DD241533A3 - PROCESS FOR THE BIOTECHNICAL MANUFACTURE OF A SERINE PROTEASE - Google Patents

Abstract

The aim of the invention is to develop a technologically simple method for producing a serine protease. The object is achieved according to the invention by the use of Bacillus licheniformis 41 p - ZIMET 10 911, wherein the fermentation is preferably carried out in a medium in which the glucose content of 200 mg / ml and the amino acid content of 20 mmol / ml are not exceeded in the product formation phase and the p H value during the fermentation in the weakly acidic range and increases in the phase of intensive protease synthesis up to 7.0. At a fermentation temperature of 34 to 37C, the degree of spillover of Bacillus licheniformis 41 p - ZIMET 10 911 - in the product formation phase at the o. G. Conditions kept below 10%.

Description

Field of application of the invention

The invention relates to a method for the biotechnical production of a serine protease with a Bacillus licheniformis strain. The enzyme can be used in the detergent industry and to degrade various proteins, e.g. used in the leather and film industries, in the preparation of cosmetics and in the food industry.

Characteristic of the known technical solutions

Microbial serine proteases are synthesized by a variety of microorganisms. Among the bacteria several species of the genus Bacillus are capable of such. Bacillus subtilis, Bacillus licheniformis, Bacillus firmus, Bacillus cereus, Bacillus alcalophilus, Bacillussacchariticus and others.

The production of serine proteases with Bacillus licheniformis is mentioned in several patents, for example: DE-OS 2044161, SU-PS 400116, DE-OS 2018451, US-PS 3623451, DE-OS 2063988, DE-OS 2925427, DE-OS 2101803 , GB-PS 2024830, DE-OS 2121 397, DE-OS 1 940488, US-PS 3871 963, CH-PS 533679, GB-PS 1 303633, CH-PS 626916, CH-PS 547858, GBPS 1 263765, DE -OS 2926808.

The known technical methods have the disadvantage that pH adjustments or pH regulations must be made by additions directly or indirectly pH-changing substances.

Object of the invention

The aim of the invention is to develop a technologically simple method for producing a serine protease.

Presentation of the essence of the invention

The invention has for its object to produce with a strain of the species Bacillus licheniformis under certain technological conditions, a serine protease according to the object of the invention. According to the invention, Bacillus licheniformis 41 ρ-ZIMET10911, which differs from both the B. licheniformis ATCC 14580 type strain and other B. licheniformis strains for the production of serine protease, is used for the production of the serine protease. With reference to the in o.g. Patents mentioned B. licheniformis strains ATCC 21471, ATCC 21424, ATCC 12759, NCIB 10402, P 300, Bacillus sp. ATCC 21964, the following cultural-morphological and biochemical differences occur compared to the B. licheniformis 41 ρ according to the invention:

- anaerobic gas formation from nitro bouillon,

- z.T. asporogenicity,

- abundant growth on glucose-asparagine agar,

Hairy outgrowths on nutrient agar,

- the biotin and vitamin B 1 neediness and

The optimum of the H-ion concentration during the production of protease.

In addition, the strain according to the invention 41 ρ - ZIMET 10911 differs from the type B. licheniformis ATCC 14580 s. BERGEY's Manual of Determinative Bacteriology (1974) also by

That it grows on proline as the sole source of C and N,

By acid formation from xylose, mannose, fructose, ribose, esculin, raffinose, rhamnose,

By resistance to reference phages 0-29, SP50, SP60, SP70, SP82, SP109 and BPS1.

The strain Bacillus licheniformis 41 ρ according to the invention was deposited in the central strain collection of the GDR, in the Central Institute for Microbiology and Experimental Therapy in Jena, under the number ZIMET 10911.

Characteristics of Bacillus licheniformis 41 ρ - ZIMET 10911

morphology

Shape of the spore: oval

Location of the spore: central

Sporulation Behavior: 95% (Barley Grain 10d / 37 ° C

Agility: mobile

Gram behavior: Grampositive short rods

Colony morphology on different nutrient media

Blood plate: Colony light beige, milky, dull, lobed, ß-hemolysis after 48 H / 37 ⁰ C.

Nutrient Agar (NA): Colony light beige, milky, matte, lobed

NA + Glue. + Cas.-Acid: colony maude / rose, matte-glossy, dye formation

Biochemical features

Acid formation from: D (+) - xylose, glucose, d-mannose, d-fructose, maltose, trehalose, sucrose,

Raffinose, rhamnose, D-mannitol, d-sorbitol, D-ribose, aesculin

no acid formation from: L-arabinose after 24 h, d-galactose, lactose, dulcite

Indole Detection: -

VPR: +

Citrate recycling: +

Urea detection: -

Arginine dihydrolase: +

Growth behavior and biochemical features

Growth in nutrient broth: Skin formation, cloudiness, sediment Growth on glucose-nitrate agar: strong growth Growth on glucose asparagine agar: weak growth Growth in NaCl Broth: 3-7% NaCI good growth 10% NaCI weak growth Gas formation from nitrate under anaerobic Conditions: - Nitrate reduction: + Starch hydrolysis: + HZ 2 mm Casein hydrolysis: + HZ 1mm Gelatin: + HZ4mm Lezithinase: - HZ = hydrolysis zone

The biotechnological production of the enzyme with the strain according to the invention can be carried out in the submersible process in forced-ventilated, sterilizable production tanks under known technological conditions.

However, it has been found that process conditions adapted to the properties of the strain result in higher enzyme yields.

The Bacillus isicheniformis 41 ρ-ZIMET 10911 is preferably cultured in those media in which, in the phase of intensive protease synthesis, the content of glucose is below 200 μg / ml and the amino acids are below 20 μΜΜ / ml of culture solution and the pH is at 7.0 increases.

The C and N sources commonly known for fermentations of alkaline protease also meet the requirements of Bacillus Iicheniformis 41p - ZIMET 10911.

Crucial for a high protease accumulation by Bacillus isicheniformis 41 ρ, however, is that the C and N sources of the nutrient medium are combined so that by the metabolic action of the culture the o.g. Values for glucose and amino acid levels in the culture solution in the phase of intensive protease synthesis should not be exceeded.

With appropriate nutrient supply, Bacillus licheniformis 41 ρ forms amylolytic side activities in amounts that release the assimilable sugars from the polymeric carbohydrates required for growth and product synthesis only to such an extent that at the time of intense protease synthesis, no repressible glucose concentrations (greater than 200 μg / ml).

Furthermore, the N-offer is designed so that no protease-repressing amino acid concentrations can occur neither at the start of fermentation nor by the metabolic activity of the cells during the fermentation process in the culture solution.

The synthesis of the alkaline protease by Bacillus licheniformis ρ 41 - ZIMET 10911 -is χ by cystine and tyrosine at initial concentrations of 2 10 ^-3 mol / L or by an amino acid mixture with a starting concentration of 2 x 10 ~ ^z mol / l or by a total concentration of about 20μΜοΙ / ΓηΙ repressed in the product formation phase.

It was further found that, while maintaining the abovementioned conditions fermentation temperatures of 34 ⁰ C to 37 ⁰ C set such a metabolic position of the Bacillus licheniformis 41 ρ that at maximum protease accumulation is a degree of sporulation of less than 10%.

An advantage of the preferred method of the invention is that the fermentation process in the weakly acidic pH range (preferably between pH 5.9 to 7.0) without pH settings, buffering, controls or additives of directly or indirectly pH-changing C and / or N sources.

Furthermore, the preferred method of the invention is characterized in that the nutrients of the culture solution are degraded by the metabolic conversion so advantageous that the subsequent treatment process can be carried out in an effective manner by known methods to product forms suitable for application.

The invention will be explained in more detail by way of examples.

embodiments

example 1

The bacterial lawn of a 48 h at 37 ⁰ C brewed slant culture on casein agar is washed off with sterile tap water and transferred to 50ml of a preculture medium in 500ml round bottom flask. After incubation for 7 h at 37 ° C. on a shaker with 220 strokes / min, approximately 4 to 6 × 10 ³ cells / ml are achieved. Thus, the production media are inoculated so that about 3 x 10 ⁷ cells / ml of culture solution are present.

The fermentation process using a production medium listed below was carried out in a laboratory fermenter with 1.5 l of net volume. The fermentation temperature was 37 ° C, the air intake was 1 vvm with one revolution of the stirrer of 700 U / min. The initial pH was 6.5. At the time of dismantling after 40h.

an H-ion concentration of pH 7.0 was achieved. Under these conditions, the ZIMET of the invention forms 10911

220 _Hb / ml.

Composition of the nutrient media

Casemagar: glucose 0.5% casein 0.5% Na ₂ HPO ₄ -2H ₂ O 0.6% KH ₂ PO ₄ 0.1% Agar Agar 2.5% pre-culture medium: glucose 2.0% Dry yeast 0.5% Wheat fine meal 0.5% Na ₂ HPO ₄ -2H ₂ O 0.4% MgSO ₄ -7H ₂ O 0.1% Production medium: glucose 0.5% starch hydrolysis 5.0% soybean meal 3.0% casein 1.6% Na ₂ HPO ₄ -2H ₂ O 0.5% MgSO ₄ -7H ₂ O 0.05 ° /

Example 2

The cultivation takes place as in Example 1.

The fermentation medium has the following composition:

glucose 0.5% starch hydrolysis 6.0% soybean meal 3.0% casein 1.8% Na ₂ HPO ₄ -2H ₂ O 0.5% MgSO ₄ -7H ₂ O 0.05%

The natural initial pH of the nutrient medium is 6.5, at the time of degradation after 44h it is 7.0. The cultivation conditions are the same as in Example 1

The protease yield is 280 _Hb / ml.

Example 3

The cultivation takes place as in Example 1.

The fermentation medium has the following composition:

glucose 0.5% starch hydrolysis 5.5% field beans 3.0% casein 2.0% Na ₂ HPO ₄ -2H ₂ O 0.45% MgSO ₄ -7H ₂ O 0.05%

The natural initial pH of the medium is 6.45. At the time of degradation after 40 h, it is 7.0.

The cultivation conditions are the same as in Example 1.

The protease yield is about 30% below that of Example 2.

The determination of the proteolytic activity is carried out by a modified Anson method (M.L. Anson, J. Gen. Physiol.

22 [1939], pp. 79-89).

The method is based on the photometric determination of peptide fragments soluble in a precipitant (consisting of 0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid) resulting from the proteolytic degradation of denatured hemoglobin.

The resulting fragments are based on 1 μΜοΙ tyrosine and the activity is expressed in tyrosine units per ml = TEnb / ml.

A TE _H b is the enzyme activity, which from a hemoglobin solution under defined and optimal conditions as much in the precipitant soluble substrate fragments per min. releases that their absorption corresponds to 1 μΜοΙε tyrosine.

Claims (3)

Invention claim: 1. A method for the biotechnical production of a serine protease, characterized in that the Bacillus licheniformis 41p - ZIMET 10911 is used. 2. The method according to item 1, characterized in that the fermentation is carried out in a medium in which in the product formation phase, the glucose content of 200 pg / ml and the amino acid content of 20 μΜοΙ / ml is not exceeded and the pH during the fermentation is in the weakly acidic range and increases to 7.0 in the phase of intensive protease synthesis. 3. Method according to items 1 and 2, characterized in that the degree of spillover of Bacillus licheniformis 41 ρ - ZIMET10911 - is kept below 10% in the production formation phase by the fermentation temperature of 34 ⁰ C to 37 ° C.