US3833474A - Asporogenic protease-producing strains of bacillus subtilis - Google Patents

Abstract

A new mutant strain of Bacillus subtilis which has the property of being substantially asporogenic while showing proteolytic activity similar to that of the native strain from which it was derived. The process for preparing the new mutant strain is based on the discovery that the non-sporulated cells of Bacillus subtilis are not resistant to chloroform vapor whereas the spores are resistant.

Description

United States Patent Aubert et al.

[451 Sept; 3, 1974 ASPOROGENIC PROTEASE-PRODUCING STRAINS OF BACILLUS SUBTILIS Inventors: Jean-Paul Aubert, Montrouge;

Robert Longin, Meudon La Foret; Jacqueline Millet, Paris, all of France Agence Nationale de Valorisation de la Recherche (Anvar), Paris, France Filed: July 25, 1972 Appl. No.: 275,069

Assignee:

Foreign Application Priority Data July 28, 1971 France 71.27671 US. Cl. 195/65, 195/78, l95/103.5 R,

195/96 Int. Cl. ClZk l/00, Cl2d 13/10 Field of Search 195/65, 66 R, 96

[56] References Cited UNITED STATES PATENTS 3,740,318 6/1973 Churchill et al. 195/65 FOREIGN PATENTS OR APPLICATIONS 2,018,451 10/1970 Germany 195/66 R Primary Examiner-Lionel M. Shapiro Attorney, Agent, or Firm-Edward J. Brenner 57 ABSTRACT 3 Claims, No Drawings ASPOROGENIC PROTEASE-PRODUCING STRAINS OF BACILLUS SUBTILIS This invention relates to the production of enzymes from strains of Bacillus subtilis.

It is known that the proteolytic enzymes obtained by fermenting strains of Bacillus subtilis are widely used in industry, especially in the detergent industry.

However, workers exposed to the commercial-grade dust of proteolytic enzymes have been found to suffer from allergic reactions (asthma, coughing, chest pains). Similarly, certain workers have been found to suffer from allergies affecting the skin.

Although this has not been demonstrated with any degree of certainty, it is generally thought that these allergic reactions are caused by the presence of spores in commercial-grade enzymes. The standards established by common agreement between enzyme manufacturers and manufacturers of domestic detergents limit the number of spores per gram of commercial enzymes although, hitherto, these standards have been difficult to attain.

According to one of its aspects, the invention relates to a new mutant strain of Bacillus subtilis which has the property of being substantially asporogenic whilst showing proteolytic activity similar to that of the native strain from which it is derived.

The invention also relates to a process for mutating a native strain of Bacillus subtilis and isolating the asporogenic strain according to the invention.

The mutant strain obtained by this process has the same identification characteristics as the original native strain except that it; does not sporulate. Its identification which is thus already possible by comparison with the well-known and defined native strain is facilitated by the fact that the mutant strain has been deposited in the collection of the Pasteur Institute in Paris and given the identification number 71-l00l.

By virtue of the fact that the mutant strain according to the invention is no different from the native strain already used in the production of protease, i.e., an extracellular enzyme used in detergents, apart from its asporogenic character, this mutant can readily be subjected to known processesfor commercially growing the native strain with a view to obtaining enzymes suitable for use in household detergents.

In addition to the fact that they do not give rise to any of the allergic reactions referred to above, the enzymes thus obtained by applying a conventional process to the new mutant strain according to the invention also have the advantage of not giving off the unpleasant odour attending the use of detergents containing enzymes obtained from spore-forming strains. Finally, the absence of spores avoids any subsequent microbial growth.

Preparation of the mutant strain according to the invention is based on the following observation:

We have found that the non-sporulated cells of Bacillus subtilis are not resistant to chloroform vapour whereas the spores are resistant. It is thus possible to mark the colonies of asporogenic strains (sp) because these'colonies are not replicated after exposure to chloroform vapours.

Accordingly, the process comprises (A) subjecting the spores of the native strain to a mutagenic treatment, (B) spreading the spores subjected to the mutagenic treatment over an agar-containing nutrientbroth accommodated in Petri dishes to obtain after incubation spore-containing colonies and (C) isolating colonies of the asporogenic strain by comparison between the homologous Petri dishes of one series emanating from operation (B) and having been subjected to treatment with chloroform vapours followed by replication and incubation, and a series having undergone the same operations except for exposure to the chloroform vapours, and finally (D) selecting the mutant strain satisfying the required sporulation conditions from the mutant strains isolated showing proteolytic activity.

In one embodiment of this process, the mutagenic treatment (A) of the spores of the native strain of Bacillus subtilis comprises initially heating a suspension of spores for 10 minutes to C., followed by germination in Difco nutrient broth in the presence of chloramphenicol ug/ml), the culture being stirred for 2 hours at 39C., and finally by the actual mutagenic treatment with N-methyl-N'-nitro-N-nitrosoguanidine (50 tog/ml), the culture being stirred for 1 hour at 37C. The culture medium used for germination has the following composition:

for 1 litre of distilled water NaOH quantity sufficient to adjust pH to 6.8 7.0

A dilute form of the suspension of spores germinated same composition as that used for germination, but additionally contains 20 g/litre of agar. After incubation for 48 hours at 37C., the sp colonies contain spores.

Isolation (C) which includes a treatment with chloroform vapours applied to certain colonies comprises the following operations:

a. colonies of series I are replicated in other Petri dishes containing the same medium (series II);

b. colonies of series I are then exposed to chloroform vapours for 45 minutes at the temperature of the laboratory, after which the colonies thus treated are replicated in a new series of Petri dishes (series III);

c. series II and III are incubated for 24 hours at 37C.;

d. the homologous dishes of series II and III are compared.

The sp colonies are absent from series lIl but present in series Il because the cells are killed by the chloroform vapours. Samples of the sp colonies are taken from series II and then isolated again. After the second isolation, the strain retained for their sp character are tested for their proteolytic activity. To this end, the medium used is'preferably agar-containing Difco nutrient broth with casein added. For example, a Difco medium of the following composition is used:

for 1 litre of distilled water Difco nutrient broth 16 g MgSO '7H,O 0.50 g KCl 2.0 g MnCl,4H O l' M 2 ml FeSO -7H,O l0M 2 ml CaCl -2H,O 1.5% 20 ml NaOH quantity sufficient to adjust pH to 6.8 7.0 agar 20 S The medium is mixed in equal volumeswith a solution containing 20 g of casein per litre of distilled water, the pH-value being adjusted to 6.8 7.0

The colonies showing proteolytic activity (Pr are surrounded by a proteolysis ring.

Selection (D) of the mutant strain is made as follows:

Out of 35,000 colonies examined in series 1, seven mutant strains were isolated. They were studied in liquid Difco nutrient broth with the same composition as the broth previously used for germinating the spores in order accurately to determine their sporulation rate fer. After the mixture has been incubated for 30 minutes in. a water bath at 37C., the non-hydrolysed azocasein is precipitated with 2 ml of 10% trichloroacetic acid. The mixture is filtered. 1.5 ml of 0.5 N NaOH are added to l .5 ml of the filtrate, followed by a reading at 440 nm. One unit (UA) corresponds to 1 mg of azocasein hydrolysed for 30 minutes at 37C. under the conditions specified above. I

The results are set out in Table l below:

strain y proteolytic sporulation 4 activity sp/ml.

UA/ml B native 14 8.10 B l2 l4 0 B 22 l 0 B 6| 9 r 5.10 Mutants B 82 16.3 1.4 l0

B I ll 2.8 0 B I 12 l l 4.4 l0 B143 14.5 l.2' 10 Labile rod (width 0.8 to l.0 ,u; length 2.4 to 2.6 t).

In the exponential growth phase, the cells are in the form of more or less long chains (4 to 10 cells). After growth, the chains are shorter (2 to 4 cells) and the cells are less long.

Gram-positive colouration.

The spores formed are oval (0.8 1.4/1.1 11.).

Mutant strain ldentical, but does not form spores.

, 2. Appearance of colonies on the solid agarcontaining Difco nutrient broth having the composition specified for treatment (B) with the mutagenic agent.

Native strain:

Round, white, dull smooth-edged colonies flattened at the centre and opaque. After 48 hours at 37C., a yellow pigment is excreted.

Mutant strain Identical colonies except that, after 3 to 4 days at 37C., the colonies become translucent (destruction of the cells). Yellow pigment is again excreted.

b. Physiology Growth in Difco nutrient broth having the same composition as the media used for the germination operations (A), for examining the mutant strains in phase (D) and for examining the morphology of the cells (a) above.

Native strain Mutant strain Growth rate at 30C #1 0.49 t, 0.49 (division/hour) 0.32 0.32 Number of cells (maximum/ml) 9.10 9.!0 Proteolytic activity in UA/ml -14 -14 Number of spores/ml 810 0 Prototrophy c. Comparison of the properties of the enzymes excreted by the nativestrain and the mutant strain 1. Heat resistance.

a. Having reached maximum proteolytic activity, the cells of one culture are eliminated by centrifuging. Aliquot portions of the supernatant phase are heated in a water bath for 10 minutes at different temperatures (40, 45, 50, 55 75C). The residual proteolytic activity is measured by the process described above. The percentage ofresidual proteolytic activity is recorded on a graph as a function of temperature for the two types of enzyme. It is found that, in both cases, the,

temperature at which 50 percent of the original activity log A/Ao kt log e for a temperature of C. The gradient of each straight line [k log e] makes it possible to determine the velocity constant of the denaturation' reaction (k). If more than one straight line is obtained,

it means that there is more than one enzyme in the supernatant phase. The gradient of each straight line is characteristic of an enzyme.

The gradients observed for the enzymes of the mutant and of the native strain suggest that the supernatant phases contain two proteolyticenzymes of which one shows hardly any resistance to heat.

So far as the most resistant enzymes are concerned, the gradients k log e are the same for the enzyme emanating from the mutant and for the enzyme emanating from the native strain. For these enzymes, the velocity constant of the denaturation reaction is:

k (70C) 0.1 min are not inhibited by o-phenantroline hydrochloride b. The degree of inhibition caused by diisopropyl fluorophosphate IO M) is'of the order of 95 percent for the mutant and the native strain. Accordingly, the principal enzyme would be seryl-protease in both cases.

Overall, the foregoing examination shows that the mutant strain according to the invention is an asporogenie mutant of the native strain of Bacillus subtilis which does not have any effect either upon production or upon the properties of the extracellular protease, i.e., the enzyme produced by fermentation from the strain, except in regard to the formation of spores.

The enzyme produced by fermentation of the Bacillus subtilis strain is present in solution in the aqueous phase of the culture broth. The following procedure can be adopted for producing the enzymes according to the invention on a commercial scale:

A fermentation process is carried out in a variablesized fermenter aerated in depth, agitated and equipped with a heat-regulating system. The culture medium contains organic nitrogen compounds such as distillery residues, maize maceration liquor, soya flour, casein, mineral elements etc. This culture medium is inoculated with an asporogenic Bacillus subtilis culture (strain according to the invention as deposited) obtained in agitated flasks or from an inoculum fermenter. i

The aeration required amounts for example to between 100 and 500 m" per hour for a fermenter with a useful capacity of m.

Fermentation is carried out at 37C. However, the fermentation temperature can be in a range from 30 to 38C. The pH-value is adjusted to 7.0 at the beginning of fermentation.

After having been inoculated, the culture medium is sterilised with live steam for 30 minutes at 121C.

The air injected at the bottom of the fermenter is sterilised by passage through a cylinder filled with glass wool.

On completion of fermentation, the liquid phase is separated from the bacteria and solid particles by fermentation, generally in a filter press comprising :1 Nylon cloth. A filtration additive is added to the fermentation juice to be filtered.

The filtrate is precipitated with a solvent such as ethanol, butanol, or even with ammonium sulphate. The precipitate obtained is centrifuged in a centrifuge, for example of the Sherpless type and filtered in a filter press. The wet cake obtained is introduced in trays into an oven in which it is dried in vacuo at a temperature not exceeding 60C.

The powder obtained does not contain any spores. It can be used in detergents made up in accordance with the usual formulations and-does not produce any of the allergic reactions observed with enzymes obtained from sporular strains.

We claim: 1. A process for selectingasporogenic bacteria from the genus Bacillus subtilis which comprises:

a'pmutating germinated spores of wild Bacillus and spreading thespores subjected to the mutagenic treatment over an agar containing nutrient broth accommodated in a series of Petri dishes to obtain after incubation colonies containing spores;

b. replicating each Petri dish of said series to obtain a first homologue series of Petri dishes;

c. submitting each Petri dish of said first mentioned series to chloroform vapor and replicating it to obtain a second homologue series of Petri dishes;

d. incubating said first homologue series and said second homologue series of Petri dishes; I

e. by comparison between homologue Petri dishes of said first homologue series and said second homologue series, selecting the asporogenic mutant strains which are strains of said Petri dishes of said first homologue series which are not in the Petri dishes of said second homologue series; and

f. testing proteolytic activities of these selected strains on casein.

2. Process for preparing protease without spore which comprises culturing in a known manner for the wild Bacillus subtilis an asporogenie bacteria selected by the process of claim 1.

3. Process according to claim 2 wherein asporogenic bacteria is a Bacillus subtilis of the type identified by No. 71-1-00] deposited in Institut Pasteur de Paris.

Claims (2)

1. 2. Process for preparing protease without spore which comprises culturing in a known manner for the wild Bacillus subtilis an asporogenic bacteria selected by the process of claim 1.
2. 3. Process according to claim 2 wherein asporogenic bacteria is a Bacillus subtilis of the type identified by No. 71-I-001 deposited in ''''Institut Pasteur de Paris.''''