CZ258097A3 - Psychrophilic protease and psychrophilic bacteria - Google Patents

Description

Psychrophilic protease and psychrophilic bacteria

Technical field

The invention relates to a protease having high activity in the low temperature range, to its use, and to a psychrophilic bacterium that produces the protease.

BACKGROUND OF THE INVENTION

Psychrophilic bacteria have been known for a long time and their existence under low temperature conditions has been confirmed in a number of cases. Psychrophilic bacteria can be isolated from a variety of low-temperature environments, such as soil, fish products or dairy products, as well as from an artificially low-temperature environment. There are studies of psychrophilic bacteria in food microbiology, but these studies essentially relate to the phylogenetics of microorganisms.

It is believed that enzymes derived from psychrophilic bacteria will also be psychrophilic, that is, their optimum temperature will be found in the low temperature range. In general. that a psychrophilic enzyme that acts efficiently at low temperatures can be added, for example, to a detergent which can then be used in water at low temperature. It is also believed that such an enzyme can be used in a chemical reaction in the presence of an organic solvent that is volatile at room temperature, as well as to improve the quality of the food at a temperature that does not spoil the food. Study of enzymes

·. . · ·· ·· ···· · · · .... · · ·

... ······ · z · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · interesting also in terms of physiological function and adaptation mechanisms related to low temperatures in psychrophilic bacteria.

Summary

A new bacterial strain has been found which produces a new psychrophilic protease.

Accordingly, the present invention provides a new psychrophilic protease.

It is a further object of the present invention to provide a novel microorganism which produces this psychrophilic protease. (

Another object of the present invention is to provide a process for the preparation of this

psychrophilic proteases by said novel microorganism. ,

The psychrophilic protease of the present invention has the following physicochemical properties.

- Specific activity and substrate specificity: the protease breaks down casein and dimethyl casein but does not affect ribonuclease.

Optimal temperature: the optimal temperature of this enzyme is approximately 40 ° C.

- Temperature stability: under storage conditions at pH 7 for 1 hour at 30 ° C, the protease is virtually inactivated, but at 40 ° C it loses approximately 40% of its activity, and at 50 ° C is rapidly inactivated so that enzyme activity is completely lost within 15 minutes.

According to a preferred embodiment of the invention, this protease also has the following physicochemical properties:

··················

- Optimal pH: the protease works optimally at pH 7.5;

PH stability of the enzyme: the protease is stable in the pH range of 6.0 to 10.0 under storage conditions at 20 ° C for 1 hour;

Molecular weight: approximately 38 kDa (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and gel filtration method);

- Isoelectric point: approximately 4.5.

The novel microorganism of the invention is Flavobacterium balustinum, which has the ability to produce a psychrophilic protease as described above.

The method of preparing a psychrophilic protease of the invention comprises culturing a Flavobacterium balustinum microorganism and obtaining a psychrophilic protease from a microbial culture.

Short description of the display

Giant. 1 shows the result of Example 2, which shows the relationship between temperature and activity of P104 strain proteases and Subtilysin Carlsberg protease.

Giant. 2 shows the result of Example 4 (2), which shows the effect of initial pH on extracellular protease activity and growth

Flavobacterium balustinum P104.

Giant. 3 shows the result of Example 4 (3), which shows the effect of culture temperatures on extracellular protease activity and growth of Flavobacterium balustinum P104. Giant. 3 (A), (B) and (C) show the results obtained at 10, 20 and 30 ° C.

Giant. 4 shows the gel filtration elution result of Example 5 (2) (c).

• ·

Giant. 5 shows the result of chromatographic elution in Example 5 (2) (c).

Giant. 6 shows the result of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to determine the molecular weight in Example 6.

Giant. 7 shows a calibration curve for the determination of the molecular weight in Example 4.

Giant. 8 shows a calibration curve for gel filtration to determine the molecular weight in Example 6.

Giant. 9 shows the result of isoelectric focusing in Example 7. {

Giant. 10 shows the isoelectric J focusing curve of Example 7

Giant. 11 shows the result of Example 8, which shows the effect of pH on the enzyme reaction performed by the enzyme of the present invention.

Giant. 12 shows the result of Example 9, which shows the pH-stability of the enzyme according to the invention.

Giant. 13 shows the result of Example 10, which shows the effect of temperature on the enzyme reaction performed by the enzyme of the invention.

Giant. 14 depicts the result of Example 11 showing the thermal stability of the enzyme of the invention.

Giant. 15 shows the result of Example 13, which shows the effect of the sodium modifier sodium dodecyl sulfate on the enzyme of the invention.

Giant. 16 shows the result of Example 13, which shows the effect of a urea protein modifier on an enzyme of the invention.

• · · · · · · ·

Giant. Figure 17 is a Lineweaver-Burk plot of the enzyme of the invention as determined in Example 16. FIG. 17 (A) shows a change in the value of 1 / v in the range of 0 to 2.0; FIG. 17 (B) in the range of 0 to 0.2.

SUMMARY OF THE INVENTION

Bacteria producing a new protease

The novel protease of the invention is produced by microorganisms belonging to the genus Flavobacterium and having the ability to produce a protease having the properties described above.

A specific example of microorganisms having the ability to produce a protease of the invention preferably comprises Flavobacterium balustinum P104. This strain is a microorganism isolated from salmon gut and was deposited with the National Institute of Bioscience and Human Technology, Ageney of Industrial Science and Technology under number FERM BP-5006 on

February 17, 1995.

The bacteriological properties of Flavobacterium balustinum P104 according to the invention are listed below.

(1) Morphological properties

The strain takes the form of short bacilli of 0.4 to 0.5 x 1.7 to 1.9 microns.

(2) Nature of the culture medium

The strain grew on agar medium and produced a yellow pigment.

(3) Optimal pH growth conditions: the strain grew in the pH range of 5 to 9, and the optimal pH growth was approximately neutral.

Temperature: The strain grew between 10 and 30 ° C, and the optimal growth temperature was approximately 20 ° C.

(4) Affiliation to aerobic and anaerobic bacteria: aerob.

(5) Gram staining: Gram negative.

(6) Biochemical properties

Main biochemical properties of microorganism

Flavobacterium balustinum P104 are shown in Tab. 1.

Table 1

Tested properties Result Galactosidase - Arginindihydrolasa - Lysine decarboxylase - Ornitindecarboxylase - Use of citric acid - Production H 2 S - Ureasa + Trypto fanaminasa - Indole production + Gelatinase + Glucose - D-mannitol - Inositol - D-sorbitol - Rhamnosa - Sucrose - D-melibiosis - D-amygdalin - L-arbinol  - Oxidase

Although the strain appeared to be Flavobacterium indolgenes according to these properties, it was classified as Flavobacterium balustinum by comparing the DNA sequence encoding 16S ribosomal RNA with that of a known microorganism as described in Example 3.

The culture medium used for culturing the microorganism is either liquid or solid, but generally a shake culture or an aerated culture containing a liquid medium is used.

Any culture medium suitable for the growth and production of protease may be used. Specifically, examples of carbon sources include glucose, trehalose, fructose, maltose, sucrose, starch and malt oligosaccharides. Examples of the nitrogen source include yeast extract, soybean meal, cottonseed meal, corn steep, various amino acids and their salts, and nitrates. Synthetic media or natural media containing suitable inorganic salts such as magnesium, calcium, sodium, potassium, iron and manganese phosphates, and other nutrients as appropriate, may also be used.

Culture conditions such as pH and temperature can be determined within the range of protease production, shake culture on liquid medium or aerated shake culture are preferably performed at neutral pH and a temperature of about 20 ° C.

The protease of the invention is produced in the cell wall of the bacterial cell, inside the cell and in the culture supernatant and is therefore in the form of a bacterial wall, a crude enzyme derived from a bacterial cell, or culture supernatant on liquid medium, or in the form of extracted and purified enzyme. The enzyme may also be in the form of a protease immobilized on a substrate by a known method.

Enzyme isolation

Well-known methods, either singly or in combination, can be used to isolate and purify the enzyme of the invention from culture on a liquid medium.

The protease of the invention is predominantly secreted extracellularly, especially in liquid medium cultures, therefore

bacterial cells can be readily removed, for example, by filtration or centrifugation to obtain a coarse enzyme solution. The crude enzyme solution can be further purified by known methods. These methods preferably include salting out using a salt such as ammonium sulfate, a precipitation method with organic solvents such as methanol, ethanol, or acetone, a crude starch adsorption method, an ultrafiltration method, and various chromatographic methods such as gel filtration chromatography or ion exchange chromatography. Typical examples of preferred methods are given in the Examples below.

Protease properties

The properties of the protease of this invention were examined and the results are shown below.

(1) Activity and substrate specificity

This enzyme cleaves macromolecules well! proteins such as casein or dimethyl casein or denatured proteins. It also cleaves gelatin, a denatured collagen protein in the range of about 50% compared to casein. It has little effect on other natural proteins and has no effect on ribonuclease.

Industrial proteases such as subtilisin do not have substrate specificity and act on most proteins. Conversely, the enzyme of the present invention only acts on macromolecular proteins or on denatured proteins into which it is relatively easy to enter.

The protease, which is similar to the enzyme of the present invention and is derived from the psychrophilic bacterium Pseudomonas fluorescens, cleaves well macromolecular proteins such as casein or dimethyl casein or denatured proteins. However, this protease, which differs from the enzyme of the invention, also cleaves natural globular proteins such as hemoglobin and bovine serum albumin in the range of about 40% compared to dimethyl casein. Therefore, the enzyme of the invention is likely to have a higher substrate specificity than known enzymes.

(2) Optimal temperature and temperature stability

The enzyme of the invention acts at a temperature of 40 ° C.

When the enzyme is stored at pH 7 for 1 hour, it is rarely inactivated at temperatures up to 30 ° C, but at 40 ° C it loses its activity. At 50 ° C, it is rapidly inactivated, losing all activity within 15 minutes.

Thus, the enzyme of the invention is a psychrophilic enzyme exhibiting effective low temperature catalytic activity.

(3) Optimal pH and pH range when the enzyme is stable

The enzyme of the invention has an optimum pH of 7.5.

It is stable over a pH range of 6.0 to 10.0 under storage conditions at 20 ° C for 1 hour.

Thus, the enzyme is a neutral protease that does not operate within an extremely acidic or basic pH range. The enzyme is inactivated when stored in an extremely acidic or basic pH range.

(4) Molecular weight

The enzyme of the invention has a molecular weight of 38 kDa, which was measured by polyacrylamide gel electrophoresis methods in the presence of sodium dodecyl sulfate and gel filtration.

(5) Isoelectric point · 1 · 1 · 1 · 1 · · · «« · ···· · · ··· ·

The enzyme of the present invention has an isoelectric point of 4.5, which was measured by isoelectric focusing.

(6) Inhibition of activity

The enzyme protease activity is not inhibited by phenylmethylsulfonylfluoride or iodoacetamide, but is greatly inhibited by ethylenediaminetetraacetic acid, 2,2-bipyridyl, citric acid or oxalic acid. Thus, the protease activity depends on the metal ions, so the enzyme of the invention is probably a metalloprotease. The inhibition of protease activity by citric and oxalic acid results in a dependence of enzyme activity on calcium ions.

In addition, the enzyme activity is greatly inhibited by metal ions such as Ag ', Cu ² ', Zn ^2-1 , Co ²⁴ and Fe ²⁺ , among them in particular Ag ''. However, neither Mg ² 'nor Ca ^{2 1} / (7) enzyme reaction kinetics are inhibited

The enzyme of the invention exhibits a type of Michaelis-Menten reaction rate depending on the concentration of the substrate such as kaseih. The K values decrease and the V values increase with m and r with increasing temperature. In addition, the enzyme exhibits remarkably high values in the range of 10 to 40 ° C. Enzymes are generally inhibited by excess substrate. However, in the case of the enzyme according to the invention, the K _cat value rises when the system approaches an optimum operating temperature. Thus, the enzyme is advantageous in that no appreciable inhibition by degradation products is observed. Lineweaver-Burke dependence also suggests that the temperature-induced inhibition is a mixed form, i.e., the effect of temperature is a non-competitive or uncompetitive inhibition and the effect of decomposition products is a competitive inhibition.

• Φ φφφφ • φ φ φ φ · φ · φ φ <

• φ · · · · · • · · · · · · ·

Use of enzyme

The psychrophilic protease of the invention has an optimal temperature in the low temperature range. Thus, with the psychrophilic protease of the invention, proteins can be cleaved at low temperature. For example, a detergent that can also be used in water at low temperature is prepared by adding the protease of the invention to the detergent composition. Except for the inclusion of the psychrophilic protease of the invention, the detergent composition may be prepared by conventional methods. Briefly, it can be prepared by combining the protease of the invention with a conventional detergent component, such as a detergent surfactant, bleach or filler.

The enzymatic reaction of the psychrophilic protease of the invention takes place at low temperature. Thus, even if the reaction system contains an organic solvent that is volatile at room temperature, the reaction may proceed at a low temperature where the organic solvent does not evaporate. Moreover, when we intend to improve the quality of the foodstuffs using the protease of the present invention, it is preferable to use this protease because the reaction takes place at a low temperature at which the foodstuffs are effectively protected from decomposition.

As the protease of the present invention is available, we expect progress in further studies of the physiological mechanisms of psychrophilic bacteria and their application mechanisms at low temperatures.

The invention is described in more detail below with reference to specific examples, but the scope of the invention is not to be construed as being within the scope of the invention. · · · · · · · · · · · · · · · · · · · · · · · · · · ·

In this context, proteins were quantitated by the Bio-Rad Protein Assay staining method and protein fractions of the eluate obtained by chromatography were measured using ultraviolet absorption at 280 nm unless otherwise indicated.

In addition, protease activity was measured as follows. _(A) Decomposing protein activity measured with azocasein

0.05 ml of enzyme solution was added to 0.3 ml of 0.067 M phosphate buffer containing 1% (W / V) azokassein (pH

7.0) and the mixture was maintained at 30 ° C for 30 minutes. |

The reaction was quenched by the addition of 6% trichloroacetic acid 1 solution. After 15 minutes, the reaction mixture was centrifuged at rt

000 rpm at room temperature for 5 minutes. ,

The absorbance of the supernatant was measured at 340 nm. Enzyme I activity was defined on the basis of AGU (azocasein digestion unit), which represents an increase in absorbance of 0.001 per minute at 340 nm.

(b) Protein degradation activity determined by the modified Anson method

0.05 ml of the enzyme solution was added to 0.3 ml of 0.067 M phosphate buffer containing 1% (W / V) protein concentration (pH 7.0) and the mixture was then held at 30 ° C for 30 minutes. The reaction was quenched by the addition of 7.5% trichloroacetic acid solution. After 30 minutes, the reaction mixture was centrifuged at 14,000 rpm at room temperature for 5 minutes. Absorbance of the supernatant was measured at 280 nm.

Enzyme activity was defined based on the AU (Modified Anson Unit) that it represents

- _o · · · · · · · · · · · · · · · · · · · · · 1 · 1 · 1 · 1 · 1 · 1 · 1 · 1 · 1 mole per minute.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 (1) Search for new bacterial strains

Isolation of the new bacterial strain was performed on agar medium. An isolated sample of salmon internal organs was resuspended in saline and the supernatant was used as a stock solution from which a 100-fold diluted solution was prepared. Volumes of 0.2 ml stock solution and 100 times dilute solution were sprayed onto strain-finding agar medium (polypepton 3 g / l, yeast extract 10 g / l, Na casein 10 g / l, MgSO 4 .7H ₂ 0 0, 2 g / l, 2.0 g / l agar (9 cm diameter petri dishes) and agar plates were cultured at 10 ° C for 3 days. Well-growing isolates were selected from agar-grown colonies for subculturing and inoculation on agar plates to form stock cultures.

Exoenzyme activity was determined on an agar medium. The isolated bacterial strain was inoculated in lanes on agar medium for screening as described above and cultured at 10 ° C for 3 days. 10% trichloroacetic acid was then sprayed onto the agar plate on which bacteria were grown and the protease producing bacteria were detected by clear staining.

(2) Cultivation of isolated bacterial strain and enzyme production

The isolated bacteria from the stock medium were inoculated into 25 ml of pre-culture medium (polypepton 10 g / l, endoextract 10 g / l, MgSO4.7H2O 0.2 g / l, pH 7.0 in a 100 ml Erlenmeyer flask) and cultured on a rotary shaker at 10 ° C and 150 rpm for 48 hours in TAITECNR-80 to stabilize bacterial growth activity. Typically, 0.25 ml of pre-culture solution was inoculated into 25 ml of enzyme production medium (polypepton 5 g / l, yeast extract 2.5 g / l. Casein 5 g / l, MgSO ₄ .7H _and 0 0.2 g) (PH 7.0 in a 100 ml Erlenmeyer flask) and cultured on a rotary shaker at 10 ° C and 150 rpm for 72 hours. The culture medium was previously steam sterilized at 121 ° C (1.2 kgf / cm ² ) for 15 minutes.

The isolated bacterial strain was stored on agar medium for storing bacteria at 10 ° C and subcultured after 2 weeks to 1 month.

(3) Measurement of protease activity

The culture solution obtained by the previous procedure (2) was centrifuged (17,000 x g, 4 ° C, 15 minutes). The supernatant was used as a crude enzyme solution. Protease activity was measured by decomposition of azocasin. 0.05 ml of coarse enzyme solution was added to 0.3 ml of a 0.067 M phosphate solution containing 1% (W / V) azokasin (pH 7.0) and the mixture was incubated at 20 ° C for 30 minutes. The reaction was then quenched with 6% trichloroacetic acid and after 15 minutes the reaction mixture was centrifuged at 14,000 rpm at room temperature for 5 minutes. The absorbance of the supernatant was measured at 340 nm using a spectrophotometer (Beckman DU640). Enzyme activity was determined on the basis of ACU (azokasein digestion unit), which *

• · · · · · · · · · · · φ · · ·

Φ ·· · · φφ φ represents the increase in absorbance ο 0.001 per minute at 340 nm.

Steps (1) to (3) resulted in the isolation of the bacterial strain P104 with protease activity. The bacterial strain exhibited protease activity as shown in the following table.

The strain growth rate shown in the table was obtained by comparison with the growth of the Cytophaga xantha strain IFO 14972.

Table 2

Bacterial strain with protease activity

Strain Growth rate Protease activity (x 10 ³ ACU / ml) P104 +++ 0,982

Example 2

Protease activity in P104

The effect of temperature on enzyme activity was investigated in a culture solution obtained by culturing a protease producing strain P104 in a temperature range of 0 to 60 ° C. The temperature dependence of enzyme activity was investigated in the same way for Subtilisin Carlsberg (Sigma), a commercially available enzyme protease derived from Bacillus licheniformis.

The result is shown in Figure 1, in which the specific activity of strain P104 is shown by squares and the specific activity of Subtilisin Carlsberg triangles.

Optimum temperatures were 40 ° C for oxoprotease strain P104 and 60 ° C or more for Subtilisin Carlsberg. Exoprotease of strain P104 retained at 20 ° C and at an optimum temperature of 40% or more protease activity. Proteasa Subtilisin

Carlsberg retained only about 10% of protease activity at optimal temperature.

In the range of 10 to 40 ° C, the activation energy of the enzyme reaction was calculated for these exoproteases. The results are shown in the following table.

Table 3

Bacterial cell Activating energy (kJ / mol) P104 39.8 Bacillus subtilis 58.3

Example 3

Identification of strain P104 based on the sequence fits in the DNA for 16S ribosomal RNA.

A sample of the culture solution obtained according to Example 2 was collected in a 1.5 ml microtube and the bacteria were collected by centrifugation. Genomic DNA was extracted to amplify the DNA sequence of bases encoding 16S RNA by PCR (polymerase chain reaction). The base sequence was then determined using the Sanger method and compared to a Genbank database for identification. The list of primers used is shown below as IF-Link and 5R-Link were used as PCR.

Primers:

1F-Link i 5'-TGTAAAACGACGGCCAGTAGTTTGATCATGGCTCAG-3 ';

3R-Link: 5'-CAGGAAACAGCTATGACCCGTCAATTCATTTGAGTT-3 ';

3F-Link: 5'-TGTTAAAACGACGGCCAGTGTAGCGGTGAAATGCGTA-3 ';

5R-Link: 5'-TGTAAAACGACGGCCAGTAAGTCCCGCAACGAGCGCAA-3 '.

• ·

The results of the comparison with the Genbank database are shown in the following tables. In these tables, Query represents the 16S ribosomal RNA gene originating from the P104 strain and the Subject represents the 16S ribosomal RNA from Flavobacterium balustinum (FVBRR16SH). The bacterial strain was identified as Flavobacterium balustinum. Thus, the P104 strain is hereinafter referred to as Flavobacterium balustinum P104.

·····································

Aο ··· ······ · ···· · · · · · · · · · · · (a) Primer used IF -Link

Identity = 185/204 (90%), Similarity = 185/204 (90%)

Qucry: l ACG CTA GCGGGAGGC 31 GATNA ACG CTA G G G G A G G C Qucry: 21 CTAAC ACA T G C AAG CCG AGC 51 CTAACACA T G C AG AG Query: 40 GGTATTTG • «« «» T C T and TTCGGGACA (and aaa 71 GGTATAGA T T C T TCGGA ATC Qucry: 6i GAGAG agc G G A G G A G G G T G 91 TAGAG AGC G G C GTACGGGTC Query: Sl CGGAACAC GTG TGCAACCTA Sbjctl 11 CGGAACAC GTG TGCAACCTA Query: 101 CCTTTATC AGG GGGATAGCC Sb jer131 CCNTTATC AGG GGGATAGCC Query: 120 TTTCG AAA G G A AGA T TA AT A Sbjcrl51_ T T C C G A A A G G A AGATTAATA Query: I4i CCCCATAA T A T ATTGATTGG 171b: 171 CCCNATAA T A T ATTGACTGG Query: 16l CATCAGTT ACT ATTGACAAC a i a a a a «a« Collection: 191 CATCAGTC GAT ATTGAA aac Query: 180 TCCGGTGG A T A GAGATGGTC Coll .: 211 TCCGGTGG A T A GAGATGGGC Query: 201 A C G C «A <1 Collection: 231 A C G C

• • • • • • • • • • • • • • • • • • 9

(B) 3F-Link primer used

Identity = 208/330 (63%), Similarity = 208/330 (63%)

Query: l G A T A

Sbjcr.695 G A T A

Query: 21 T G C «« I *

Sbjct: 715 T G C

Query: 4l G Τ Ν T

I · »

Sbjci: 735 Τ Τ T

Qucry: 61 C C G A * 4

Sbjct: 755 A G G A

Query: 81 G A G

TTACTTAG:::; : i::

TTACTTAG G A G G G A N G

GAAGGCAG N A 'C C G G T

AACACCAA

TAACTGAC

AAGTGNGN • · · S i

AAGCGTGG A T T A O T T N

AAC ACCAA Τ T C A C T A T :::::::

GTCACTAT GCN G A T G N * · 4-444

GCTGATGG

TGAGTGAA • t * «44

4 * 4 4

GG A G C A G CCATGGTC

775

QucryrlOl

795

Query: 120

C A G G

G N C C 4 4

4 4

G T C C

T A T C

A Τ T A

A C O N; i:

A C G C

T C G N

G A T A

C G Τ N

4

C G T A

. Τ Ν T

C C C T

C A C N <*

A A C G

G G G A

G G A A Τ Ν T

I *

A T G C

Τ T A N

Sbjct: 815 T

Query: 41 N A

835 G G

Qucryrlol A G • I

Sbjci: 843 and G

Qucry: 181 N C

Coll .: 865 G G

Query: 20l G

Sbjct: 885 G

A C T C G T

G Τ N C A G * 4 14 4

4 «14

G Τ T C A G

Τ N G T A T <4 4

T G A T A A

G N G T C N * * 4

4 4

G A G T A C

A:: T C A::: i:

A A C T C A

G Τ Τ T G G

C G A G T A

A G A C TA

G Ν A G N i I »<4 4

G Τ T A G C

G C T C G C

G C Τ Τ T A

A C A N A G

A G C G A A

C A C C G »I« ·

4 * 4

N C C T G

A G G Τ Τ T

A A G A A T

T G G C G G

I 4

Τ T G A C G · V V V V V V V

Qucry: 201 NTGGCCNNTACACNCTGTGN

II «·· <I ··

I «11« I «II

Sbjct: 9O3 GGNNCCNG CACAAG.CGGTGG

Query: 22l T T T T T T T G T N T A A G G T T I · I I I «« «« I I lili «II III ·» I

Sbjct: 925 ATTATGTGGTTTAATTCGAT Query: 24l NATNCNAGAGGGTCCTNACC:: i:: j: j::::

Sbjci: 94i OATACGCGAOOAACCTTACC Query: 261 ANGNTTNNTTNGGGTCNTCC «· I 11« | • I t «II |

Sbjct: 965 AAGGCTTAAATGGGAATTGA Qucry: 281 CAOCCTTCGTCTNTACTTGT

I «I lil l i

I «« I »« I

Sbj’Ci: 982 CAGGTTTAGAAATAGACTTT Query: 3O1 GNTTCGGACA

SbjcclOOS TCTTCGGACA • · · · · · (C) 3R-Link primer used

Identity = 128/162 (79%), Similarity = 128/162 (79%)

Query * 162 T T T T G G G A A A A A G G G C | 1 «» »« «« «« | Sbjct 552 Query: 140 tttattgggttta aagggtc CNTCNGCGGACCTGTAAATC No. 572 Query: 122 CGTAGGCGGATCTGTAAGTC ATTGGT GATATC T C A G A G 5: 592 Query: 101 AGTGGT GAAATCTCAT AG, CT tgtctatgggactaccattg 612 Qucry; 82 taactatgaaactgccattg atgctccaggtcatgagtct 632 Query: 62 atactgcaggtcttgagt AA agcaggagtggctggaataa 656 Query 42 agtagaagtggctggaataa gtactgtaacg gtgtaatgc 5bj «: 672 Qucry: 22 gtagtgtagcggtgaaa TGC atagatattactcagaaccc 692 Qucry: 2 AT AGAT ATT ACT AGAAC AC C A

Sbjci: 712 CA · · 12 · φ φ φ 7 7 7 7 7 7 7 7 7 7 7 7 7 (D) Primer used 5R-Link

Identity = 237/273 (86%), similarity

237/272 (86%)

Query: 273 C C G G T and AND and C N C « C 1 C T T and G and G G <a C C and a AND and C AND (L) 93 C C C T T and AND • C G C and T T and G G G and a c c AND C and AND Query: 255 C A C G T AND AND T AND C AND AND T C N C A AND G N Sbjct: 1213 C A C G T AND AND T AND C AND AND T G G C C AND G T Query: 233 A C • t AND and G and AND O N G T AND G C N A C C A G N C 1233 A C AND G AND G G G C AND • G and C T A C C A G G C Query: 212 G N C T G G AND T G C G AND AND T C T C G AND AND 1233 G A C T G G AND T G C G AND AND T C T C G AND AND Qoery: 193 A N 1 C τ 1 G C 1 Ň C T C AND G AND N C G G AND N T Sbjct 1273 1 A G C T N and G N C T C AND G T T C G G AND τ τ Qucry: 173 G G 1 1 AND 1 G and T 1 C « T < G and C and AND 1 AND 1 C and T and C G and a A C 1! τ 1 E T Sbjct: J293 G G AND G T C T G C AND AND C T C G A C T C T Query: 136 A T 1 1 G AND AND and AND 1 AND C 1 T G and G and AND and T N C G C T AND G T No. 1313 A T G AND AND G C T G G AND AND τ C G g τ AND G τ

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Query: 136 AND AND T c G C A T A T c A G N C A T G A T Sbjct: 1333 AND AND T c G C A T A T G A G C G A T G A T Que; y: 13 N C AND G G T G A A N A C G T T G C G N G G Sbjct: 1333 G C G G T G A A T A C G T T C C C N G G Qucry: 93 C C T T G N A A A C A C C G C C C G T C Sbjct: I373 C C T T G T A C A C A C C G C N C G T C Qoery: 73 AND 1 AND < C C C A T 0 G A A G T T G G G G T Sbjc «: 1393 AND AND G C C A T G G A A G T T T c O G G 7 Query: 52 AND C C T G N A G T C G G T G A C C OTA 1413 AND C C T G A A G T G Q G T G A C C OTA Query 33 AND C N G G A C C T N C C T G G G T A N

I433 ACAGGAGCTGCCTAGGGTAA Query: 13 NACAAGTAACTAG

Sbjci: J433 AACAAGTAACTAG 9 9 9 9 9 9 9

99 * ·· 9 9

9

9 9 · • ···· · · · · ·

9999 · ···

Example 4

Culture Flavobacterium balustinum PI4 (1) Measurement of bacterial cell concentration

The culture solution obtained according to Example 2 was diluted with saline to ensure a value of 0 to 5 cells per bacterial counter cell. Cells were counted using an optical microscope. To obtain a correlation between cell number and cell suspension turbidity, turbidity of individual dilutions of the culture solution was measured spectroscopically at 6.50 nm. The relationship between turbidity and bacterial cell concentration of Flavobacterium balustinum P104 is described by the following equations:

(Cells / ml) = 1.13 x 10® x Abs.660 nm, where 1.13 x 10® is a factor obtained from a calibration curve.

(2) Effect of pH

The bacterial strain was cultured at various initial pH values of culture media for protease production ranging from 5 to 9 in order to investigate the effect of initial pH on exoprotease activity and growth.

The results are shown in Fig. 2.

At alkaline pH, bacterial growth was significantly reduced and protease activity was also reduced. Non-significant differences, either in bacterial growth or in protease activity, were observed at acid pH. Bacterial strain growth was highest and protease activity reached the highest level at neutral pH.

(3) Influence of cultivation temperature

Flavobacterium balustinum PI04 was cultivated at <RTIgt; </RTI> o · ······ At different temperatures ranging from 10 to 30 ° C to investigate fluctuations in exoprotease activity and growth during cultivation.

The results are shown in Figure 3.

While the bacterial strain grew well at temperatures of 10 and 20 ° C, the overall biomass increase at 30 ° C was about half compared to the steady state at the end of growth at 10 and 20 ° C.

The strain showed the highest growth rate at 20 ° e, so the optimal temperature for cultivation is probably 20 ° C.

Example 5

Purification of Flavobacterium balustinum P104 protease (1) Culturing a bacterial strain

The isolated bacterial strain obtained from the following bacterial culture medium was inoculated into 25 ml of pre-culture medium (in a 100 ml Erlenmeyer flask) and cultured on a rotary shaker at 150 rpm in TAITEC NR-80 at 10 ° C for 48 hours. hours. The actual culture was prepared by inoculating 0.25 ml of pre-culture solution into 25 ml of the actual culture medium in a 100 ml Erlenmeyer flask and cultured on a rotary shaker at 150 rpm and 10 ° C for 72 hours.

Storage medium:

polypepton 3 g / l enzyme extract 2,5 g / l

Casein 20 g / l

MgSO 4 .7H _and 0 0.2 g / l agar 20 g / l pH 7.0 · · · · · · · · · · · · · ·

Recultivation medium:

polypepton enzyme extract

Casein

MgSO. 7H O ³ 4 2 g / L

2.5 g / l i g / l 0.2 g / l pH 7.0

Culture medium:

polypepton 3 g / i enzyme extract 2.5 g / l Casein 5 g / l KH MO .7H 0 2 4 2 3 g / l MgSO. 7H 0 4 2 0.2 g / l

pH 7.0

The culture media was steam sterilized under high pressure in an autoclave at 120 ° C (1.2 kgf / cm ² ) for 15 minutes.

Flavobacterium balustinum P104 was stored on agar plates at 10 ° C. The strain was subcultured after 2 weeks to 1 month.

(2) Purification of the protease (a) Salting out with ammonium sulfate

The culture solution obtained in the previous step (1) was centrifuged (17,000 x g, 4 ° C, 15 minutes). The supernatant was used as a crude enzyme solution. Ammonium sulfate was added to the crude enzyme solution so that the solution contained a final concentration of ammonium sulfate equal to 50% of its saturation concentration. After slowly stirring for 1 hour, the solution was centrifuged (17,000 x g, 4 ° C, 15 minutes) to give a.

Q <r ··· · · · ·

J the corresponding saturation fraction (0 to 50%). The amount of ammonium sulfate added was calculated at a saturation concentration at 25 ° C.

(b) Gel filtration

Gel filtration was performed on a HiLoad 16/60 Superdex 200 prep grade preparative column. All column chromatography operations were performed at 4 ° C using the HiLoad System 50 (Farmacia Biotec Co.).

The HiLoad 16/60 Superdex 200 prep grade column was equilibrated by flowing 20 mM Bis-Tris buffer (pH 6.0) at a linear velocity of 60 cm / hour (ratio to gel volume was at least 3, i.e., 400 mL).

A ml sample of the enzyme solution after salting out with ammonium sulfate was applied to a column equipped with a Superloop. The column was eluted with 20 mM Bis-Tris buffer (pH 6.0) at a linear rate of 60 cm / hour and 5 ml fractions were collected.

The elution curve is shown in Figure 4.

The transparent, high molecular weight yellow protein contained in the culture solution was eluted as the exclusion limit. The following eluted fraction contained a colorless transparent protein exhibiting UV absorption at 280 nm. The fragment attached to the protease was probably removed due to the presence of protease activity in this fraction.

(c) Column chromatography

Ion exchange chromatography was performed on a Sepharose Q HP column. A 0.7 x 12.5 cm column made of five HiTrapQ (1 ml) columns connected in series was used as the chromatography column. The column was equilibrated with a flow rate of 20 mM Bis-Tris buffer (pH 6.0) at a linear velocity of 35 cm / hour (the ratio of the gel volume to at least 5, i.e. 25 ml).

A sample of the enzyme solution of the gel eluting fraction having protease activity was loaded onto a column equipped with Superloop at a linear rate of 17.5 cm / hr. The column was then washed with 80 ml of 20 mM Bis-Tris buffer containing a linear gradient of 0 to 150 mM NaCl at a linear rate of 35 cm / hour and 2 ml fractions were collected. A linear NaCl gradient was prepared by adding 1 M NaCl to said buffer to increase its ionic strength.

The elution curve is shown in Figure 5.

In addition, the purification method described above is summarized in the following table.

Table 4

Summary of protease purification

Amount (ml) Protein Enzyme Spec. Efficiency asset activity, purification Degree of purification (x) (mg) (x10 ³ ACU) (XLO ³ CU.mg ^-1; (%) ) Kultiv. material 270 13.0 365 28.1 100 ALIGN! 1 Salting out (NH j SO 5 3.38 273 80.8 74.8 2.9 Gelová filtration 25 0,752 77.7 103 21.3 3.7 Ion exchange chromatography 18 0.154 29.3 190 8.03 6.8

Example 6

Determination of protease purity and molecular weight (1) Electrophoresis (a) Polyacrylamide gel electrophoresis in the presence of sodium

9 9999

9 99,999 9

9 9 · 9

9 9 9 9 9 9 dodecyl sulfate (SDS-PAGE)

A 10% polyactylamide gel of 1 mm thickness was used.

Electrophoresis was carried out using an electrical current of 20 mA until bromophenol blue (BPB) reached the lower limit of the gel. The gel plate was stained with an aqueous solution containing 30% methanol, 10% acetic acid and 0.02% Coomasie Briliant Blue R250 for 1 hour and then bleached overnight with a bleach (30% methanol and 10% acetic acid).

The protease molecular weight was determined by SDS-PAGE using phosphorylase, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and alpha-lactalbumin as markers.

The results and the SDS-PAGE calibration curve are shown in FIG.

and 7. The enzyme has a single band and can therefore be considered as a single protein without subunit structure.

(b) Gel filtration

Molecular weight was determined by a gel filtration method using a Hiprep 16/60 Sephacryl S-100 HR column. The column was equilibrated by flowing 50 mM phosphate buffer containing 0.15 M NaCl (pH 7.0) at a linear velocity of 30 cm / hour (ratio to gel volume was at least 3, i.e., 400 mL). A 1 ml sample of the enzyme solution was loaded onto the column and eluted with the same buffer at a linear rate of 30 cm / hour and 2 ml fractions were collected. Free volume was determined for Blue Dextran 2000 with albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) as protein standards.

QQ ··· ······

ZO ············· · · · · · · ···· · ··· · · · · ·

The results are shown in Figure 8. In addition, the UV absorption values of the protein at 280 nm and the enzyme activity were correlated together. The protein absorption curve in the UV region of the spectrum showed maximum absorption at 278 nm and no absorption in the visible light region. Based on these observations, the protein can be considered sufficiently pure to investigate its properties. ?

Example 7

Isoelectric focusing i

Isoelectric focusing was performed using the Phast system j (Farmacia-Biotec Co.). IEF3-9 gel was used and the sample was applied to the gel at a location 3 cm from the anode. Electrophoresis was performed under the following conditions: 2000 V, 2.5 mA, 15 ^e C, 410 Vh. The gel plate was fixed in 20% TCA at 20 ° C for 5 minutes and then washed with rinse and destaining solution (30% methanol and 10% acetic acid) for 2 minutes. The plate was finally rinsed and stained with a solution containing 0.02% Coomassie Brilliant Blue R250 (50 ° C, 10 minutes). The Broad Pi Calibration kit (Farmacia-Biotec Co.) was used as the pi marker.

The results and the isoelectric focusing curve are shown in Figures 9 and 10.

The enzyme was stained as a single band with isoelectric point at pH 4.5.

Example 8

Influence of pH on enzyme reaction

Azokassein was cleaved by the enzyme at various pH values.

Buffer solutions in the reaction mixture had a concentration of 67 mM and contained acetate buffer (pH 4-5.5), KH 2 PO ₄ -Na 2 PO 4 (pH 6.0-8.0), Na ₂ B ₄ O _in -HCl (pH 8.0-9.0), Na 2 B 4 -NaOH (pH

9.5-10.0) and Na 2 HPO 4 -NaOH (pH 10.5-12.0).

The results are shown in Figure 11.

The relative enzyme activity at pH 7.5 as optimal pH was maintained at 80% in the pH range of 6.0 to 10.0. Thus, the enzyme functions over a relatively wide range around neutral pH. However, the enzyme did not function satisfactorily at pH 5.5 and below or at pH 10.0 and above, and was inactivated at pH 12 and lost protease activity.

Example 9

PH-dependent protease stability

The enzyme was assayed in various pH buffers using Econo-Pac (Biorad Co.). Buffer solutions were 67 mM and contained acetate buffer (pH 4-5), KH 2 PO 4 -Na 4 HPO 4 (pH 6-8), glycine-NaOH (pH 9-10) and Na 4 HPO 4 -NaOH ( pH 11-12). After buffer exchange, the enzyme was incubated at 20 ° C for 1 hour to evaluate residual protease activity.

The results are shown in Figure 12.

The enzyme was found to be stable between pH 6.0 and 10.0 at 20 ° C for 1 hour but was inactivated at pH 4.0 and 12.0 under the same conditions.

• · ·· ····

9 • 9 9 9 • 99 9 9 9

Example 10

Influence of temperature on enzyme reaction

Azokassein was cleaved by the enzyme at various pHs in the temperature range of 0 to 70 ° C.

A similar reaction was performed with commercially available enzymes such as Subtilisin Carlsberg, V8 protease (isolated from Staphylococcus aurcub V8), Sabinase (Novonordisc), and Alkalasa (Novonordisc).

The results, assuming that the substrate was present in excess and all the enzyme was present in the form of enzyme-substrate complex, are shown in Figure 13, where 0 represents our enzyme, V8 protease, O Subtilisin Carlsberg, A Sabinase and V Alkalas.

The enzyme has been found to have a temperature optimum at 40 ° C and is rapidly inactivated at a reaction temperature above its optimum temperature. It was also found that all of the commercially available proteases have optimum temperature of 50 ° C or higher, and that the value of K _<; us-presented _at the enzyme is in the range 10 to 40 ° C higher than any of the comparable proteases.

Example 11

Thermal stability of the protease

The enzyme was incubated at temperatures from 20 to 50 ° C. Changes in activity versus incubation time are shown in Figure 14, where Q represents changes at 20 ° C, φ at 30 ° C, 0 at 40 ° C, and Z \ at 50 ° C.

The enzyme was not practically inactivated at 20 or 30 ° C, but «ΰ« β »», »».

After one hour of incubation at 40 ° C, its activity gradually decreased to 60% of the activity observed at 20 or 30 ° C, and at 50 ° C. C, the enzyme was completely inactivated within 15 minutes.

The enzyme can be considered thermally unstable because the above temperatures are lower than the optimal protease temperatures used in the previous experiment for comparison.

Example 12

Effect of inhibitors

Phenylmethylsulfonyl fluoride (PMSF) acting on serine proteases, iodoacetamide (IAA) acting on cysteine proteases, ethylenediaminotetraacetic acid (EDTA) acting on metalloproteases, o-phenanthroline, 2,2'-bipyridyl, and citrate and oxalate were used as inhibitors Ca ² * ions. After adding inhibitors at various concentrations to the enzyme reaction system, this system was incubated at 20 ° C for 1 hour to determine residual protease activity. The results are shown in the following table.

Table 5

Effect of inhibitors

Inhibitor Concentration Residual Activity (%) No inhibitor 10 mM 100 ALIGN! PMSF 10 mM 99 Iodoacetamide 10 mM 94 EDTA 10 mM 8 o-Phenantrolin 10 mM 91 2,2'-Bipyridyl 10 mM 39 Citrate 10 mM 100 mM 41 0 Oxalate 10 mM 100 mM 45 0

• · · · · · ···· ♦

The protease activity of the enzyme was not inhibited by PMSF or IAA, but significantly inhibited by EDTA, 2,2'-bipyridyl, citrate and oxalate. Thus, it was found that the protease activity is dependent on the presence of metal ions and thus that the enzyme studied is a metalloprotease. Inhibition by citrate and oxalate suggests that the protease activity is calcium dependent.

Example 13

Influence of protein denaturants

Sodium dodecyl sulfate (SDS) and urea were used as protein denaturants. After the addition of various concentrations of protein tanning agents, the enzyme system was incubated at 20 ° C for 1 hour to determine residual 1 protease activity.

The results obtained with SDS and urea are shown in Figures 15 and 16.

Protease was inhibited even at low SDS concentrations and complete inactivation was achieved using 0.25% SDS. Protease was not inhibited by urea up to concentration

M, using 3 M urea, the residual activity was 60% and 4 M urea completely inhibited the protease activity.

Example 14

Influence of metal ions

AgNO ₃ , CuSO ₄ , ZnSO ₄ , CoSO ₄ , FeSO ₄ , MnSO ₄ , CaCl ₄ and MgSO 4 were used as a source of metal ions. After addition of salt

9999

9 9 «· · 999999 8

9 9 9

DD · 9999 9 9

9 9 9 9 9

The enzyme system was incubated at 20 ° C for 1 hour and the residual enzyme activity was determined.

The results are shown in the following table

Table 6

Influence of metal ions

Metal ion Residual Activity (%) No metal ion 100 ALIGN! Ag * 11 Cu ^2M 44 Zn ²⁺ 61 Co ² * 63 Fe ²⁺ 76 Mn ² 95 Ca ² ' 100 ALIGN! Mg ² * 100 ALIGN!

An enzyme was significantly inhibited by Ag-ions, Cu ^2+, Zn ^2+, Co ² *, and Fe ^2+. With Ag 1 inhibition, the residual activity was only 10%. The enzyme was not inhibited by Mg ²⁺ ions and Ca * ² '.

Example 15

Substrate specificity

Casein (Hammarsten), dimethyl casein, gelatin, hemoglobin, bovine serum albumin and ribonuclease were used as substrate proteins to measure proteolytic activity by the modified Anson method. Azokassein and azoalbumin were used as azoprotein-modifying proteins.

The results are shown in the following table.

································ 9 9 9 9 9

9999 9 999 99 99

Table 7

Substrate specificity

Substrate (1%) Hydrolysis rate (%) Casein (Hammarsten) 100 ALIGN! Dimethylkasein 93 Gelatine 53 Hemoglobin 18 Beef serumalbumin 17 Ribonukleasa 0 Azokasein 100 ALIGN! Azoalbumin 20 May

The enzyme of the invention degrades well

high molecular weight proteins and denatured proteins such as three

for example casein and dimethyl casein and also decomposes gelatin j. . . as a denatured collagen protein with 50% efficiency compared to casein. The enzyme practically does not decompose other natural proteins and in particular not ribonuclease. 1

Example 16

Kinetics of enzyme reaction with casein

The Lineweaver and Burk plots were obtained at various temperatures from 5 to 40 ° C with a solution containing 0.05 to% casein as enzyme substrate. The plots are shown in Fig. 17, where the top graph (A) shows a change in the value of 1 / v in the range of 0 to 2.0 and the bottom graph (B) shows a change in the value of 1 / v in the range of up to 0.2. In Figure Q, the plot is at 5 C, φ at ° C, O at 20 ° C, at 30 ° C and at 40 ° C.

The kinetic constant of the enzyme reaction was obtained from Lineweaver and Burk plots as shown in the table below.

9 · 9

Table 8

Substrate specificity

Substrate (1%) Hydrolysis rate (%) Kasein (Hammarsten) 100 ALIGN! Dimethylkasein 93 Gelatine 53 Hemoglobin 18 Beef serumalbumin 17 Ribonukleasa 0 Azokasein 100 ALIGN! Azoalbumin 20 May

As shown in the table, the enzyme exhibits a Michaelis-Menten-type reaction rate for various casein concentrations. The value of K decreased and the value of V increased with increasing m max with temperature. Enzyme activity was inhibited at high casein concentrations at 5 and 10 ° C. In general, the enzyme tended to be inhibited at excess substrate concentration. However, the enzyme was not inhibited at a casein concentration of 1% at an elevated reaction temperature. We believe that the reason for this behavior was an increase in value as the temperature approached the optimum value and there was little inhibition of the casein degradation products. Based on the plots by Lineweaver and Burk, we also believe that thermal inhibition is a mixed type. That is, the effect of temperature is considered a non-competitive or acompetitive inhibition and the effect of casein decomposition products as a competitive inhibition.

Industrial applicability

The psychrophilic protease of the invention has an optimal temperature in the low temperature range. Thus, with the psychrophilic protease of the invention, proteins can be cleaved at low temperature. For example, a detergent that can also be used in water at low temperature is prepared by adding the protease of the invention to the detergent composition. Except for the inclusion of the psychrophilic protease of the invention, the detergent composition may be prepared by conventional methods. Briefly, it can be prepared by combining the protease of the invention with a conventional detergent component, such as a detergent surfactant, bleach or filler.

The enzymatic reaction of the psychrophilic protease of the invention takes place at low temperature. Thus, even if the reaction system contains an organic solvent that is volatile at room temperature, the reaction may proceed at a low temperature where the organic solvent does not evaporate. Moreover, when we intend to improve the quality of the foodstuffs using the protease of the present invention, it is preferable to use this protease because the reaction takes place at a low temperature at which the foodstuffs are effectively protected from decomposition.

Claims (10)

1. A psychrophilic protease having the following physicochemical properties: Specific activity and substrate specificity: the enzyme breaks down casein and dimethyl casein but does not break down ribonuclease; - Optimum temperature: the enzyme has an optimum effect temperature of 40 ° C; and - Temperature stability: under storage conditions at pH 7 for 1 hour, the enzyme is not practically inactivated at temperatures up to 30 ° C but loses approximately 40% of its activity at 40 ° C, and is rapidly inactivated at 50 ° C so that the activity disappears completely after 15 minutes. The psychrophilic protease according to claim 1, having the following physicochemical properties: - Optimal pH: works optimally at pH 7.5; and - pH-dependent stability: the enzyme is stable at a pH of between 6.0 and 10.0 under storage conditions at 20 ° C for 1 hour. The psychrophilic protease of claim 1, wherein the protease has a molecular weight of 38 kDa as measured by polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate and by gel filtration method. The psychrophilic protease of claim 1, wherein the protease has: 9 9 9 9 9 9 9 98 9 9 9 9 999 9 9 • ··· 9 9 9 9 9 9 99 99 9999 9 99 9 99 99 9 isoelectric point 4.5 as measured by isoelectric focusing. An isolated microorganism belonging to the species Flavobacterium balustinum which is capable of producing a protease according to claims 1 to 4. The isolated microorganism belonging to the species Flavobacterium balustinum according to claim 5, wherein the microorganism grows preferably at temperatures ranging from 10 to 20 ° C. AND The isolated microorganism belonging to the species Flavobacterium j balustinum according to claim 5, which is a strain of Flavobacterium balustinum P104 (FERM BP-5006). 8. A process for the preparation of a psychrophilic protease comprising: culturing a microorganism Flavobacterium balustinum according to claim 5, and isolating the psychrophilic protease according to claim 1 from a microbial culture. 9. A method of preparing a psychrophilic protease comprising: culturing a microorganism Flavobacterium balustinum according to claim 6, and isolating the psychrophilic protease according to claim 1 from a microbial culture. · E e e e e e e e e e e e e e e e e e e e e e e e e e · 10. A method of preparing a psychrophilic protease comprising: culturing a Flavobacterium balustinum microorganism of claim 7, and isolating the psychrophilic protease of claim 1 from a microbial culture.