engineering methods.

Endoxylanases break down xylan, the main component of hemicelluloses. In this regard, endoxylanases are of great interest in both industrial and applied biotechnological aspects. Endoxylanases find industrial application in the paper industry in bleaching processes, in the food and feed industry, and other fields (FEMS Microbiology Reviews, 1999, v.23, p.411). Various fungal producers of endoxylanases are known: Trichoderma reesi, Trichoderma harzianum, (J Biotechnol, 1997, 57 (1-3): 137.), Thermoascus auranticus, (US Pat. No. 4,966,850); Aspergillus tubigensis (U.S. Patent 5,358,864), Humicola insolens (U.S. Patent 5,610,048).

As the closest analogue of the claimed invention, consider a strain of the fungus P.canescens VKPM F-832, which is the producer of secreted endo (1-4) beta-xylanase (RF patent No. 2197526).

This strain accumulates up to 770 activity units of secreted endo (1-4) beta-xylanase in 1 ml of culture fluid.

The disadvantage of the VKPM F-832 strain is its presence of satellite cellulolytic activity (1.4 u / ml), which inhibits the use of the xylanase enzyme preparation based on this strain.

The objective of the present invention is to obtain a strain of fungus P.canescens - producer of secreted endo (1-4) beta-xylanase with a reduced level of cellulolytic activity.

To solve the problem, a strain of P.canescens XYL-dEGL fungus was constructed - producing secreted endo (1-4) beta-xylanase, which has deletions for the structural genes egl2 (endoglucanase gene of family A cellulases, family 5 glycosyl hydrolases) and egl3 (gene endoglucanases of the family N of cellulases, family of 12 glycosyl hydrolases) endoglucanases - an enzyme that exhibits cellulolytic activity. The strain is deposited in the All-Russian collection of industrial microorganisms (VKPM) under the number VKPM F-912.

The inventive strain of P.canescens VKPM F-912 has the following characteristics.

Cultural and morphological characters on various nutrient media after 10 days of growth at 30 ° C.

On wort agar having the composition (in wt.%): Wort - 40, agar - 2, water - the rest, the strain forms colonies with a diameter of 6.5-7.5 cm, the reverse side of which is orange-dark brown; conidiophores 430-450 × 3.0-3.2 microns in size, conidia 2.0-2.2 microns, spherical, smooth at a young age, rough in the old, formed in chains, connected into columns, decaying over time.

On a Rowlen-Tom agar medium with sucrose having the composition (in wt.%): Sucrose - 2, corn extract - 1, KH ₂ PO ₄ - 0.2, K ₂ SO ₄ - 0.02, MgSO ₄ × 7H ₂ O - 0.02, agar - 2, a mixture of trace elements: MnSO ₄ × 5H ₂ O - 0.008, CuSO ₄ × 5H ₂ O - 0.04, ZnSO ₄ × 7H ₂ O - 0.0088, Co (NO ₃ ) ₂ × 6H ₂ O - 0.01, FeSO ₄ × 7H ₂ O - 0.1, H ₃ BO ₃ - 0.006, (NH ₄ ) ₂ MoO × 4H ₂ O - 0.03, CaCl - 0.1, water - the rest, colony diameter 3.8-4.8 cm, colony felt or slightly fluffy in the center, slightly convex, with radial grooves, white, painted at the edges in a gray-blue tone, the edges are uneven, lobed, the reverse side of a gray-beige tone.

On an agarized Chapek medium with glucose having the composition (in wt.%): NaNO ₃ - 0.2, KH ₂ PO ₄ - 0.1, MgSO ₄ × 7H ₂ O - 0.05, KCl - 0.05, FeSO ₄ - 0.001, glucose - 2, agar - 2, water - the rest, the diameter of the colonies is 2.8-4.0 cm, the colonies are gray, folded, with a smooth edge, conid formation is intense, the color of conidia is light gray. The reverse side of colonies with radial folds of a weak cream tone.

Physiological and biochemical characteristics.

Mesophile, grows at 25-37 ° С, optimal growth temperature is 29 ° С. Growth optimum at pH values of 4.1-5.8. It weakens gelatin slightly.

Relation to carbon sources. It is well absorbed by mono-, di- and polysaccharides, with the exception of arabinose, which is poorly utilized.

Attitude to nitrogen sources. It is well absorbed by ammonium and nitrate nitrogen, peptone, urea.

Enzymatic activity. The inventive strain accumulates up to 800 units of activity of secreted endo (1-4) beta-xylanase and not more than 0.13 units of cellulase activity in 1 ml of culture fluid.

The invention is illustrated by the following figures of a graphic image:

Figure 1. Map of plasmid pPCdelEG3niaD

Figure 2. PPCEG2NM plasmid map

Example 1. Construction of the inventive strain.

Recipient strain P.canescens PCA10 (Current Genetics, 1995, v. 28, p. 474) transforms (Biotechnology, 1999, N3, p. 3-13) 1 μg of DNA of the plasmid pPCdelEG3niaD that we constructed (Fig. 1) containing 5 'non-translated region of the egl3 gene of P.canescens (5' UTP egl3), incomplete coding 5 'region of the egl3 gene (CDS 1 egl3), Aspergillus niger nitrate reductase gene (niaD) (Gene, 1992, v.111, p.149-155 ) as a selective marker, the incomplete coding region of the 3 'region of the egl3 gene (CDS 2 egl3) and the 3'-untranslated region of the egl3 gene (3' UTP egl3) in the pUC57 plasmid vector (Gene, 1985, v. 33, p.103-119 ) linearized at the SmaI restriction site and 10 μg of the DNA of the PCR construct fragment of the plasmid pPCEG2NM (Fig. 2) that we described, including the 5'-untranslated region of the egl2 P.canescens gene (5 'UTP egl2), the incomplete coding 5' region of the egl2 gene (CDS I egl2), the incomplete coding 3 'region of the egl2 gene (CDS 2 egl2) and the 3'-untranslated region of the egl2 gene (3 'UTP egl2) in the plasmid vector pBluescript II KS (+) (Nucleic Acids Res., 1989, v.17, 9494) obtained using a pair of primers:

EG2-5: 5'-TCATTTAAGCAGGGGTTGTCGGGTTTGTTAGATTC-3 '

EG2-3: 5'-TCGGATTTCGAAATGCTTCCGTATGTATCATCAC-3 '

After 4-5 days of incubation at 30 ° C, the resulting cotransformants are screened for deletions using PCR from conidia of individual transformants.

To identify strains with the Δegl2 genotype (deletion in the egl2 gene), PCR is performed using a pair of primers:

EG2ID: 5'-ACCCTGAAAACCACTACAACTG-3 ';

EG2IR: 5'-GCTCTTGTATTTGGTCGCAA-3 '.

To identify strains with the Δegl3 genotype (deletion in the egl3 gene), PCR is performed using a pair of primers:

EG3ID: 5'-TGGTCAAGGACAAAAAGTCGA-3 ';

EG3IR: 5'-GGCGTACCAATCAAATACTGAG-3 '

Analysis of cotransformants EG23-1 and EG23-5 with the genotype Δegl2, Δegl3 is carried out by measuring the level of xylanase and cellulase activity in the culture fluid during deep cultivation on a medium with beet pulp (as in example 2). The measurement results are presented in Table 1.

Table 1. Xylanase and cellulase activities during fermentation in flasks. Strain Genotype Xylanase activity, units / ml Cellulase activity, units / ml EG23-1 Δegl2, Δegl3 145 1.02 EG23-5 Δegl2, Δegl3 143 1.05 RSA10 niaD ^- 143 1.40

Strain EG23-1 was selected for further manipulations as having lower cellulase activity while maintaining a high level of xylanase activity.

To obtain mutants of the nitrate reductase gene (niaD), which are necessary for the amplification of the structural gene encoding xylanase, 10 ⁶ conidia of P. canescens strain EG23-1 are treated with nitrosoguanidine (0.4 mg / ml) for 30 min at 30 ° C and plated on an agar plate minimal medium (Biotechnology. 1999. N3. C.3-13) containing 0.45 M chlorate and as the sole nitrogen source 10 mM NH ₄ Cl. The plates are incubated for 4-5 days at 30 ° C and the grown colonies are tested on plates with agarized minimal medium, with additives as the sole source of nitrogen: a) 10 mM NaNO ₃ ; b) 0.45 M chlorate, 10 mM NH ₄ Cl; c) 5 mM hypoxanthine; g) - 10 mm NH ₄ Cl. The strains selected by their ability to grow on media with ammonium chloride, chlorate and hypoxanthine and the lack of growth ability on media with NaNO ₃ determine their xylanase and cellulase activities by the method described in example 2. Among them, strains with productivity of xylanases and cellulases are selected, close to strain EG23-1. The result is a strain of EG23-1 / niaD.

The inventive strain P.canescens VKPM F-912 - producer of endo (1-4) -β-xylanase containing deletions Δegl2, Δegl3, obtained by introducing additional copies of the xylA gene. endo (1-4) -β-xylanase P.canescens (Appl. Biochem. MicrobioL, 2002, v. 38 (5), p. 495-501) to strain EG23-1 / niaD using cotransformation with plasmid pPCXYLA (Patent RF No. 21197526), carrying the P. canescens xylA gene, with the transforming plasmid pSTA-10 (Gene, 1989, v. 78, p. 157) containing the Aspergillus niger niaD selective nitrate reductase gene.

Example 2. Evaluation of the enzymatic activity of the claimed strain.

Inoculum is obtained by culturing a strain of P.canescens VKPM F-912 on Rowlen-Tom agar medium (see Example 1) at 30 ° C for 5-7 days. Then, an aqueous suspension of conidia thus obtained (10 ⁷ conidia / ml) was inoculated with 100 ml of a pectin-containing medium of the following composition (wt.%): Beet pulp - 3, peptone - 5, KH ₂ PO ₄ - 2.5, water - the rest.

Cultivation is carried out on a circular rocking chair at 240-250 rpm in 750 ml rocking flasks at 30 ° C for 120 hours. At the end of the fermentation, the culture fluid is separated from the mycelium and solid residues by centrifugation (10,000 g, 15 min) and xylanase and cellulase activities are determined in the supernatant.

Xylanase activity is determined by the amount of reducing sugars released during the hydrolysis of xylan from larch. Incubation of the substrate and the enzyme is carried out at 50 ° C for 10 minutes The sample includes 0.45 ml of a 1% solution of xylan in 0.1 M acetate buffer solution (pH 5.0) and 0.05 ml of the enzyme in an appropriate dilution. The unit of activity is the amount of enzyme releasing 1 μmol / min of reducing sugars under the above conditions. Reducing sugars are determined by the Shomody-Nelson method (J. Biol. Chem. 1952. V.195. N1. P.19-28, J. Biol. Chem. 1944. V.153. P.375-380).

To determine the activity of cellulases using colored carboxymethyl cellulose (CMC OS-41). Prepare a 1% solution of CMC OS-41 in 0.1 M acetate buffer, pH 4.5. 0.15 ml of 0.1 M acetate buffer (pH 4.5) was added to 0.25 ml of this solution, and it was heated for 5 min at 40 ° С. Add 0.1 ml of sample warmed up for 5 min at 40 ° С diluted in the same buffer. Incubated for 10 min at 40 ° C, then add 1 ml of a 1 M solution of CaCl in 80% ethanol. The suspension is centrifuged (3000 g, 5 min) and the absorbance of the supernatant is measured at 490 nm against a “blind” sample. The preparation of a “blind” sample is carried out in the same way as the test sample, but incubation is passed for 10 min at 40 ° C.

Activity (u / ml) = 0.611 × OD ₄₉₀ × 5 × R / t;

Where:

0.611 - correlation coefficient;

5 - dilution of the sample in the reaction mixture;

OD ₄₉₀ — optical density at 490 nm;

t is the incubation time;

R is the initial dilution of the sample.

The inventive strain of the fungus P.canescens VKPM F-912 under the conditions described above exhibits xylanase activity of 800 units / ml and cellulase activity of 0.13 units / ml.

Claims (1)

The strain of the fungus Penicillium canescens VKPM F-912 is a producer of secreted endo (1-4) beta-xylanase.

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