WO1997030172A1

WO1997030172A1 - Cold-active protease cp-58 and psychrotrophic bacteria

Info

Publication number: WO1997030172A1
Application number: PCT/US1997/002437
Authority: WO
Inventors: A. K. M. Quamrul Hasan; Eiichi Tamiya
Original assignee: The Procter & Gamble Company; Japan Advanced Institute Of Science And Technology
Priority date: 1996-02-16
Filing date: 1997-02-14
Publication date: 1997-08-21
Also published as: JPH09224666A; AR005831A1; EP1007722A4; EP1007722A1

Abstract

A cold-active protease is here disclosed which has the following physicochemical properties: (a) specific activity and substrate specificity: the protease acts on casein, gelatin, albumin and hemoglobin to specifically decompose them in the order of casein, gelatin, albumin and hemoglobin; (b) optimal pH: 7.5 to 8.0; (c) pH stability: the protease is stable at a pH in the range of 5.5 to 10.5 at 20 °C for 1 hour; (d) optimal temperature: 20 °C at pH 10.5 and 40 °C at pH 8.0; (e) temperature stability: at pH 10.5 for 1 hour, the protease is scarcely inactivated at a temperature of 10 °C to 30 °C, but it is inactivated at 40 °C as much as about 30 % and completely inactivated at 50 °C; (f) enzyme activity: the protease has about 60 % or more of its maximum activity at 20 °C; (g) the active center of the enzyme is a metallic ion; and (h) the molecular weight of the protease is about 58 kDa as measured by SDS-PAGE.

Description

COI D-ACTrVE PROTEASE CP-58 AND PSYCHROTROPHIC BACTERIA

BACKGROUND OF THE MVENTION

(V, Field ofthe Invention

The present invention relates to a protease having a high activity at a low temperature, its utilization, and a microorganism producing the protease, fii. Description ofthe Related Art Psychrophilic bacteria have been known for a long time, and their existence can be confirmed extensively in low temperature circumstances. For example, psychrophilic bacteria can be isolated from soils, fishery products, milk products as well as artificial low temperature circumstances. Researches on psychrophilic bacteria have been conducted in accordance with food microbiological requirements, but they have principally been confined to those with respect to the phytogeny of microorganisms.

Enzymes obtained from psychrophilic bacteria are expected to be cold-active enzymes having an optimal temperature in a low temperature range. The cold-active enzyme which acts efficiently at the low temperature is considered to be capable of being incorporated into, for example, a detergent which can be used even in water at the low temperature. It is also considered to be employed for a chemical reaction in the presence of an organic solvent which is volatile at ordinary temperature and for the quality improvement of foods at the low temperature at which the foods do not rot. Furthermore, the investigation on the enzyme derived from the psychrophilic bacteria is fairly interesting to reveal the physiological functions and the adaptation mechanism to the low temperature ofthe psychrophilic bacteria. SUMMARY OF THE INVENΗON

The present inventors have now found that a protease can be isolated from the supernatant liquid of a culture medium of a Serratia marcescens AP3801 strain and then purified, and that the isolated and purified protease has activity at a low temperature. The present invention is based on such knowledge. Therefore, an object of the present invention is to provide a cold-active protease, a microorganism producing the protease, a method for preparing the above-mentioned cold-active protease by the use of the microorganism, and a peptide comprising an amino acid sequence present at the N teπninal ofthe cold-active protease.

The protease according to the present invention has a part or all of the following physicochemical properties.

- Specific activity and substrate specificity: The protease acts on casein, gelatin, albumin and hemoglobin to specifically decompose them in the order of casein, gelatin, albumin and hemoglobin. - Optimal pH: 7.5 to 8.0

- pH stability: The protease is stable in the range of pH 5.5 to pH 10.5 at 20°C for 1 hour. The protease according to the present invention further has a part or all of the following physicochemical properties.

- Optimal temperature: 20°C at pH 10.5 and 40°C at pH 8.0.

- Temperature stability: At pH 10.5 for 1 hour, the protease is scarcely inactivated at a temperature of 10°C to 30°C, but it is inactivated at 30°C as much as about 40% and completely inactivated at 50°C.

- Enzyme activity: At 20°C, the protease has a maximum activity at pH 10.5 and about 60% or more of its maximum activity at pH 8.0.

- The enzyme belongs to a metallo-protease or metal-activated protease.

- The molecular weight ofthe protease is about 58 kDa as measured by SDS-PAGE. - The isoelectric point is about 7.4 as measured by an isoelectric focusing.

- The protease according to the present invention consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l, or a protein containing a part or all of an amino acid sequence described in SEQ ID NO: 1 at its N terminal.

A microorganism according to the present invention is one belonging to Serratia genus which is capable of producing the protease.

A method for preparing the above-mentioned protease according to the present invention comprises the steps of culturing the microorganism, and then collecting the protease from its culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a drawing illustrating the results of the purification of an enzyme according to the present invention.

Fig. 2 is a drawing illustrating a calibration curve for measuring the molecular weight of the enzyme according to the present invention.

Fig. 3 is a drawing illustrating the influence of pH on an enzyme reaction of the enzyme according to the present invention. A white square represents MES (pH 5.5-6.5), a white circle represents MOPS (pH 6.5-8.0), a white triangle represents TAPS (pH 8.0-9.0), a black circle represents CHES (pH 9.0-10.0), a black square represents CAPS (pH 10.0-11.0), and a black triangle represents Glycine-NaOH (pH 11.0-13.0).

Fig. 4 is a drawing illustrating the pH stability of the enzyme according to the present invention. A white circle represents MES, a black square represents MOPS, a black triangle represents TAPS, a black circle is CHES, a white square represents CAPS and a white triangle represents Glycine-NaOH.

Fig. 5 is a drawing illustrating the effect of temperature on the enzyme reaction ofthe enzyme according to the present invention. A white circle is at pH 10.5 and a black square is at pH 8.0. 75 Fig. 6 is a drawing illustrating the temperature stability of the enzyme according to the present invention. A white circle is at 10°C, a white square is at 20°C, a white triangle is at 30°C, a black circle is at 40°C, a black square is at 50°C, and a black triangle is at 60°C. Fig. 7 shows the results of isoelectric focusing.

Fig. 8 shows the effect of a culture temperature on the growth of an AP3801 strain and the 80 activity of the protease.

DETAILED DESCRIPTION OF THE INVENTION Microorganisms for producing cold-active protease

A cold-active protease according to the present invention can be produced by the use of microorganisms. The production microorganisms belong to a Serratia genus, and any 85 microorganisms are usable, so far as they have an ability for producing the protease. Furthermore, as described in Example 12, the microorganisms can grow well at 10 to 25°C.

A preferable typical example of the microorganisms having the ability for producing the protease according to the present invention is a Serratia marcescens AP3801 strain. This strain is isolated from soil (about 1,000 meters above sea level) by the present inventors, and they are

90 deposited in Agency of Industrial Science and Technology, Biotechnology Research Institute under

Deposition No. FERM BP-5401 on February 15, 1996.

The bacteriological characteristics of the Serratia marcescens AP3801 strain according to the present invention are as follows.

(1) Morphological properties

95 The strain has mobility, and its morphology is single bacillus.

(2) Properties on culture medium

The strain grows on an agar medium and a liquid culture medium, and it assumes white or light yellow.

(3) Optimum growth conditions

100 The strain suitably grows in the range of at least 10 to 25°C, but at 37°C or more, it does not grow any more.

(4) Protease released from bacteria

From any bacteria which have grown at 10 to 25°C, the protease is released. The protease derived from the bacteria which have grown at 10°C has a higher protease activity than the protease 105 derived from the bacteria which have grown at 25°C.

(5) Distinction between aerobe and anaerobe

The strain is judged to be facultative anaerobic from the results of a biochemical test

(6) Gram stain

The strain is judged to be Gram-negative from the results of Gram stain. 110 (7) Biochemical properties The main biochemical characteristics of the Serratia marcescens AP3801 strain are as shown in Table 1.

Test items Results β-Galactosidase +

Arginine dihydrolase -

Lysine decarboxylasse -

Ornithine decarboxylase +

Utilization of citric acid -

Production of hydrogen sulfide -

Urease -

Tryptophan deaminase -

Production of indole -

Production of asetoin +

Gelatinase +

Glucose +

D-Mannitol +

Inositol -

D-Sorbitol +

L-Rhamnose -

Sucrose +

D-Melibiose +

D-Amygdalin +

L-Arbinose +

1 Oxidase test -

The results were given by the use of Profile Index (API 20, NTHON Bio Merieux_Biotech Co.,

Ltd.)

On the basis of the above-mentioned characteristics, the microorganisms obtained by the present invention can be judged to be Serratia marcescens. and also comparing these microorganisms with known microorganisms in points of the base sequence of a DNA for coding 16S ribosome RNA, this strain can properly be judged to belong to Serratia marcescens. Culture of microorganism

In culturing the strain which can be used in the present invention, a culture medium may be liquid or solid, but a shake culture or an aeration spinner culture using a liquid culture medium is usually used. As the culture medium for culturing the microorganisms therein, any medium is usable, so far as it can produce the protease. That is to say, as a carbon source, there can be used, for example, glucose, trehalose, fructose, maltose, sucrose, starch and malt oligo-saccharide. As a nitrogen source, there can be used, for example, peptone, yeast extract, malt extract, meat extract, soybean powder,

130 cotton seed powder, cone steep liquor, various amino acids and their salts, and nitrates. A synthetic medium or a natural medium which can be used in the present invention suitably contains the above¬ mentioned carbon source and nitrogen source, inorganic salts such as magnesium phosphate, calcium, sodium, potassium, iron and manganese as well as other nutrients, as needed.

Culturing conditions such as the pH and the culture temperature of the culture medium can be

135 suitably altered, so far as they permit the production of the protease, but in the case of the shake culture or the aeration spinner culture, it is preferred that the pH is about neutral and the culture temperature is from 10°C to 20°C.

The protease of the present invention is present in cell walls of bacteria, cells of the bacteria and the supernatant of a culture medium, and it may be used in any form such as bacterial cells, a

140 crude enzyme obtained from the bacterial cells or the supernatant of the culture medium, or an extracted and purified enzyme. Alternatively, the protease immobilized by a known method can also be used.

In order to collect and purify the protease of the present invention from the culture medium, known purification methods can be utilized singly or in combination thereof.

145 Since the protease according to the present invention is mainly excreted extracellularly, namely into the culture medium, a crude enzyme solution can easily be obtained by removing the bacterial cells with the aid of filtration or centrifugation.

This crude enzyme can be further purified by a known purification method. Examples of the known preferable purification method include a salting-out method using a salt such as ammonium

150 sulfate, a precipitation method using an organic solvent (e.g., methanol, ethanol or acetone), an adsorption method using raw starch, an ultrafiltration method, and various chromatographical methods such as gel filtration chromatography and ion exchange chromatography. Typical embodiments of the preferable purification methods will be described in the undermentioned examples.

The characteristics of an enzyme according to the present invention are as follows.

(1) Specific activity and substrate specificity

The enzyme according to the present invention acts on casein, gelatin, albumin and hemoglobin to specifically decompose them. The substrate specificity of the enzyme decreases in the 160 order of casein, gelatin, albumin and hemoglobin.

(2) Optimal pH The optimal pH of the enzyme according to the present invention is 7.5 to 8.0. Furthermore, the enzyme retains about 50% or more of a maximum activity in the range of pH 5.5 to pH 10.5.

(3) pH stability

165 The enzyme according to the present invention is stable at 20°C for 1 hour in the range of pH

5.5 to pH 10.5.

(4) Optimal temperature

The optimal temperature of the enzyme according to the present invention is 20°C at pH 10.5 and 40°C at pH 8.0. At a temperature of 30°C, the enzyme retains about 80% of the ma»irtnim 170 activity at pH 10.5 and pH 8.0. At a temperature of 10°C, the enzyme retains about 50% of the maximum activity.

With regard to savinase which is a commercially available enzyme, its optimal temperature is 60°C. In addition, with regard to most of known cold-active enzymes, their optimal temperatures are about 40°C. Therefore, the enzyme according to the present invention can be considered to be a cold- 175 active enzyme.

(5) Temperature stability

At pH 10.5 for 1 hour, the enzyme according to the present invention is scarcely inactivated at a temperature of 10°C to 30°C, but it is inactivated at 40°C as much as about 30% and completely inactivated at 50°C. Therefore, the enzyme according to the present invention can be considered to 180 be a cold-active enzyme.

(6) Enzyme activity

At 20°C, the enzyme according to the present invention a maximum activity at pH 10.5 and has about 60% or more of its maximum activity at pH 8.0.

In consequence, according to another aspect of the present invention, we provide a protease 185 having about 60% or more of its maximum activity at 20°C.

(7) Inhibition of activity

The protease activity of the enzyme according to the present invention is not inhibited by any of pepstatin, L-trarts-epoxysuc nylleucylarmd 4-guamάirκ>butane (E-64), and phenylmethanesulfonyl fluoride (PMSF) but it is notably inhibited by 1,10-phenanthroline, and 190 ethylenediaminetetraacetic acid (EDTA). In addition, the present enzyme has homology with a metallo protease as much as about 50%. In view of this fact, it can be suggested that the enzyme according to the present invention is a metallo protease or metal-activated protease. Therefore, the active center of the enzyme according to the present invention can be considered to be a metallic ion.

(8) Molecular weight

195 The enzyme according to the present invention has a molecular weight of about 58 kDa as measured by SDS-PAGE.

(9) As a result of the isoelectric focusing, isoelectric point (PI) is about 7.4. It is considered that the value is higher than that of other known enzymes. (10) Amino acid sequence at N terminal

200 The amino acid sequence at the N terminal of the enzyme according to the present invention is described in SEQ ID NO: l. Accordingly, the protease according to the present invention may consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO: 1 or a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l at its N terminal.

205 With regard to the amino acid sequence at the N terminal of the enzyme according to the present invention, its homology with each amino acid sequence of known proteins was inspected by the use of a data bank Εntrez". As a result, it was apparent that the amino acid sequence at the N terminal had homology with the metal protease as much as about 50%.

Furthermore, according to still other aspects of the present invention, we provide a protease

210 consisting of a protein containing a part or all of an amino acid sequence described in SEQ ID NO: 1, and a protease consisting of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l at its N terminal. This protease may have such characteristics as described in the above-mentioned (1) to (9).

Here, "the protein containing a part or all of an amino acid sequence described in SEQ ID

215 NO: 1" includes a protein in which an optional amino acid sequence is added to an N terminal and/or a C terminal of a part or all ofthe amino acid sequence described in SEQ ID NO:l. Utilization of enzyme

The psychrophilic protease according to the present invention has an optimal temperature in a low temperature range. Thus, the psychrophilic protease of the present invention permits the

220 decomposition reaction of a protein to be carried out in low temperature environments. For example, a detergent utilizable even in low temperature water can be prepared by adding the protease according to the present invention to a detergent composition for clothes. This detergent composition can be prepared in accordance with a conventional method except that the psychrophilic protease according to the present invention is added. That is to say, the detergent can be formed by blending the protease

225 of the present invention with ordinary detergent components such as a surface active agent for the detergent, a bleach, a builder and the like.

Furthermore, the psychrophilic protease according to the present invention enables the reaction to proceed at a low temperature. Therefore, even if an organic solvent which is volatile at ordinary temperature is present in the reaction system, the reaction can be carried out at a low

230 temperature at which the organic solvent component is not volatilized. Moreover, when it is attempted to improve the quality of a food by the use of the protease according to the present invention, the reaction proceeds advantageously at a low temperature, whereby the food can be effectively prevented from rotting. In addition, since the protease according to the present invention is provided, it can be expected to advance the elucidation of the physiological mechanism of

235 psychrophilic bacteria and their application mechanism to a low temperature. Protein having amino acid sequence at N terminal

According to another aspect of the present invention, we provide a peptide consisting of a part or all of an amino acid sequence described in SEQ ID NO:l, a protein comprising a part or all of an amino acid sequence described in SEQ ID NO: 1, and a protein comprising a part or all of an amino

240 acid sequence described in SEQ ID NO: 1 at its N terminal. This protein may have a protease activity.

This peptide or protein consists of a part or all of an amino acid sequence present at the N teπninal of the enzyme according to the present invention, or comprises a part or all of the amino acid sequence (preferably at the N terminal). Therefore, the above-mentioned peptide or protein is useful as an antigen in forming an antibody to the enzyme according to the present invention. 245 The present invention will be described in more detail with reference to examples, but the scope ofthe present invention should not be limited to these examples.

Next, the present invention will be described in more detail with reference to specific examples, but needless to say, the scope of the present invention should not be limited to these examples. 250 Test Procedures

In this context, proteins were quantitatively determined by the use of Bio-Rad protein assay (made by Bio-Rad Co., Ltd.) which was a protein staining method, unless otherwise specified.

Furthermore, the detection of the proteins in an eluate of chromatography was accomplished by measuring an absoφtion at an ultraviolet position of 280 nm. 255 In addition, the activity of a protease was measured by the following procedure (a) or (b).

(a) Decomposition activity of protein with azocasein

0.05 ml of a sample enzyme solution was added to 0.3 ml of a 500 mM glycine-sodium hydroxide buffer solution (pH 10.5) containing 1% (W/V) azocasein, and the mixture was then kept at 20°C for 30 minutes. Afterward, the reaction was terminated with 1 ml of a 6% trichloroacetic 260 acid solution, and the solution was allowed to stand at room temperature for about 30 minutes and then centrifuged (15,000 rpm, room temperature, 10 minutes). Next, the absorbancy of the resultant supernatant liquid at 340 nm was measured by the use of a spectrophotometer.

(b) Phenol reagent method

20 ul of a sample enzyme solution was added to 130 μl of a 100 mM glycine-sodium hydroxide 265 buffer solution (pH 10.5) containing a 1% (W/V) substrate solution, and the mixture was kept at 20°C for 30 minutes. Afterward, the reaction was terminated by adding 150 μl of a trichloroacetic acid solution (0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid). After allowed to stand at room temperature for 30 minutes, the reaction solution was centrifuged (10,000 rpm, room temperature, 10 minutes), and 500 μl of a 0.5 M sodium carbonate solution and 100 μl of a phenol 270 reagent solution twice diluted with distilled water were added to 100 μl of the resultant supernatant liquid. After the solution was allowed to stand at room temperature for 1 hour, the absorbency ofthe solution at 660 nm was measured. Example 1 (Screening of novel microorganisms)

The isolation of novel microorganisms was carried out on an agar plate culture medium. 0.1 g 275 of a soil sampled in the vicinity of Kuzuryu Dam, Izumi village, Fukui Prefecture, Japan was suspended in a physiological saline, and its supernatant liquid was used as a stock solution. Furthermore, a lCr dilute solution of this stock solution was prepared therefrom. Next, the stock solution and the 10² dilute solution were each sprayed on an agar plate culture inium for screening (5 g/liter of glucose, 5 g liter of yeast extract, 1 g/liter of sodium casein, 0.2 g/liter of MgS0 7H₂0, 280 1.0 g/liter of K₂HS0₄, 10 g liter of Na₂C0₃ and 1.5 g/liter of agar), and then cultured at 10°C for 3 to 4 days. Among colonies which had grown on the agar medium, well grown colonies were selected, subcultured, and then inoculated into a retention culture medium.

It was confirmed on the agar medium that a protease was released from the bacteria. The isolated microorganisms were inoculated into the above-mentioned agar medium for screening, and

285 then cultured at 10°C for 72 hours. Afterward, a 10% trichloroacetic acid solution was sprayed on the agar medium on which the bacteria grew. From the presence of transparent plaques around the colonies, it was confirmed that the protease was released from the bacteria.

In order to stabilize the growth activity of the bacteria, a bacterial strain was inoculated into 150 ml of the undermentioned culture medium (each 25 ml of the medium was poured into six 100- 290 ml Erlenmeyer flasks), and rotary shaking culture was then carried out at 10°C for 72 hours at 140 rpm by the use of a triple shaker NR-80 (Tietec Co., Ltd.). As a main culture, 150 ml of the pre- culture medium was inoculated into 3 liters ofthe undermentioned culture medium, and rotary culture was then done at 10°C for 96 hours at 140 rpm by the use of a laboratory fermenter LS-5 (Oriental Yeast Co., Ltd.). 295 Composition of culture medium

Glucose 0.5%

Yeast extract 0.25%

Casein sodium 0.1%

K₂HP0₄ 0.1%

300 MgSθ4_7H₂0 0.025%

Na₂C0₃ 1.25%

(pH 10.5) The culture medium and the like were sterilized with high-pressure vapor for 15 minutes under 1.2 kgf/cm²G (121°C) by an autoclave. Furthermore, a Serratia marcescens AP3801 strain was 305 subcultured in an agar plate medium for a period of two weeks to one month, and then preserved at 10°C.

Example 2 (Purification of enzyme)

All the operations of protease purification were carried out at 4°C.

(a) Ion exchange chromatography 310 The culture medium obtained in the above-mentioned (a) was centrifuged (8,000xg, 4°C, 15 minutes) for separation of the bacteria and a crude enzyme. This crude enzyme solution was subjected to ion exchange chromatography to purify the same. As a column, there was used an INdEX 100 column (Pharmacia Biotec Co., Ltd.) filled with 2 liters of a DEAE Sephalose Fast Flow anion exchanger (Pharmacia Biotec Co., Ltd.). Into the above-mentioned column, a 20 mM tris-

315 hydrochloric acid buffer solution (pH 8.0) was introduced at a linear velocity of 150 cm hr in an amount five times (10 liters) or more as much as a gel volume to equilibrate the column.

The crude enzyme solution was introduced into the column at a linear velocity of 100 cm/hr. Elution was carried out at a linear velocity of 100 cm/hr by the use of 3 liters of each of the tris- hydrochloric acid buffer solutions (pH 8.0) containing 0.2 M, 0.4 M and 0.6 M NaCl, respectively,

320 and only portions in which a protein was detected by a UV meter were fractionated.

(b) Salting out with ammonium sulfate

Ammonium sulfate was added to the thus obtained fraction under ice cooling so that the fraction might be saturated as much as 80% with ammonium sulfate. After the solution was slowly stirred at 4°C overnight in a cold chamber, centrifugation (18,000xg, 4°C, 30 minutes) was carried

325 out to precipitate an enzyme, thereby obtaining a saturated fraction. The amount of ammonium sulfate to be added was an amount necessary to achieve a saturated concentration at 25°C.

(c) Gel filtration

Next, gel filtration was carried out through a HiLoad 16760 Superdex 200 prep grade column (Pharmacia Biotec Co., Ltd.). As a device, there was used HiLoad System 50 (Pharmacia Biotec Co., 330 Ltd.).

A tris-hydrochloric acid buffer solution (pH 8.0) was caused to flow through the HiLoad 16/60 Superdex 200 prep grade column at a linear velocity of about 60 cm/hr to equilibrate the column, the amount of the tris-hydrochloric acid buffer solution being three times or more (400 ml) as much as the gel volume. Afterward, 5 ml of the sample enzyme solution which had been subjected to the 335 salting out with ammonium sulfate was introduced into the column by the use of a Superloop. Then, elution was carried out at a linear velocity of 60 cm hr by the use of the tris-hydrochloric acid buffer solution (pH 8.0) as an eluent to collect fractions every 5 ml.

Example 3 (Measurement of purity and molecular weight of purified enzyme) The purity and the molecular weight ofthe purified enzyme according to the present invention 340 were measured by the use of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 7.5% polyacrylamide gel having a thickness of 1 mm was used as a support Electrophoresis was carried out by applying 20 mA of an electric current to the gel until Bromophenol Blue (BPB) reached a lower end. The gel plate was stained with an aqueous 30% methanol-10% acetic acid solution containing 0.02% of Coomassie Brilliant Blue R250 for 1 hour, and then decolored with a 345 decolorant (a solution of 30% of methanol and 10% of acetic acid) overnight. The results and the calibration curve ofthe SDS-PAGE are shown in Figs. I and 2, respectively. Example 4 (Effect of pH on enzyme reaction)

The decomposition reaction of azocasein was carried out with the enzyme of the present invention at various pH values. Each of buffer solutions for a reaction solution had a concentration of 350 100 mM and they were an MES (2-morpholinoethanesulfonic acid monohydrate) buffer solution (pH 5.5-6.5), an MOPS(3-moφholinopropane-sulfonic acid) buffer solution (pH 6.5-8.0), a TAPS (N- tris(hydroxymetJrιyl)methyl-3-aminopropanesulfonic acid) buffer solution (pH 8.0-9.0), a CHES (N- cyclohexyl-2-aminoethanesulfonic acid) buffer solution (pH 9.0-10.0), a CAPS (N-cyclohexyl-3- aminopropanesulfonic acid) buffer solution (pH 10.0-11.0) and a glycine-sodium chloride-sodium 355 hydroxide buffer solution (pH 11.0-13.0). The results are shown in Fig. 3.

The relative activity ofthe enzyme in the range of pH 7.5 to 8.0 which were optimal pH values was maintained as much as about 50% or more in the range of pH 5.5 to pH 10.5. Thus, it is apparent that the enzyme of the present invention can act in a considerably wide pH range where a neutral pH is a center, but the enzyme does not function sufficiently in an alkaline pH range of pH

360 11.0 or more, and the protease activity of the enzyme rapidly deteriorates in the alkaline pH range.

Example 5 (pH stability of enzyme)

The enzyme according to the present invention was maintained in each of the above- mentioned buffer solutions at 20°C for 1 hour to inspect a remaining protease activity. The results are shown in Fig. 4. 365 The enzyme according to the present invention had a residual activity of 80% or more in the range of pH 6.0 to pH 9.5 at 30°C for 1 hour, but at pH 11.0 or more, the residual activity was 40% or

Example 6 (Effect of temperature on enzyme reaction)

The decomposition reaction of azocasein was carried out with the enzyme according to the 370 present invention at various temperatures in each of a 50 mM glycine-sodium hydroxide buffer solution (pH 10.5) and a tris-hydrochloric acid buffer solution (pH 8.0). The reaction temperatures were changed from 10°C to 60°C. The results are shown in Fig. 5.

The optimal temperature of the enzyme according to the present invention was 20°C at pH 10.5 and 40°C at pH 8.0. Even at a temperature of 30°C, the enzyme of the present invention could 375 maintain about 80% ofthe activity at either pH. At a temperature of 40°C or more, the activity of the enzyme rapidly decreased, and at 60°C, it was completely lost Comparing the optimal temperature of the enzyme of the present invention with the optimal temperature (60°Q of Savinase which is known as a commercially available protease enzyme, it can be considered that the enzyme according to the present invention possesses the activity at the low temperatures. 380 Example 7 (Temperature stability of enzyme)

The enzyme according to the present invention was maintained at 10 to 60°C for 1 hour. The variation of its activity with time is shown in Fig. 6. The enzyme according to the present invention was scarcely inactivated, when maintained at 10°C, 20°C and 30°C for 1 hour. However, it was inactivated at 40°C to about 70% of the activity, 385 and afterward, it was gradually inactivated. At 50°C and 60°C, the enzyme was rapidly inactivated, and it was completely inactivated and the residual activity was 10% or less. Example 8 (Effect of inhibitor on enzyme)

As inhibitors, there were used pepstatin acting on an aspartic protease, L-trans- epoxysuccinylleucylamido-4-guanidinobutane (E-64) acting on a cysteine protease, 390 phenylmethanesulfonyl fluoride (PMSF) acting on a serine protease, 1,10-phenanthroline and ethylenediaminetetraacetic acid (EDTA) acting on a metal protease and a metal-dependent protease. These inhibitors were each added to an enzyme reaction system so as to become various final concentrations, and the reaction system was then maintained at 20°C for 1 hour to inspect the residual protease activity. The results are shown in Table 2. 395

Table 2

Inhibitor Concentration Residual activity(%)

None . 100

PMSF I mM 100 10 mM 113

Pepstatin 1 M 93 10 M 65

E-64 10 M 105 100 M 87

1, 10-Phenanthroline I mM 15 10 mM 7

EDTA I mM 51 10 mM 40

The protease activity of the enzyme according to the present invention was not inhibited by pepstatin, L-trans-«poxysucdnylleucyι mido-4-guanidinobutane (E-64) and phenylmethanesulfonyl fluoride (PMSF), but it was notably inhibited by 1,10-phenanthroline and ethylenediaminetetraacetic acid (EDTA). From these results, it is implied that the enzyme according to the present invention is the metal protease. Accordingly, the active center of the enzyme can be considered to be a metallic ion.

Furthermore, the residual activity is higher in the case that ethylenediaminetetraacetic acid is used than in the case that 1,10-phenanthroline which inhibites the metal protease is used. This reason would be considered to be that et ylenediarninetetraacetic acid acts as a chelating agent to the metal ions forming the stereostructure ofthe protease, though this reason is not restrictive. Example 9 (Substrate specificity of protease)

The proteolytic activities ofthe protease were measured in accordance with a phenol reagent method by the use of casein, hemoglobin, albumin and gelatin as water-soluble substrate proteins and by the use of two (derived from human hair and the hoof of a cow) of keratins, elastin and collagen which were water-sparingly soluble or water-insoluble substrate proteins. The results are shown in

Table 3.

Table 3

I Substrate (1%) Hydrolysis rate(%) <water soluble>

Casein (by Hammarsten's method) 100

Gelatin 84

Albumin (from cow) 8

Hemoglobin (from cow) 5

Collagen (from cow Achilles tendon) 3

Elastin (from cow neck ligament) 3

Keratin (from human hair) 14

Keratin (from cow hoof) 4

The substrate specificity of the enzyme according to the present invention is most active to casein at a low temperature, and the specificity decreases in the order of gelatin, albumin and hemoglobin. Furthermore, with regard to the insoluble substrates, the enzyme shows the substrate 420 specificity to the keratin derived from the human hair.

Example 10 (Determination of amino acid sequence at N teπninal)

In the enzyme according to the present invention, 36 residues ofthe amino acid sequence at its N terrninal were determined. The results are shown in SEQ ID N0:1.

The a ino acid sequence at the N terrninal of the enzyme according to the present invention 425 was determined, and its homology with known amino acid sequences was inspected by the use of a data bank Εntrez". As a result it was apparent that there was the homology with the metal protease as much as about 50%.

Example 11 (Measurement of isoelectric point by isoelectric focusing)

Isoelectric focusing was carried out by the use of a Phast system (Pharmacia-Biotec Co.). 430 First, electrophoresis was done under constant voltage and under conditions of 2.5 mA, 15°C and 410 Vh. Next a gel plate was stained, fixed with a 20% trichloroacetic acid solution, and then washed with a decoloring solution (30% methanol and 10% acetic acid). The results are shown in Fig. 7.

As a result of the isoelectric focusing, the enzyme according to the present invention was stained as a substantially single band, and the measured isoelectric point was pI-7.4. 435 Example 12 (Effect of culture temperature on AP3801 strain and the like)

The microorganisms according to the present invention were cultured under the same conditions as in Example 1 except that culture temperatures were set to 10°C, 25°C and 37°C. Afterward, the turbidities of the liquid culture media was measured. The results are shown in Fig. 8.

Furthermore, the enzymes according to the present invention were purified from the liquid 440 culture media obtained at the respective culture temperatures in accordance with a procedure described in Example 2, and the enzyme activities of these enzymes were then measured. The results are shown in Fig. 8. As a result it is apparent that the optimal culture temperature of the microorganisms according to the present invention is preferably at least 37°C or less, more preferably in the range of 445 10 to 25°C. In addition, it is definite that the cold-active enzymes produced by the microorganisms can also be produced at a temperature of at least 37°C or less. List of Sequence Sequence No.: 1 Sequence length: 36 450 Sequence type: Amino acid

Strandedness: Single strand Topology: Linear Molecule type: Peptide Fragment type: N terminal 455 Original source:

Organism: Serratia marcescens Strain: AP3801 strain Sequence description: Ser Leu Asn Gly Lys Thr Asn Gly Tφ Asp Ser Val Asn Asp Leu Leu 460 1 5 10 15

Asn Tyr His Asn Arg Gly Asx Gly Leu Thr Ile Asn Asn Lys Pro

20 25 30

Ser Phe Asp Ile Ala 35 465

Claims

WHAT IS CLAIMED IS:

1. A protease having about 60% or more of its mavimiim activity at 20°C.

2. A protease having the following physicochemical properties:

(a) specific activity and substrate specificity: the protease acts on casein, gelatin, albumin and hemoglobin to specifically decompose them, and the substrate specificity decreases in the order of casein, gelatin, albumin and hemoglobin;

(b) optimal pH: 7.5 to 8.0; and

(c) pH stability: the protease is stable in the range of pH 5.5 to pH 10.5 at 20°C for 1 hour.

3. The protease according to Claim 2 which further has the following physicochemical properties:

(d) optimal temperature: 20°C at pH 10.5 and 40 C at pH 8.0;

(e) temperature stability: at pH 10.5 for 1 hour, the protease is scarcely inactivated at a temperature of 10°C to 30^CC, but it is inactivated at 40°C as much as about 30% and completely inactivated at 50°C; and

(f) enzyme activity: the protease has about 60% or more of its maximum activity at 20°C.

4. The protease according to any one of Claim 2 or 3 whose molecular weight is about 58 kDa as measured by SDS-PAGE.

5. The protease according to any one of Claims 2 to 4 whose isoelectric point is about 7.4 as measured by an isoelectric focusing.

6. The protease according to any one of Claims 1 to 5 which consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l.

7. The protease according to any one of Claims 1 to 5 which consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO:l at an N terminal.

8. A protease which consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO: 1.

9. A protease which consists of a protein containing a part or all of an amino acid sequence described in SEQ ID NO: 1 at an N terminal.

10. A microorganism belonging to Serratia genus, which is capable of producing the protease according to any one of Claims 1 to 9.

11. The microorganism according to Claim 10 which grows well at 10°C to 25°C.

12. The microorganism according to Claim 10 or 11 which is deposited under Deposition No. FERM BP-5401.

13. A microorganism which is deposited under Deposition No. PERM BP-5401.

14. A method for preparing the protease according to any one of Claims 1 to 9 which comprises the steps of culturing the microorganism according to any one of Claims 10 to 15, and then collecting the protease according to any one of Claims 1 to 9 from its culture medium.

15. A peptide which consists of a part or all of an amino acid sequence described in SEQ ID

NO:l.

16. A protein which comprises a part or all of an amino acid sequence described in SEQ ID NO:l.

17. A protein which comprises a part or all of an amino acid sequence described in SEQ ID NO: l at its N teπninal.