Title of Invention

AN ISOLATED OR RECOMBINANT PROTEASE AND A METHOD FOR PURIFYING THE SAME AND ISOLATED POLYNUCLEOTIDE

Abstract The present invention relates to a protease, and more specifically to a protease derived from Aranicola proteolytics, a gene coding for said enzyme, a gene expression system for said protease, a process for purifying the protease, and the uses of said protease in industrial applications, such as for example, detergents, cosmetics, leather processing agents, chemicals for laboratory research, solubilizing or softening agents for food, meat modifier, feed or food additives, or oil and fat separating agents, as well as pharmaceutical compositions.
Full Text Technical Field
The present invention relates to a protease, and more specifically to a protease derived
from Aranicola proteolyticus, a gene coding for said enzyme, a gene expression
system comprising said gene, a process for purifying the protease, and the use of the
protease in industrial applications.
Background Art
Protease is an enzyme which catalyzes hydrolysis of peptide bond in proteins or
peptides, exists in all organisms and plays a variety of physiological roles. Most of
proteases from microorganisms are secreted to the extracellular environment and their
activities are inhibited or activated by carbon sources or nitrogen sources. In addition,
most of microbial proteases have their origin to pathogenic microbes to animals or
plants, or the proteases have a pathogenic property.
Microbial proteases are classified according to such characteristics as temperature,
optimal pH, and the residues at the active site and have different industrial applications
accordingly. For example, proteases are classified into thermostable, mesophilic or
thermophilic based on temperature; into acidic, weak acidic, neutral or basic based on
optimal pH; and into serine protease, cysteine protease, aspartate protease or
metalloprotease based on the residues at the active site.
The enzymatic activity of proteases is regulated by metallic cations such as Ca2+, Zn2+,
Mg2+, Mn2+ found at the active site of the enzymes. Most proteases are zinc-containing
proteins in which zinc is essential for activity. The representative example for the
protease is serrapeptase produced by Serratia marcescens(ATCC 21074) isolated from
the intestine of Bombyx mori and this enzyme can be used as an anti-inflammatory
agent because it has a fibrin degrading ability and a hydrolysis activity for bradykinin
and histamine which are inflammatory peptides. Bacterial proteases which have been
cloned and characterized hitherto include those derived from Vibrio proteolyticus (See,
David, V. A., A. H. Deutch, A. Sloma, D. Pawlyk, A. Ally, and D. R. Durham. Gene.
112:107-112. 1992), Erwinia chysanthemi B374 prtA (See, Ghigo, J. M., and C.
Wandersman. Mol. Gene. Genet. 236:135-144. 1992), Psudomonas aeruginisa LasB
(See, Doung, F., A. Lazdunsk, B. Cami, and Murgier. Gene. 121:47-54. 1992),
Serratia marcescens PrtSM (See, Braunagel, S. C, and M. J. Benedik. Mol. Gen.
Genet. 222:446-451 1990), Bacillus thuringiensis (See, Lovgren, A., M. Zhang, A.
Engstrom, G. Dalhammar, and R. Landen. Mol. Microbiol. 4:2137-3146. 1990), etc.
Disclosure of Invention
Hitherto, the present inventors have succeeded in isolating a novel microorganism
having an ability to degrade proteins from the intestine of Nephilia clavata and named
it as Aranicola proteolyticus HY-3(KCTC accession number: 0268BP). . Specifically,
the present inventors have isolated and identified a protease-producing strain of
Aranicola proteolyticus from the intestine of Nephila clavata [Korean Patent
Publication No. 10-220091 dated June 18, 1999] and succeeded in purifying the
protease which is stable at temperature between 20 to 40 °C and a pH range between 6
to 10 and having an apparent molecular weight of 51.5 kD. However, the publication
was focused on the identification and characterization of the microbe in morphological
and taxonomic aspects. The present inventors have continued the research on the
microorganism and the protease, and as a result, found that the protein is inhibited by
metalloprotease inhibitors, and shows a quite increased protein degrading activity
either at low temperatures , a broad range of pH or under a high salt concentration,
and that it can effectively be obtained by the use of a genetically modified expression
system or a purification process fitted to the specific microorganism. The inventors
have also found that the protease has a broad range of industrial applications, such as
for example, detergents, cosmetics, leather processing agents, chemicals for laboratory
research, solubilizing or softening agents for food, meat modifier, feed or food
additives, or oil and fat separating agents, as well as pharmaceutical compositions
which can be used as a digestive enzyme for improving alimentary diseases, digestive
ailments or abnormal conditions after operation of a digestive organ, thromobolytic
agents which lyse fibrin by directly acting onto thrombus and an anti-inflammatory
enzyme for eliminating inflammatory materials or necrosis tissues which serves as an
in vivo protective system or as an anti-inflammatory agent for alleviating edema after
surgery and trauma, and completed the present invention.
Therefore, an object of the present invention is to provide a novel gene coding for the
protease comprising the sequence described in SEQ ID No. 2 and a mutant or a variant
thereof which can produce said protease or its functional equivalents
Another object of the present invention is to provide a novel expression system which
comprises the above gene, a mutant or a variant thereof which codes for the above
protease or its functional equivalents, a constitutive promoter or a regulative promoter,
as a selection marker a nutrient deficient gene such as URA3 (orotidine-5'-phosphate
decarboxylase) or an antibiotic resistant gene such as Ap(Ampicilin) resistant gene,
and a transcriptional terminator, and a microorganism harboring the expression
system.
Further object of the present invention is to provide a protease having aforementioned
characteristics and comprising the amino acidic sequence as set forth in SEQ ED NO.
1.
Still further aspect of the present invention is to provide a process for purifying said
protease which comprises
a) cultivating Aranicola proteolyticus in a culture media;
b) filtering the culture media to give a supernatant; and
c) purifying the protease contained in the supernatant with a resin.
Still further aspect of the present invention is to provide the uses of the protease and
the gene therefor in industrial applications, such as for example, detergents, cosmetics,
leather processing agents, chemicals for laboratory research, solubilizing or softening
agents for food, meat modifier, feed or food additives, or oil and fat separating agents.
In addition, the present invention provides pharmaceutical compositions which can be
used as a digestive enzyme for improving alimentary diseases, digestive ailments or
abnormal conditions after operation of a digestive organ, thromobolytic agents which
lyse fibrin by directly acting onto thrombus and an anti-inflammatory enzyme for
eliminating inflammatory materials or necrosis tissues which serves as an in vivo
protective system or as an anti-inflammatory agent for alleviating edema after surgery
and trauma, and which comprises as an active agent the protease, enzyme variant or
mutant enzyme thereof and a pharmaceutically acceptable carrier.
Further objects and advantages of the invention will become apparent through reading
the remainder of the specification.
Brief Description Of Drawings
The present invention is further illustrated by reference to the accompanying drawings,
in which;
Fig. 1 represents inhibitory effects of the protease derived from Aranicola
proteolyticus by various inhibitors. Protease was prepared according to the procedure
described in Example 1, using azokazein as a substrate;
Fig. 2 represents heat stability of the protease from Aranicola proteolyticus. Protease
was prepared according to the procedure described in Example 1, using azokazein as a
substrate;
Fig. 3 represents activity and stability of the protease at different pH. Protease was
prepared according to the procedure described in Example 1, using azokazein as a
substrate; and
Fig. 4 represents a DNA sequence coding for the protease of the invention and the
corresponding amino acid sequence of the protein.
Best Mode For Carrying Out The Invention
Hereinafter, the invention will be illustrated in more detail.
The present invention, in an aspect, provides a novel gene coding for the protease
comprising the sequence described in SEQ ID NO. 2 and a mutant or a variant thereof
which can produce said protease and its functional equivalents.
The gene of the invention has a nucleotide sequence as set forth in SEQ ID NO. 2 of
the attached sequence listing and its size is 2.48 kb. The gene has an ORF(open
reading frame) consisting of 1,461 base pairs, a -35 region (TGTGCA) and a -10
region (TATAAT) with a space of 16 base pairs in the upstream, and Shine-Dalgarno
(SD) sequence known as a ribosome binding site before the initiation codon. TAA is
used as a stop codon and a palindromic sequence suspected as the transcription
termination site appears in the downstream. This genesis isolated from Aranicola
proteolyticus HY-3.
The term used herein, "a mutant or a variant thereof is meant by a mutated gene in
which a single base or two or more bases have been changed by a mutation such as, for
example, substitution, deletion, addition or insertion, but still can produce the same
protease or its functional equivalents. Therefore, those skilled in the art would easily
appreciate that the mutant or variant can be encompassed within the scope of the
present invention. Generally, a mutant or variant having about 80% or more of
homology in the case of the protease and the gene of the invention, and preferably
90% or more of homology can be encompassed in the contexts of the invention, a
mutant or a variant thereof.
The present invention, in an another aspect, provides a novel expression vector which
comprises a gene coding for the above protease, a mutant or a variant thereof which
codes for the protease or its functional equivalents, a constitutive promoter or a
regulative promoter, as a selection marker, a nutrient deficient gene such as URA3
(orotidine-5'-phosphate decarboxylase) or an antibiotics resistant gene, such as
Ap(Ampicilin) resistant gene, and a transcriptional terrninator and a microorganism
transformed with the expression vector.
The vector system containing the gene coding for the protease and the transformant
can be prepared according to the conventional recombinant DNA technology known to
those skilled in the art, in which, for example, a DNA fragment containing the
sequence coding for the aforementioned protease is isolated from the wild type strain
such as Aranicola proteolyticus and is cloned into a suitable regulatory elements
together with a suitable expression signal, and the resulting vehicle is then introduced
into an autonomously replicating plasmid or into a chromosome of bacteria.
A series of such steps constituting the conventional recombinant processes have
essentially been known in the art, and for example one can easily accomplish the
purpose by employing those processes taught by Maniatis et al., Molecular Cloning: A
Laboratory Manual 8.11-8.13 (The 2nd ed., Cold Spring Harbor Laboratory Press
(1989)].
The present invention further provides a protease having the aforementioned
characteristics and comprising the amio acid sequence as set forth in SEQ ID NO. 1, or
an enzyme variant or a mutant enzyme thereof.
The tenn used herein, "an enzyme variant or a mutant enzyme" is meant by the
functionally same enzyme as the protease in which a single amino acid or a plurality of
the amino acids have been modified or changed due to the changes in DNA nucleotide
sequence(s) of the parental gene or the derivatives thereof. Though preferable protease
according to the invention can be derived from Aranicola proteolyticus, for example,
Aranicola proteolyticus HY3-1, the functionally equivalent protease can be obtained
from a form of a variant or a mutant thereof using a vector system and a suitable host
microorganism which may essentially not be the same organism from which the
parental gene has been derived.
The present inventors isolated and purified the protease produced from Aranicola
proteolyticus, analyzed enzymological characteristics thereof, and carried out gene
cloning and sequencing therefor. As a result, the amino acid sequence of the protease
as set forth in SEQ ID NO: 1 showed 92.6% similarity to the protease derived from
Serratia marcescens SM6. In addition, amino acid sequence at the Zn2+ binding site
and the active site which exist in most metalloproteases was found to be well
conserved. The relative activity of the protease became lowered in the presence of
equal concentrations of metalloprotease inhibitors, for example EDTA and
phenanthroline (See, Fig. 1). These observations support that the protease is a zinc-
binding metalloprotease requiring zinc ion(Zn2+) for its activity. Besides, according to
the test in which its relative activity was measured at different temperatures ranging
from 4 °C to 80 °C, the maximum activity was observed at 37°C and at least 75% of
relative activity was observed at temperatures between 20 °C to 40 °C (See Fig. 2).
Relative activity measured for a substrate, azokazein at different pH showed the
maximum activity at pH 8.0; whereas at least 80% of the maximum acitivity at pH 7.0
to 9.5(See Fig. 3). SDS-PAGE(Sodium dodecyl sulfate-poly acrylamide gel)
electrophoresis of the enzyme showed that the protease band had an apparent
molecular weight of about 51.5 kDa.
Therefore, the protease according to the present invention has an apparent molecular
weight of 51.5 kDa and the optimal temperature for activity at 20°C to 37°C (at pH 7.6)
and an optimal pH at 7.0 to 9.5 (at 37°C). The protease retains high activity under high
salt concentrations and inhibited by metalloprotease inhibitors such as EDTA and
phenanthroline.
The present invention further provides a process for purifying the protease, which
comprises
a) cultivating Aranicola proteolyticus in a culture media;
b) filtering the culture media to give a supernatant; and
c) purifying the protease contained in the supernatant with a resin.
In one embodiment of the present invention, the protease can be produced by
cultivating Aranicola proteolyticus in a suitable nutrient medium containing a nitrogen
source and inorganic salts followed by recovering the protease, or by using the
conventional recombinant DNA technologies.
Aranicola proteolyticus HY-3 is a mobile aerobic Gram-negative bacterium. It is
globular in shape with 0.5 to 0.S mm in diameter. Its growth response was positive in
the presence of catalase and negative in the presence of oxidase.
The microorganism can be cultured in a medium containing assimilable carbon
sources and nitrogen sources with other essential nutrients. The culture medium can
be prepared in a conventional manner. The extracellular protease produced in the
fermentation broth can be recovered and purified by the conventional method known
to the art.
In order to isolate the protease of the invention, Aranicola proteolyticus is first
cultured as aforementioned. The bacterial pellet and the supernatant are separated and
the supernatant is then concentrated. The concentrated solution is purified using resins.
In a preferred embodiment, the purification of the concentrated solution is carried out
by an ion exchange resin using DEAE-cellulose and a gel filtration exchange resin
using Sephadex G-75 which is pretreated with Tris-HCl buffer. Finally, a
preservative is optionally added to the purified protease.
In another embodiment of the present invention, the protease can be produced by the
conventional recombinant DNA technology in which, for example, a DNA fragment
containing the sequence coding for aforementioned protease is isolated from the wild
type strain such as Aranicola proteolyticus and is cloned into a suitable regulatory
elements together with a suitable expression signal, and the resulting vehicle is then
introduced into an autonomously replicating plasmid or into a chromosome of bacteria.
The bacteria is then cultured in a medium optimized to express the protease and the
protease is recovered from the medium by aforementioned method. Preferable host
organism, but not limited thereto, includes, for example, E. coil, Bacillus, Aspergillus,
Streptomyces or Saccharomyces.
The present invention, in a still further aspect, provides the uses of the protease in
industrial applications, such as for example, detergents, cosmetics, leather processing
agents, chemicals for laboratory research, solubilizing or softening agents for food,
meat modifier, feed or food additives, or oil and fat separating agents, as well as
pharmaceutical compositions which can be used as a digestive enzyme for improving
alimentary diseases, digestive ailments or abnormal conditions after operation of a
digestive organ, thromobolytic agents which lyse fibrin by directly acting onto
thrombus and an anti-inflammatory enzyme for eliminating inflammatory materials or
necrosis tissues which serves as an in vivo protective system or as an anti-
inflammatory agent for alleviating edema after surgery and trauma.
Proteolytic property of tThe protease of the invention can be used in detergent
industry. In a preferred embodiment of the protease utility, the present invention
provides a detergent composition comprising the protease according to the invention,
other enzymatic ingredients and additives.
The enzymatic ingredients known to the art, but not limited thereto, include one or
more other enzymes selected from the group consisting of amylases, lipases,
cellulases, oxidases, peroxidases and/or the mixture thereof.
The detergent composition of the invention may comprise one or more surfactants,
which may be of an anionic, non-ionic, cat-ionic, amphoteric or zwitterionic type, or a
mixture of these. Typical examples of anionic surfactants include linear alkyl benzene
sulfonates (LAS), allcyl sulfates (AS), alpha olefin sulfonates (AOS), alcohol ethoxy
sulfates (AES) and alkali metal salts of natural fatty acids. Examples of non-ionic
surfactants include alkyl polyethylene glycol ethers, nonylphenol polyethylene glycol
ethers, fatty acids esters of sucrose and glucose, and esters of polyethoxylated allcyl
glucoside.
The detergent composition of the invention may also contain other detergent
ingredients known in the art such as builders, bleaching agents, bleach activators, anti-
corrosion agents, sequestering agents, anti soil-redeposition agents, perfumes,
stabilizers for the enzymes and bleaching agents, formulation aids, optical brighteners,
foam boosters, chelating agents, fillers, and fabric softeners.
The detergent compositions of the invention can be formulated in any convenient
form, such as powders, liquids, etc.
The protease of the invention may be included in a detergent composition by adding
separate additives containing the detergent protease, or by adding a combined additive
comprising different detergent enzymes.
The additive of the invention can be formulated as e.g. granules, liquids, slurries, etc.
Preferred detergent additive formulations are non-dusting granules, liquids, in
particular stabilized liquids, slurries, or protected enzymes. Dust free granulates may
be produced e.g. according to GB Patent Publication No. 1,362,365 or U.S. Pat. No.
4,106,991, and may optionally be coated by methods known in the art. The detergent
enzymes may be mixed before or after granulation. Liquid enzyme preparations may,
for instance, be stabilized by adding a polyol such as e.g. propylene glycol, a sugar or
sugar alcohol, lactic acid or boric acid, according to established methods. Other
enzyme stabilizers are well known in the art. Protected enzymes may be prepared
according to the method disclosed in EP Patent Publication No. 238,216. In one useful
embodiment the protease of the invention may be incorporated in detergent
formulations according to e.g. EP Patent Publication Nos. 342,177; 368,575; 378,261;
and 378,262.
In another preferred embodiment, the present invention provides a pharmaceutical
composition which comprises as an active agent the protease of the invention, enzyme
variant or mutant enzyme thereof and a pharmaceutically acceptable carrier.
The pharmaceutical composition of the invention can be used as a digestive enzyme
for improving alimentary diseases, digestive troubles, or abnormal conditions after
operation of a digestive organ, thromobolytic agents which lyse fibrin by directly
acting onto thrombus and an anti-inflammatory enzyme for eliminating inflammatory
materials or necrosis tissues which serves as an in vivo protective system when foreign
toxic agents attack, or as an anti-inflammatory agent for alleviating edema after
surgery and trauma, those having ordinary skill in the art can readily identify
individuals who are suspected of suffering from such diseases, conditions and
disorders using standard diagnostic techniques.
Pharmaceutical compositions according to the invention comprise a pharmaceutically
acceptable carrier in combination with the protease of the invention. Pharmaceutical
formulations are well known and pharmaceutical compositions comprising the
protease may be routinely formulated by one having ordinary skill in the art. The
mode of administration of the protease may determine the sites in the organism to
which the protease will be delivered. For parenteral administration, i.e., intravenous,
subcutaneous, intramuscular. Intravenous administration is the preferred route. They
are best used in the form of sterile aqueous solution which may contain other solutes,
for example, sufficient salts, glucose or dextrose to make the solution isotonic. For oral
mode of administration, the protease of the present invention may be used in the form
of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and
suspension, and the like. Various disintegrants such as starch, and lubricating agents
may be used. For oral administration in capsule form, useful diluents are lactose and
high molecular weight polyethylene glycols. When aqueous suspensions are required
for oral use, certain sweetening and/or flavoring agents may be added. Other suitable
pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A.
Osol, a standard reference text in this field, which is incorporated herein by reference.
The concentration of the active ingredient, protease is about 10D~100 ?/? body
weight per day. However, those skilled in the art would appreciate that the dosage
varies depending upon known factors such as the pharmacodynamic characteristics of
the particular agent, and its mode and route of administration; age, health, and weight
of the recipient; nature and extent of symptoms, kind of concurrent treatment,
frequency of treatment, and the effect desired. See Gennaro, Alfonso, ed., Remington's
Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Co., Easton Pa.
The present invention, in a further preferred embodiment, provides a cosmetic
composition which comprises the protease of the invention and/or acid buffer, acid
protease or inorganic acid together with a cosmetically acceptable carrier, vehicle or
excipient.
The cosmetically acceptable carrier, vehicle or excipient component of the acidic
buffer is selected from the group consisting of lotions, tinctures, creams, emulsions,
gels, ointments, water, water-workable cream, polyvinyl alcohol, hydroxyethyl
cellulose, cellulose, hydrophilic acrylic polymer, emollients, skin moisturizing
components, enzyme stabilizers, glycerol, surfactants, preservatives, hydrophilic
thickening agents used in pharmaceutical formulations and mixtures thereof. Acid
protease is selected from the group consisting of fungal proteases, bacterial proteases
or mammalian proteases or mixtures thereof or selected from the group consisting of
pepsin, cathepsin, human urinary acid protease, rhizopuspepsin, penicillopepsin,
endothiapepsin or mixtures thereof. The representative examples of the inorganic acid
is selected from the group consisting of phosphoric acid, pyrophosphoric acid,
triphosphoric acid, polyphosphoric acid, sodium bisulfate, potassium bisulfate or
mixtures thereof.
Further, the protease of the invention, in another preferred embodiment, can be used as
leather processing agents. Therefore, it is possible to provide an acqueous enzyme
preparation, or a composition which is particularly suitable for the leather industry
which handles anhydrous organic solutions.
The use of protease accounts for a large part of the leather manufacturing process.
Hereinbleow, the use of the protease of the invention in connection with the leather
processing industry will be explained in more detail.
In order to manufacture leather products, a raw materal is subjected to a soaking
process, a liming process, a deliming process, a bating process, a pickling process, a
tanning process, a dyeing process, a drying process and a finishing process. Among
these processes, protease is used in the deliming process and bating process. The
deliming process and the bating process comprise steps of neutralization and acid
treatment, the condition of which being adjusted so that collagen protein in the leather
skin leave undestructed. In other words, directly subjecting dehaired skin to the
pickling process and the tanning process, skipping the deliming process and the bating
process, may cause fatal destruction or denaturation of proteins constituting the leather
skin.
The deliming process removes components in the leather skin that are unnecessary for
leather processing by protease digestion. The bating process provides softness and
elasticity in the skin by enzyme treatment, supplementing the deliming process which
is insufficient to relax fiber tissue in the leather skin. It is this deliming process that
the action of protease is considered most important.
The protease digests and elutes epidermal cells, hair roots, glandulae sudoriferae,
calcium soaps, emulsified fats which left in the dehaired skin after partial hysrolysis
with lime pickling, and it also clears and smooths the surface of dehaired skin. In
addition, it digests and elutes various unnecessary proteins and acts on elastin fiber or
reticulin fiber to modify their physicochemical properties to certain extent.
The uses of protease for laboratory research can be contemplated in various
embodiments depending on its specific purposes. For example, it can be used to
remove unnecessary proteins in DNA isolation and purification procedures. It can also
be used in research involving protein chemistry such as amino acid sequencing.
Examples
Hereinbelow, the invention will be explained in more detail by way of the following
examples. However, it is apparent that the scope of the invention shall not be limited
by the examples.
Example 1: Production of protease
In order to purity the protease of the invention, Aranicola proteolyticus HY-3 was
cultured in a culture medium (Bactotryptone 0.5%, Yeast extract 0.5%, NaCl 0.1%,
KC1 0.05%, CaCl2 0.02%, MgCl2 0.02%) at 22°C for 18 hours. A supernatant was
separated from the culture broth using 2 0 membrane filter, and men concentrated
using 10 kDa membrane filter. As the protease of the invention is basically an anionic
protein, the protease was purified by an ion exchange resin using DEAE-cellulose
pretreated with 50 mM Tris-HCl buffer (pH 7.6) and subsequently a gel filtration
exchange resin using SephadexG-75 pretreated with 20 mM Tris-HCl buffer (pH 7.6).
Subsequently, the thus purified enzyme solution was analyzed by 10% SDS-PAGE to
identify its band pattern. As a result, it was confirmed that the protease of the
invention was a monomer having an apparent molecular weight of about 51.5 kDa.
Example 2: Sequence determination of the gene of the protease of the inventin
Partial amino acid sequences of the protease obtained in the Example 1 were
sequenced by an amino acid sequencing analyzer (Precise Protein Sequencing System,
Applied Biosystems). Based on the amino acid sequence thus obtained as described
by SEQ ID NO: 3 (Ala Glu Gin Gin Gin Gin Ala) and SEQ ID NO: 4(Ile Gly His Ala
Leu Gly), PCR primers as described by SEQ ID NO: 5 (forward primer: gcggaacagc
agcagcaggc) and SEQ ID NO: 6(reverse primer: gcccaacgca tggccaat) were designed.
Using synthetic primers, PCR was performed in the presence of genomic DNA
purified from Aranicola proteolyticus HY-3 as a template. As a result, a DNA
fragment of 2.48 kb in length was obtained and the sequence of the PCR product was
then determined. On the basis of the thus determined sequence, PCR primers as
described by SEQ ID NO: 7 (ataatggccg ggacgatcct ggctgtagtt ac) and SEQ ID NO: 8
(cttacgcctt cctgccgaac accatttatc ag) were designed. Using synthetic primers, reverse
PCR was performed in the presence of genomic DNA purified from Aranicola
proteolyticus HY-3 as a template.
The gene of the invention has a nucleotide sequence as set forth in SEQ ID NO. 2 of
the attached sequence listing and its size is 2.48 kb. The gene has an ORF(open
reading frame) consisting of 1,461 base pairs, a -35 region (TGTGCA) and a -10
region (TATAAT) with a space of 16 base pairs in the upstream, and Shine-Dalgamo
(SD) sequence known as a ribosome binding site before the initiation codon. TAA is
used as a stop codon and a palindromic sequence suspected as the transcription
termination site appears in the downstream.
The amino acid sequence of the protease as set forth in SEQ ID NO: 1 showed 92.6%
similarity to the protease derived from Serratia marcescens SM6. In addition, amino
acid sequence at the Zn2+ binding site and the active site which exist in most
metalloproteases was found to be well conserved.
Example 3: Experiment on enzymatic characteristics of the novel protease
(1) The effect of protease inhibitors on the protease
Enzyme activity of the protease was measured according to the method described by
Braun & Shmitz (Braun, V. & Schmitz, G., Arch. Microbiol. 124, 55-61, 1980). A
substrate solution was prepared by dissolving 0.24 g of azocasein in 10 ml of 50 mM
phosphate buffer (pH 7.5). The 300 D of the substrate solution was mixed with 100 D
of bacterial culture solution and reacted at 37°C for 30 minutes. Then, 300 ? of 10%
trichloride acetate was added to the reaction solution and incubated at ambient
temperature for 1 hour. The reaction solution was centrifuged at 7,000 rpm until pellet
and supernatant was separated substantially. After adding 30 D of 10% sodium
hydroxide (NaOH) to the supernatant, the absorbance was measured at 420 nm. The
enzymatic titer was defined as 1 unit by the 1.0 fold increment of absorbance value.
In order to examine the effects of protease inhibitors on the activity of the protease, the
enzyme solution was added by 1 mM each of inhibitors enumerated below and
incubated at 37°C for 5 minutes before measuring relative enzyme activity. The
inhibitors tested include antopain which is an amine-peptidase inhibitor,
phosphoramidon which is a metalloendopeptidase inhibitor, pepstatin which is an
aspartate protease inhibitor, E-64(L-trans-exoxysuccinyl leucylamido(4-
fuanidino)butane) which is a cystein inhibitor, chymostatin which is a chymotrypsin
inhibitor, leupeptin, petabloc SC, aprotinin, and PMSF which are serine protease
inhibitors, phenanthroline and EDTA which are metalloprotease inhibitors. The result
showed that EDTA inhibited the enzyme activity by about 30% and phenanthroline by
about 70% (Fig. 1).
(2) The effects of salts on the protease
In order to examine effects of salts on the activity of protease, the relative enzyme
activity was measured at 37°C for 4 hours in the presence of various concentrations of
sodium chloride solution ranging from 0 to 1700 mM. The relative activity was
measured according to the method described in Example 3(1). For comparison, the
protease derived from Serratia marcescens SM6 was used as a control. The results
are shown in Table 1 below.
As shown in Table 1, the relative activity of the protease of the invention has increased
by about 1.3 to 2.0 folds in proportion to the salt concentrations.
(3) The effect of heat on the activity and stability of the protease
In order to examine the effect of heat on the activity and stability of the protease, the
relative enzyme activity was measured at temperatures ranging from 4°C to S0°C. The
measurement of the activity was performed according to the method described in
Example 3(1). The results are shown in Table 2.
As shown in Table 2, though the maximum relative activity was obtained at 37°C,
about a half of the maximum activity of the protease was maintained even as low as at
4°C to 15°C (See, Fig. 2). Moreover, 'Ea value' (hydrolysis of azocasein) which
represents a slope value indicating an activation energy in Arrenius plot to temperature
exhibited 2,432 Kcal/mole at temperatures between 4°C and 37°C. These demonstrates
that the enzyme has strong activity at low temperature.
(4) The effect of pH on the activity and stability of the protease
In order to examine the pH-dependence of the protease of the invention, the inventors
prepared various substrate solution with different pH. The each substrate solution was
buffered with citrate-phosphate(pH 3.0 to 7.0), sodium phosphate(pH 7.0 to 9.0),
Tris-HCl(pH 7.0 to 10.0), and glysine-caustic soda(pH 9.0 to 12.0), respectively. The
relative protease activity was measured after mixing enzyme with each of the above
substrate solution at 37°C for 30 minutes. The measurement of the relative protease
activity was followed by the method described in Example 3(1). The results are
shown in Table 3.
As illustrated in Table 3, the maximum relative enzyme activity was obtained at pH
8.0. At least 80% of maximum enzyme activity was observed at pHs ranging from 7.0
to 9.5, and even at pH of 5.0 to 12.0 at least 70% of the activity was obtained (See,
Fig. 3).
(5) The activity of the protease on various substrates
In order to examine the degrading ability of the enzyme against various substrates, the
enzyme reactions were performed on substrates such as albumin, casein, collagen,
elastin and hemoglobin. When the degrading ability of the enzyme on albumin in 24
hour reaction is defined as 100, all the other substrates except for hemoglobin showed
20~30 in 2 hour reaction. In 24 hour reaction, all the other substrates including
hemoglobin achieved at least 45. These results indicates that the protease of the
invention has a broad spectrum with respect to various substrates, suggesting the
utility of the enzyme in industry.
Example 5: Pharmaceutical preparations
Form of the preparation (tablet)
Protease 50 mg
Lactose 80 mg
Starch 17mg
Magnesium stearate 3 mg
Crystalline Cellulose 10 mg
One embodiment of the invention is the use of the protease as ingredient of the Tablets
whose exemplary composition is shown above. The tablets can be prepared by the
conventional method. One desirable embodiment is the tablets having the
conventional enteric coating (for example, hydroxypropylmethylcellulose pthalate),
sugar coating or film coating.
Example 6: Application of the protease in the leather industry
A 20 g of raw skin finely cut into pieces of suitable size and 14 ml of distilled water
was mixed in a 50 ml test tube. Then, 0.5% sodium bisulfite and 0.5% ammonium
sulfate on the basis of the weight of skin was added thereto and the mixture was
incubated for 15 minutes at 26°C. Subsequently, 0.2% of detergent and 0.5% of
deliming agent was added thereto and waited for 30 minutes and 1 hour, respectively,
before measuring the amount of protein eluted from the skin. In the presence of the
0.5% of protease, the protein eluted amounted to 1054 mg/ml and 1062 mg/ml after 30
minutes and 60 minutes, respectively. In the absence of the protease, the protein
eluted amounted to 304.5 mg/ml and 329.5 mg/ml after 30 minute and 60 minutes,
respectively. These results indicate the utility of the protease in the leather processing
industry.
Industrial Applicability
The protease of the invention has a stable enzymatic activity in a broad range of pH,
temperature under high salt concentration. Therefore, it has various industrial
applications, such as for example, detergents, cosmetics, leather processing agents,
chemicals for laboratory research, solubilizing or softening agents for food, meat
modifier, feed or food additives, or oil and fat separating agents, as well as
pharmaceutical compositions.
We claim:
1. An isolated or recombinant protease comprising an amino sequence as described
in SEQ ID NO: 1 and having at least 70% of activity at pH of 5 to 10.
2. The isolated or recombinant protease as claimed in claim 1, which is purified from
Aranicola proteolyticus.
3. The isolated or recombinant protease as claimed in claim 2, wherein the Aranicola
proteolyticus is Ararlicola proteolyticus HY-3 deposited under accession number of
KCTC 0268 BP.
4. The isolated or recombinant protease as claimed in claim 1, which is a
metalloprotease having metallic cation binding sites at active site.
5. An isolated polynucleotide encoding the protease as claimed in claim 1
6. The isolated polynucleotide as claimed in claim 5, which comprises a nucleotide
sequence as described in SEQ ID NO: 2.
7. An expression vector comprising the isolated polynucleotide as claimed in claim 2,
a constitutive promoter or a regulative promoter, at least one of selection marker
and a transcriptional terminator
8. A recombinant microorganism prepared by transforming a host microorganism with
the expression vector as claimed in claim 7
9. The recombinant microorganism as claimed in claim 8, wherein the host
microorganism is Aranicola proteolyticus.
10. The recombinant microorganism as claimed in claim 9, wherein the Aranicola
proteolyticus is Aranicola proteolyticus HY-3 deposited under accession number of
KCTC 0268 BP.
11. A process for purifying the isolated or recombinant protease as claimed in any one
of claims 1 to 4 which comprises:
a) culturing Aranicola proteolyticus in a culture media;
b) filtering the culture media to give a supernatant; and
c) purifying the protease from the supernatant with resin.
12. The process as claimed in claim 11, wherein the resin is ion exchange resin and/or
gel filtration exchange resin.
13. The process as claimed in claim 11, wherein the resin is DEAE-cellulose pretreated
with Tris-HCl buffer and the gel filtration resin is Sephadex G-75 pretreated with
Tris-HCl buffer.
14. A pharmaceutical composition comprising the isolated or recombinant protease as
claimed in any one of claims 1 to 4 as an effective ingredient and a
pharmaceutically acceptable carrier.
15. The pharmaceutical composition as claimed in claim 14, wherein the composition
is used as a digestive enzyme for improving alimentary diseases, digestive ailments
or abnormal conditions after operation of a digestive organ, thromobolytic agents
which lyse fibrin by directly acting onto thrombus and an anti-inflammatory
enzyme for eliminating inflammatory materials or necrosis tissues which serves as
an in vivo protective system or as an anti- inflammatory agent for alleviating edema
after surgery and trauma.
16. The pharmaceutical composition as claimed in claim 14, which is used for treating
digestive ailment.
17. A detergent composition comprising the isolated or recombinant protease as
claimed in any one of claims 1 to 4.
18. The detergent composition as claimed in claim 17, further comprising one or more
enzymes selected from the group consisting of amylases, lipases, celluloses,
oxidases and peroxidases.
19. The detergent composition as claimed in claims 17, which is in the form of a non-
dusting granulate, a liquid, in particular a stabilized liquid, a slurry, or a protected
enzyme.
20. A cosmetic composition comprising the isolated or recombinant protease as
claimed in any one of claims 1 to 4 as an effective ingredient and a cosmetically
acceptable carrier, vehicle or excipient.
21. The cosmetic composition as claimed in claim 20, further comprising one or more
enzymes selected from the group consisting of amylases, lipases, celluloses,
oxidases and peroxidases.
22. A leather processing agent comprising the isolated or recombinant protease as
claimed in any one of claims 1 to 4.
23. A food additive comprising the isolated or recombinant protease as claimed in any
one of claims 1 to 4.
24. A feed additive comprising the isolated or recombinant protease as claimed in any
one of claims 1 to 4.
25. A meat modifier comprising the isolated or recombinant protease as claimed in any
one of claims 1 to 4.
26. A oil and fat separating agent comprising the isolated or recombinant protease
as claimed in any one of claims 1 to 4.
27. A chemical reagent for laboratory research comprising the isolated or
recombinant protease as claimed in any one of claims 1 to 4.

The present invention relates to a protease, and more specifically to a protease derived from Aranicola proteolytics,
a gene coding for said enzyme, a gene expression system for said protease, a process for purifying the protease, and the uses of said
protease in industrial applications, such as for example, detergents, cosmetics, leather processing agents, chemicals for laboratory
research, solubilizing or softening agents for food, meat modifier, feed or food additives, or oil and fat separating agents, as well as
pharmaceutical compositions.

Documents:


Patent Number 235846
Indian Patent Application Number IN/PCT/2001/1006/KOL
PG Journal Number 36/2009
Publication Date 04-Sep-2009
Grant Date 02-Sep-2009
Date of Filing 27-Sep-2001
Name of Patentee KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY
Applicant Address 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI
Inventors:
# Inventor's Name Inventor's Address
1 PARK DO-SANG KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
2 KIM MI-GWANG KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
3 SHIN DONG-HA KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
4 PARK HO-YONG KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
5 SHIN SANG-WOON KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
6 OH HYUN-WOO KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
7 SON KWANG-HEE KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
8 PARK DO-SANG KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
9 KIM MI-GWANG KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
10 SHIN DONG-HA KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
11 PARK HO-YONG KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
12 SHIN SANG-WOON KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
13 OH HYUN-WOO KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
14 SON KWANG-HEE KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY, INSECT RESOURCES LABORATORY, 52 EOUN-DONG, YUSONG-KU, TAEJON-SHI, 305-333
PCT International Classification Number C12N 15/57
PCT International Application Number PCT/IB2000/02058
PCT International Filing date 2000-12-29
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2000/5479 2000-02-03 Republic of Korea