SI9800038A - Process for production of extracellular keratinolytically active protease - Google Patents

Abstract

A process for production of extracellular keratinolytically active protease is typical in that the growing medium containing keratinous or some other complex proteinous material as inductor of enzymatic synthesis, complex components of microbiological culture media, simple sources of carbon, and inorganic salts as a source of minerals, with its starting pH in a slightly acidic up to neutral range, is sterilized and cooled down to about 30 degrees Centigrade, then grafted with spores of the fungus Doratomyces Microsporus to obtain the culture concentration of 108 - 109 spores/L, grown in submersion aerobic conditions at the temperature of 25 up to 30 degrees Centigrade under shaking, fermented for 50 up to 60 hours, after which the slurry is filtered and the enzyme of the following characteristics is isolated from the filtrate: molecular weight 33 kDa, activity against keratinous skin in the pH range between 6 and 12, and activity against keratinous skin in the temperature range between 20 and 55 degrees Centigrade. The applied fungus Doratomyces Microsporus is preserved in the national collection of microbic cultures (Collection Nationale de Cultures de Microorganismes - CNCM, INSTITUT PASTEUR) in Paris under the designation MZKI B399 and registered on December the 23rd 1997 under reg. No. I-1963.

Description

Proteolytic enzymes that catalyze the hydrolysis of highly resistant keratin proteins are synthesized by some microorganisms and insects. These enzymes play a key role in dermatophytosis as they allow dermatophyte fungi to grow on the skin and on skin outgrowths. However, keratinolytic enzymes not only have a negative effect but can also be beneficial. Their exploitation comes into play in various fields where the degradation of keratin materials such as healthcare, cosmetics, leather and environmental protection is required.

Patents protecting keratinase production state bacteria or fungi as the production organism. Bacteria have patented species from the genera Bacillus (Shih, Williams), Micrococcus (Holland) and Streptomyces (Yarrow and Whitefield), and among fungi, dermatophytic fungi from the genera Trichophyton (Blank et al., Plempel et al.) And Microsporum (Plempel et al.). The purpose of the application is different: removal of excess keratin in acne (Yarrow and Whitefield) or garages (Blank et al.), Removal of human infected skin (Yarrow and Whitefield, Blank et al., Holland), for the dermatophytosis vaccine (Plempel et al. .), for the removal of hair in leather (Blank et al.), for the production of protein hydrolysates for feed from keratin waste, e.g. chicken feathers (Shih and Williams).

The enzymes are mainly obtained by aerobic submerged fermentation processes. However, the cultivation of the micro-organism may initially be stationary and only after a few days the culture start to shake (Blank et al.). In addition to the batch, continuous cultivation (Holland) is also described. In one case, keratinase was a by-product of the neomycin antibiotic (Yarrow and Whitefield). Batch fermentation is carried out for several days at temperatures between 25 ° C and 37 ° C with ventilation and shaking or shaking. mixing fermentation broth. The medium must contain, among other ingredients, keratin material as an inducer for the synthesis of keratinases (Blank et al., Plempel et al.) Or other complex nitrogen sources such as soy peptone and meat extract (Yarrow and Whitefield) or peptone and yeast extract ( Holland).

After fermentation, the enzymes are isolated by filtration or centrifugation and concentrated by ultrafiltration. They can be purified by various chromatographic methods, dialysed and lyophilized.

Some patents describe the characteristics of enzymes. Keratinase of Trichophyton mentagrophytes has a molecular weight of 48 kDa and an isoelectric point (pl) at 9.4 (Blank et al.). The keratinases of Micrococcus sedentarius have a molecular weight of 30 and 50 kDa, respectively, and pl are 4.6 and 2.7. Optimal activity on infected skin is at 7.1 ° C at 40 ° C and 50 ° C at pH 2.

7.5 (Holland). The enzymes are not narrowly specific for keratin but also catalyze the hydrolysis of some other proteins and peptides (Blank ef al., Holland).

Examination of the patents for the production of keratinolytically active enzymes shows that they were used as the production microorganism by most bacteria. One of the advantages of filamentous fungi over bacteria is the easy filtration of the fermentation broth after fermentation, since the fungal mycelium entangles the solids of the substrate, thereby greatly facilitating filtration. In addition, fungal hydrolytic enzymes are usually extracellular, so they can be isolated directly from the fluid and no cell breakdown or enzyme extraction is required. Some patents relate to the use of filamentous fungi for the biosynthesis of keratinases, but in these cases these are fungi that are known dermatophytes and are dangerous to humans. However, for the industrial production of enzymes, where large quantities are handled, it is desirable that workers are not exposed to harmful substances. Therefore, it seems appropriate for the production to choose a microorganism that is not pathogenic to humans.

The present patent relates to a biotechnological process for the production of extracellular keratinolytically active protease by the non-pathogenic fungus Doratomyces microsporus, enzyme isolation, preparation of a crude keratinase preparation, enzyme purification, characterization and use. The fungus was isolated in France and is stored in the Collection National de Cultures de Microorganisms (CNCM) at the Pasteur Institute in Paris under code MZKI B399, registered under number I - 1963. This fungus has not been mentioned so far in relation to production of keratinases, or any other proteases. The advantage of using this fungus is that it is not a dermatophyte fungus and is not pathogenic to humans. Its keratinase breaks down infected skin, nails, wool and also some non-keratin proteins. The fermentation process lasts from 50 to 90 h, which is much shorter than the patented fungal procedures, for example: fermentation with Trichophyton mentagrophytes lasting 12 days (Blank et al.) Or the process with various dermatophytic fungi lasting 6 to 7 days (Plempel et al.). In both of these patents, which refer to the use of fungi as production microorganisms, the medium contains keratin material of different origin. In our case, it has been shown that keratin material can be replaced by other complex protein sources such as fish meal, soybean meal, bone meal or casein, which are more readily available and reasonably priced to be suitable for fermentation medium. For larger scale cultivation, it would be difficult to obtain sufficient quantities of properly prepared infected skin or nails, respectively. hair that is difficult to reach and preparation is energy and time consuming. Fermentation broth is not viscous, filtration is easy and quick. The crude filtrate already has quite high specific activity, indicating a large proportion of keratinase among other extracellular proteins.

Preparation of keratin material for fermentation medium and enzyme substrate

Preparation of human contaminated skin

Trims of infected skin from soles are collected in 70% ethanol. They are filtered through a "black tape" filter and mixed in liquid nitrogen. Rinse well with water and dry at 50 ° C-60 ° C for 24 hours. The dry skin is ground and sieved through a 0.2 mm sieve. Particles above this size are used for the fermentation medium and the screened particles are used as the enzyme substrate for the determination of keratinase activity.

Certain keratinolytic activities of mg of contaminated skin powder in 4 ml of buffer (0.028 M Tris / HCl, pH 7.5) were incubated with 1 ml of suitably diluted enzyme solution for 30 minutes at 37 ° C with shaking 160 / min. After incubation, the enzyme reaction was stopped by the addition of 2 ml of 10% trichloroacetic acid (TCA). The blind sample was added to TCA before the enzyme was added. The resulting precipitate was centrifuged and absorbance measured at λ = 280 nm. We define one unit of enzyme activity as a change in absorption by 0.1.

Example 1: Fermentation process

The culture medium has the following composition:

infected skin 5.0 g / l kh ₂ by ₄ 1.5 g / l k ₂ hpo ₄ 1.0 g / l ZnSO ₄ x 7H ₂ O 2.0 mg / 1 MgSO ₄ x 7H ₂ O 0.2 g / l CaCl ₂ x 2H ₂ O 0.2 g / l NaCl 0.2 g / l peptone 0.4 g / l glycerol 2.0 ml / 1 malt extract 1.0 g / l glucose 1.0 g / l

Adjust the pH of the culture medium to 6.0. Pour the medium in quantities of 100 ml into a 500 ml Erlenmeyer bottle. Sterilize in an autoclave at 121 ° C and 1.2 bar and cool to 30 ° C. We vaccinate the Doratomyces microsporus MZKI B399 spore suspension so that the concentration is about 10 ⁸ -10 ⁹ spores / 1. Cultivation is performed on a shaker at 120 rpm and 30 ° C for four days. Filtration of the slurry through a black paper filter paper gives a filtrate with an average keratinolytic activity of about 6 units / ml and a specific activity of about 10 units / mg of soluble proteins.

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Example 2: Fermentation process

The composition of the culture medium;

fish meal 5.0 g / l

KH ₂ PO ₄ 1.5 g / l

K ₂ HPO ₄ 1.0 g / l

ZnSO4 χ 7H2O 2.0 mg / l

MgSO ₄ x 7H ₂ O 0.2 g / l

CaCl ₂ x 2H ₂ O 0.2 g / l

NaCl 0.2 g / l peptone 0.4 g / l glycerol 2.0 ml / 1 malt extract 1.0 g / l glucose 1.0 g / l

Adjust the pH of the culture medium to 6.0. The medium was divided into bottles, sterilized and vaccinated as in Example 1. Cultivation was performed on a shaker at 120 rpm and 30 ° C for four days. After fermentation, the broth is filtered.

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The filtrate has a keratinolytic activity of about 12 units / ml and a specific activity of about 30 units / mg of soluble proteins.

If fish meal is replaced with bone meal in the same medium, a filtrate with a keratinolytic activity of about 7 units / ml and a specific activity of about 20 units / mg is obtained.

If the fish meal in the medium is substituted with casein, a filtrate is obtained with an activity of about 10 units / ml and a specific activity of 12 units / mg.

Example 3: Fermentation process

Medium composition:

soybean meal 5.0 g / i KH ₂ PO ₄ 1.5 g / 1 k ₂ hpo ₄ 1.0 g / 1 ZnSO ₄ x 7H ₂ O 2.0 mg / l MgSO ₄ x 7H ₂ O 0.2 g / i CaCl ₂ x 2H ₂ O 0.2 g / 1 NaCl 0.2 g / 1 peptone 0.4 g / 1 glycerol 2.0 ml / 1 malt extract 1.0 g / 1 glucose 1.0 g / 1

Adjust the pH of the culture medium to 6.0. The medium was divided into bottles, sterilized and vaccinated as in Example 1. Cultivation was performed on a shaker at 120 rpm and 25 ° C for four days. After fermentation, the broth is filtered.

The filtrate has a keratinolytic activity of about 10 units / ml and a specific activity of about 33 units / mg of soluble proteins.

Example 4: Fermentation process

Medium composition: soybean meal 5.0 g / i glucose 3.0 g / i glycerol 4,8 ml / 1 peptone 0.7 g / i yeast extract 0.7 g / 1 malt extract 0.5 g / 1 KH ₂ PO ₄ 3.9 g / 1 ZnSO ₄ χ 7H2O 3.0 mg / l FeSO ₄ x 7H ₂ O 6.0 mg / l MnCl ₂ x 4H ₂ O 0.4 mg / l CUSO4 χ 5H2O 4.6 mg / l MgHPO ₄ x 3H ₂ O 4,8 mg / l CoSO ₄ 0.7 mg / l CaCl ₂ x 2H ₂ O 0.07 g / i

Set the pH of the culture medium to 6.9. The medium was divided into bottles, sterilized and vaccinated as in Example 1. Cultivation was performed on a shaker at 120 rpm and 25 ° C for three days. After fermentation, the broth is filtered to give a filtrate with an activity of about 17 units / ml and a specific activity of about 23 units / mg of soluble proteins.

Example 5: Fermentation process

Medium composition:

soybean meal 5.0 g / 1 glucose 1.0 g / 1 peptone 0.7 g / 1 yeast extract 0.8 g / 1 malt extract 0.4 g / 1 KH ₂ PO ₄ 3.2 g / 1 ZnSO ₄ x 7H ₂ O 2.0 mg / l FeSO ₄ x 7H ₂ O 0.6 mg / l MnCl ₂ x 4H ₂ O 2.1 mg / l CuSO ₄ x 5H ₂ O 2.1 mg / l MgHPO ₄ x 3H ₂ O 3.6 mg / l CoSO ₄ 2.9 mg / l CaCl ₂ x 2H ₂ O 0.06 g / 1

The starting pH is 4.4. Divide the medium into bottles, sterilize and vaccinate as in Example 1. It was grown on a shaker at 120 rpm and 25 ° C for three days. After fermentation, the broth is filtered to give a filtrate with a keratinase activity of about 20 units / ml and a specific activity of about 37 units / mg of soluble proteins.

Example 6: Fermentation process

Medium composition:

soybean meal KH ₂ PO ₄

5.0 g / l

1.5 g / l

K2HPO4 1.0 g / 1 ZnSO4 x 7H ₂ O 2.0 mg / 1 MgSO ₄ x 7H ₂ O 0.2 g / 1 CaCl ₂ x 2H ₂ O 0.2 g / i NaCl 0.2 g / 1 peptone 0.4 g / 1 glycerol 2.0 ml / 1 malt extract 1.0 g / 1 glucose 1.0 g / 1

Adjust the pH of the culture medium to 6.0. Pour into a 5 1 laboratory bioreactor with stirrer. With stirring of 200 rpm, sterilize for 35 minutes at 121 ° C. After cooling to 30 ° C, administer the spore suspension in such a way that the concentration of spores in the culture medium is about 5 x 10 ⁸ / l. It is stirred initially at 300 rpm and inflated with a flow rate of 2.5 l / min. When the partial pressure of oxygen begins to fall sharply, the stirring is increased to 400 rpm and the aeration to 5.0 l / min. After 50-60 hours, the broth was filtered. A filtrate is obtained with a keratinase activity of about 20 units / ml and a specific activity of about 30 units / mg of soluble proteins.

Characteristics of fermentation broth filtrate

The optimal pH range for exposure to infected skin is 8-9, the filtrate is inactive below pH 6.0. The optimum temperature for exposure to infected skin is 45 ° C, with no activity above 60 ° C. The filtrate acts on keratin substrates such as contaminated human skin, nails, feathers and wool. It also catalyzes the hydrolysis of non-keratin proteins such as hemoglobin, casein, volumetric serum albumin and hide powder azur (Sigma Chemie). It does not break down gelatin.

Example 7: Preparation of crude enzyme preparation

After fermentation, the filtrate is filtered through a 0.2 μτη pore filter. The filtrate thus obtained is then ultrafiltered through a membrane which retains molecules with a molecular weight above 5000 Da. The retentate was lyophilized to give a crude enzyme preparation with a keratinolytic activity of about 12,000 units / g.

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Example 8: Purification of the crude enzyme

The crude enzyme preparation was purified in the first step on a PhenylSuperose HR 5/5 chromatographic column (Pharmacia). Buffer system used: A: 50 mM phosphate buffer, pH 7.0; B: 50 mM phosphate buffer + 1 M (NH ^ SO ^ pH 7.0. The mobile phase flow is 0.5 ml / min, the pressure is maximum 2 MPa. A sample with a specific activity of about 60 units / mg soluble proteins is applied. separation gives a specific activity in the active fraction of about 160 units / mg.

In the next stage, purify the obtained fraction on the chromatographic column ΜΟΝΟ Q HR 5/5. The buffer system consists of A: 10 mM tris / HCl, pH 8.5 and B: 10 mM tris / HCl + IM NaCl, pH 8.5. The mobile phase flow is 1 ml / min at a pressure of not more than 5 MPa. After separation, the specific activity of 220 units / mg is measured in the active fraction.

In the final phase, the last impurities are removed by gel chromatography on a Sepharose 12 HR 10/30 column. Use 50mM phosphate buffer pH 7.5 with the addition of 0.2 M NaCl. A purified enzyme with a specific activity of about 600 units / mg is obtained.

The enzyme molecular weight is about 33 kDa (determined by electrophoresis)

Claims (3)

PATENT APPLICATIONS A method for producing extracellular keratinolytically active protease, characterized in that the medium containing as an inducer of enzymatic synthesis is keratin or any other complex protein material such as soybean meal, fishmeal, bone meal, casein, complex constituents of microbial media, such as is peptone, malt extract, yeast extract, simple carbon sources such as glucose and glycerol, and inorganic salts as a source of minerals, and has a starting pH in the slightly acidic to neutral range of 4.0 to 7.0, sterilized and cooled to about 30 ° C and then vaccinated with Doratomyces microsporus spores so that the concentration of the medium is 10 ⁸ - 10 ⁹ spor / L, grown under submerged aerobic conditions at 25 to 30 ° C and shaking on a shaker between 100 - 120 rpm in the course of 3 to 4 days, or in a bioreacto, with an initial stirring of 300 rpm and aeration of 0.5 1/1 / min, which are increased to 400 rpm and 1 1/1 / min during the intensive growth phase, ferment 50 up to 60 hours, at this is filtered and the enzyme isolated from the filtrate. Method according to claim 1, characterized in that the Doratomyces microsporus fungus is stored in the National Collection of Microbial Cultures (CNCM, INSTITUTE PASTEUR) in Paris under the code MZKI B399 and registered there on 23 December 1997 under number I - 1963. The enzyme material obtained according to claims 1 and 2, characterized in that it is in the form of a filtrate of fermentation broth in the form of a crude enzyme preparation or in a purified form obtained after purification using hydrophobic interaction chromatography, ion exchange and gel chromatography, and having the following characteristics: molar mass of 33 kDa, activity on infected skin between pH 6 and 12, optimum between 8 and 9, activity on infected skin between 20 and 55 ° C, optimal at 45 ° C.