JP2007189906A - Peptidase and method of using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a required new method for decomposing or reducing volume in a good efficiency in the case that jelly fishes invade a water intake of a plant utilizing sea water such as a power generating plant, etc., and fishery facilities, and become a large amount of hardly treatable waste materials, and also a required method for exterminating the jelly fishes, enabling the prevention of the invasion by the jelly fish. <P>SOLUTION: This peptidase and the method for using the same aiming at the decomposition or reduction of volume of the jelly fishes becoming hardly treatable waste materials is provided by treating them with the peptidase classified to EC 3.4, preferably the peptidase produced by Bacillus sp. 97001 strain or its similar enzyme so as to make the final treatment of the jelly fishes becoming the hardly treatable waste materials easy. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a peptidase capable of decomposing jellyfish and relates to a method for producing the peptidase.
The present invention also relates to a jellyfish decomposition method and extermination method using the peptidase.
Furthermore, the present invention relates to a preparation for degrading jellyfish containing the peptidase and a jellyfish degrading apparatus using the peptidase.

Jellyfish is a general term for the free-swimming and rodent individuals of the Coleoptera (Cnidaria) genus Hydroworm and Bee jellyfish. There are hundreds of jellyfish in Japan alone, and it is the largest plankton of marine life.
Thermal and nuclear power plants located near the coast usually take seawater to cool the steam that rotates the turbine. If a large amount of mussels (mussels), barnacles, etc. adhere to the intake pipe, problems such as an increase in the load on the intake pump will occur. In addition, if a large amount of jellyfish flows in, it becomes difficult to take in condenser cooling water, and the power generation output must be restricted. A large amount of attached organisms such as mussels and barnacles appear and clog the intake.

  Jellyfish often strike a large amount near the intake of seawater-using plants such as thermal power and nuclear power plants, obstructing the intake of seawater-using plants. The disturbance of seawater intake caused by such a massive jellyfish invasion causes extremely serious damage such as a decrease in power generation output, performance degradation of equipment, water intake and power generation stoppage. In addition, jellyfish frequently invade fishing facilities and enter fishing nets such as stationary nets, causing damage such as suffocation, death, quality degradation, and damage to the fishing nets. Examples of jellyfish that cause such problems include moon jellyfish and jellyfish jellyfish. In order to prevent such damage, countermeasure technology has been studied and implemented for a long time, but it is not possible to completely prevent inflow, and jellyfish that have been landed as a result of mass attacks are too large to carry and handle. It is a problem. For example, incineration or landfilling as general waste is a disposal method for landed jellyfish, but problems such as foul odors occur during the incineration process. Disposal is becoming difficult.

There is a need for a new method for efficiently decomposing or reducing the volume of jellyfish attacking water intakes at seawater utilization plants such as power plants and fishing facilities, resulting in a large amount of difficult-to-treat waste. There is also a need for a jellyfish extermination method that can prevent jellyfish invasion.
Conventionally, microbial methods, enzyme methods, chemical methods, physical methods, and combinations of any of these methods (see Patent Documents 1 to 5) have been proposed as means for decomposing or reducing the volume of jellyfish. ing. However, these are not always satisfactory in view of speed, efficiency, initial cost, operating cost, and the like.

In addition, for the purpose of extracting useful substances from jellyfish, there is a method of using jellyfish's own endogenous alkaline protease or externally added alkaline protease to decompose jellyfish (see Patent Document 6). Endogenous alkaline protease lacks activity to efficiently decompose or reduce jellyfish, which is a difficult-to-treat waste of the present invention, in a short time. Furthermore, although pepsin is mentioned as an example of protease added from the outside, pepsin is an expensive enzyme currently taken from pigs and cattle, and jellyfish as difficult-to-treat waste of the present invention can be decomposed efficiently or in a short time. Not suitable as a catalyst for volume reduction.
As a jellyfish extermination method, a method that is effective and has a slight adverse effect on the natural environment is desired.

JP 05-185065 A Japanese Unexamined Patent Publication No. 07-292646 Japanese Patent Laid-Open No. 11-138146 Japanese Patent Laid-Open No. 11-316008 JP 2004-255313 A Japanese Patent No. 3696018

  The present invention was devised in view of the current state of the prior art, and its purpose is that a jellyfish attacks a large amount of difficult-to-treat waste when a jellyfish attacks an intake of a seawater utilization plant such as a power plant or a fishery facility. It is an object of the present invention to provide a peptidase and a method for using the peptidase that can be used for efficiently decomposing or reducing the volume of the jellyfish.

  The present invention provides an enzyme classified as EC 3.4 and capable of decomposing jellyfish, at least a peptidase derived from Bacillus sp. 97001 or a similar enzyme, and a method for using the same. In the present invention, when a jellyfish attacks an intake of a seawater utilization plant such as a power plant, a fishing facility, etc., and the jellyfish becomes difficult-to-treat waste, this is efficiently performed within an extremely short period of time. It is possible to provide a peptidase that can be decomposed or reduced in volume and is extremely useful in the industry and a method for using the peptidase. Jellyfish that has become difficult-to-process waste is decomposed or reduced in volume by treating it with a peptidase classified as EC 3.4 and capable of decomposing jellyfish, preferably peptidase produced by Bacillus sp. The final disposal of the jellyfish that has become the difficult-to-process waste is facilitated. By the decomposition method of the present invention, the final disposal of the jellyfish is extremely easy compared to conventional incineration and the like, and it is possible to minimize the adverse effects on the operation of seawater utilization plants and fishery facilities due to the jellyfish invasion. it can.

  About 95% of the adult jellyfish is assumed to be water, and about 2% is assumed to be collagen. In order to effectively reduce the volume of jellyfish, it is considered necessary to quickly dissipate the form by decomposing this collagen fraction. However, no peptidase that can efficiently hydrolyze jellyfish-derived collagen has been known. Collagenase is generally known as an enzyme that degrades collagen, but it is very expensive. However, it is difficult to say that it can be used for waste disposal.

  What should be noted in the present invention is that the peptidase of the present invention has a function of allowing the reaction to proceed under mild conditions as compared with chemical and physical methods. Therefore, when a jellyfish is treated with a peptidase classified as EC 3.4, preferably a peptidase produced by Bacillus sp. Strain 97001 or a similar enzyme, a strong acid or a strong base may be added at normal temperature and pressure. The hydrolysis reaction can proceed without any problems and the objective can be achieved. This is overwhelmingly advantageous in terms of low energy costs and low environmental impact compared to chemical methods that require heating, adding strong acids or bases, or applying pressure. It is.

Furthermore, treatment of jellyfish with a peptidase classified as EC 3.4, preferably a peptidase produced by Bacillus sp. 97001 or a similar enzyme, is superior in speed and efficiency compared to chemical methods. Compared to equipment used for heat treatment, strong acid or base treatment, and chemical treatment that requires pressure treatment, the equipment required for the peptidase treatment of the present invention keeps the initial cost and operating cost low. Can be advantageous.
In addition, peptidases classified as EC 3.4 and capable of decomposing jellyfish, preferably peptidases produced by Bacillus sp. 97001 or similar enzymes, are overwhelmingly compared to conventionally known enzymes that degrade collagen. Because of its high production amount and decomposition activity, it is sufficiently possible for industrial use to decompose or reduce jellyfish, which are difficult-to-treat waste, by using the peptidase of the present invention.

  Therefore, the present inventors paid attention to peptidases capable of efficiently hydrolyzing jellyfish-derived collagen in order to solve the above-mentioned problems, and conducted intensive research on enzymes capable of efficiently degrading or reducing jellyfish. As a result, it was found that jellyfish can be eliminated by treating with peptidases classified as EC 3.4. Further research has been conducted and it has been found that jellyfish can be efficiently decomposed or reduced by using peptidase derived from Bacillus sp. 97001 or similar enzyme.

  Based on this knowledge, the present inventors use the enzyme in a jellyfish treatment process that has attacked water intakes and fishery facilities of seawater utilization plants such as power plants, resulting in a large amount of difficult-to-treat waste. As a result, it was conceived that the decomposition or volume reduction that was difficult in the prior art could be easily performed, and the present invention was finally completed.

  That is, the method of decomposing or reducing the volume of jellyfish that has attacked water intakes and fishing facilities of power plants and other fishery facilities and has become a large amount of difficult-to-treat waste includes the following steps (1) to (3). It becomes.

(1) The jellyfish that has attacked the intake or fishery facility of the seawater utilization plant and flowed into the intake channel will be collected by appropriate means.
(2) The jellyfish is subjected to pretreatment such as cutting and / or crushing by an appropriate means as necessary.
(3) The pretreated jellyfish is reacted with a peptidase derived from Bacillus sp. 97001 strain or a similar enzyme in a suitable container for a predetermined time.

  The jellyfish that is the subject of the present invention may be any jellyfish, for example, a jellyfish and an Echizen jellyfish. In the present invention, the term “jellyfish” can include both mature jellyfish individuals and immature jellyfish individuals.

  The peptidase of the present invention may be an enzyme capable of degrading or reducing jellyfish at least, and is a peptidase classified as EC 3.4, preferably a peptidase classified as EC 3.4.21, more preferably It is a peptidase classified as EC 3.4.21.62. For example, a peptidase derived from Bacillus sp. 97001, a peptidase produced by Bacillus sp. 97001 or a similar enzyme may be used. In addition, mutations caused by deletion, substitution or addition of one or more amino acid residues in the amino acid sequence encoding the protein or a protein having a peptidase property and / or substantially equivalent property or a salt thereof As long as it is a biologically active and stable protein. The peptidase of the present invention should be at least an enzyme capable of decomposing or reducing jellyfish, and preferably has an activity capable of decomposing jellyfish. The peptidase of the present invention is calculated from the moment when the peptidase of the present invention is brought into contact with the jellyfish. Thus, after 12 hours, the jellyfish volume should preferably be 2/3 or less, more preferably 1/2 or less, and desirably 1/3 or less of the volume immediately after contact.

  Peptidases classified as EC 3.4 of the present invention include EC 3.4.1 a-Amino-Acyl-Peptide Hydrolases, EC 3.4.2 Peptidyl-Amino-Acid Hydrolases, EC 3.4.3 Dipeptide Hydrolases, EC 3.4.4 Peptidyl Peptide Hydrolases, EC 3.4.11 Aminopeptidases, EC 3.4.12 Peptidylamino-Acid Hydrolases or Acylamino-Acid Hydrolases, EC 3.4.13 Dipeptidases, EC 3.4.14 Dipeptidyl-peptidases and tripeptidyl-peptidases, EC 3.4.15 Peptidyl-dipeptidases, EC 3.4 .16 Serine-type carboxypeptidases, EC 3.4.17 Metallocarboxypeptidases, EC 3.4.18 Cysteine-type carboxypeptidases, EC 3.4.19 Omega peptidases, EC 3.4.21 Serine endopeptidases, EC 3.4.22 Cysteine endopeptidases, EC 3.4.23 Aspartic endopeptidases, EC 3.4.24 Metalloendopeptidases, EC 3.4.25 Threonine endopeptidases, EC 3.4.99 Endopeptidases of unknown catalytic mechanism, and the like. Among these, Preferably, EC 3.4.21 Serine endopeptidases, EC 3.4.22 Cysteine endopeptidases, EC 3.4.23 Aspartic endopeptidases, EC 3.4.24 Metalloendopeptidases are mentioned.

  In addition, EC 3.4.21.1 chymotrypsin, EC 3.4.21.2 chymotrypsin C, EC 3.4.21.3 metridin, EC 3.4.21.4 trypsin, EC 3.4.21.5 thrombin, EC 3.4.21.6 coagulation factor Xa, EC 3.4.21.7 plasmin, EC 3.4. 21.9 enteropeptidase, EC 3.4.21.10 acrosin, EC 3.4.21.12 a-Lytic endopeptidase, EC 3.4.21.19 glutamyl endopeptidase, EC 3.4.21.20 cathepsin G, EC 3.4.21.21 coagulation factor VIIa, EC 3.4.21.22 coagulation factor IXa, EC 3.4 .21.25 cucumisin, EC 3.4.21.26 prolyl oligopeptidase, EC 3.4.21.27 coagulation factor XIa, EC 3.4.21.32 brachyurin, EC 3.4.21.34 plasma kallikrein, EC 3.4.21.35 tissue kallikrein, EC 3.4.21.36 pancreatic elastase, EC 3.4.21.37 leukocyte elastase, EC 3.4.21.38 coagulation factor XIIa, EC 3.4.21.39 chymase, EC 3.4.21.41 complement subcomponent C, EC 3.4.21.42 complement subcomponent C, EC 3.4.21.43 classical-complement-pathway C3 / C5 convertase, EC 3.4. 21.45 complement factor I, EC 3.4.21.46 complement factor D, EC 3.4.21.47 alternative-complement-pa thway C3 / C5 convertase, EC 3.4.21.48 cerevisin, EC 3.4.21.49 hypodermin C, EC 3.4.21.50 lysyl endopeptidase, EC 3.4.21.53 endopeptidase La, EC 3.4.21.54 g-renin, EC 3.4.21.55 venombin AB, EC 3.4 .21.57 leucyl endopeptidase, EC 3.4.21.59 tryptase, EC 3.4.21.60 scutelarin, EC 3.4.21.61 kexin, EC 3.4.21.62 subtilisin, EC 3.4.21.63 oryzin, EC 3.4.21.64 endopeptidase K, EC 3.4.21.65 thermomycolin, EC 3.4 .21.66 thermitase, EC 3.4.21.67 endopeptidase So, EC 3.4.21.68 t-plasminogen activator, EC 3.4.21.69 protein C (activated), EC 3.4.21.70 pancreatic endopeptidase E, EC 3.4.21.71 pancreatic elastase II, EC 3.4.21.72 IgA-specific serine endopeptidase, EC 3.4.21.73 u-plasminogen activator, EC 3.4.21.74 venombin A, EC 3.4.21.75 furin, EC 3.4.21.76 myeloblastin, EC 3.4.21.77 semenogelase, EC 3.4.21.78 granzyme A, EC 3.4. 21.79 granzyme B, EC 3.4.21.80 streptogrisin A, EC 3.4.21.81 streptogrisin B, EC 3.4.21.82 glutamyl endopeptidase II, EC 3.4.21.83 oligopeptidase B EC 3.4.21.84 limulus clotting factor, EC 3.4.21.85 limulus clotting factor, EC 3.4.21.86 limulus clotting enzyme, EC 3.4.21.87 omptin, EC 3.4.21.88 repressor LexA, EC 3.4.21.89 signal peptidase I, EC 3.4.21.90 togavirin , EC 3.4.21.91 flavivirin, EC 3.4.21.92 endopeptidase Clp, EC 3.4.21.93 proprotein convertase 1, EC 3.4.21.94 proprotein convertase 2, EC 3.4.21.95 snake venom factor V activator, EC 3.4.21.96 lactocepin, EC 3.4.21.97 assemblin, EC 3.4.21.98 hepacivirin, EC 3.4.21.99 spermosin, EC 3.4.21.100 pseudomonalisin, EC 3.4.21.101 xanthomonalisin, EC 3.4.21.102 C-terminal processing peptidase, EC 3.4.21.103 physarolisin, EC 3.4.21.104 mannan-binding lectin -associated serine protease-2, EC 3.4.22.1 cathepsin B, EC 3.4.22.2 papain, EC 3.4.22.3 ficain, EC 3.4.22.6 chymopapain, EC 3.4.22.7 asclepain, EC 3.4.22.8 clostripain, EC 3.4.22.10 streptopain, EC 3.4.22.14 actinidain, EC 3.4.22.15 cathepsin L, EC 3.4.22.16 cathepsin H, EC 3.4.22.24 cathepsin T, EC 3.4.22.25 glycyl endopeptidase, EC 3.4.22.26 cancer procoagulant, EC 3.4.22.27 cathepsin S, EC 3.4.22.28 picornain 3C, EC 3.4.22.29 picornain 2A, EC 3.4.22.30 caricain, EC 3.4.22.31 ananain, EC 3.4.22.32 stem bromelain, EC 3.4.22.33 fruit bromelain, EC 3.4.22.34 legumain, EC 3.4.22.35 histolysain, EC 3.4.22.36 caspase-1, EC 3.4.22.37 gingipain R, EC 3.4.22.38 cathepsin K, EC 3.4. 22.39 adenain, EC 3.4.22.40 bleomycin hydrolase, EC 3.4.22.41 cathepsin F, EC 3.4.22.42 cathepsin O, EC 3.4.22.43 cathepsin V, EC 3.4.22.44 nuclear-inclusion-a endopeptidase, EC 3.4.22.45 helper-component proteinase , EC 3.4.22.46 L-peptidase, EC 3.4.22.47 gingipain K, EC 3.4.22.48 staphopain, EC 3.4.22.49 separase, EC 3.4.22.50 V-cath endopeptidase, EC 3.4.22.51 cruzipain, EC 3.4.22.52 calpain-1 , EC 3.4.22.53 calpain-2, EC 3.4.23.1 pepsin A, EC 3.4.23.2 pepsin B, EC 3.4.23.3 gastricsin, EC 3.4.23.4 chymosin, EC 3.4.23.5 cathepsin D, EC 3.4.23.12 nepenthesin, EC 3 . 4.23.15 renin, EC 3.4.23.16 HIV-1 retropepsin, EC 3.4.23.17 Pro-opiomelanocortin converting enzyme, EC 3.4.23.18 aspergillopepsin I, EC 3.4.23.19 aspergillopepsin II, EC 3.4.23.20 penicillopepsin, EC 3.4.23.21 rhizopuspepsin, EC 3.4.23.22 endothiapepsin, EC 3.4.23.23 mucorpepsin, EC 3.4.23.24 candidapepsin, EC 3.4.23.25 saccharopepsin, EC 3.4.23.26 rhodotorulapepsin, EC 3.4.23.28 acrocylindropepsin, EC 3.4.23.29 polyporopepsin, EC 3.4.23.30 pycno .23.31 scytalidopepsin A, EC 3.4.23.32 scytalidopepsin B, EC 3.4.23.34 cathepsin E, EC 3.4.23.35 barrierpepsin, EC 3.4.23.36 signal peptidase II, EC 3.4.23.38 plasmepsin I, EC 3.4.23.39 plasmepsin II, EC 3.4. 23.40 phytepsin, EC 3.4.23.41 yapsin 1, EC 3.4.23.42 thermopsin, EC 3.4.23.43 prepilin peptidase, EC 3.4.23.44 nodavirus endopeptidase, EC 3.4.23.45 memapsin 1, EC 3.4.23.46 memapsin 2, EC 3.4.23.47 HIV- 2 retropepsin, EC 3.4.23.48 plasminogen activator Pla, EC 3.4.24.1 atrolysin A, EC 3.4.24 .3 microbial collagenase, EC 3.4.24.6 leucolysin, EC 3.4.24.7 interstitial collagenase, EC 3.4.24.11 neprilysin, EC 3.4.24.12 envelysin, EC 3.4.24.13 IgA-specific metalloendopeptidase, EC 3.4.24.14 procollagen N-endopeptidase, EC 3.4 .24.15 thimet oligopeptidase, EC 3.4.24.16 neurolysin, EC 3.4.24.17 stromelysin 1, EC 3.4.24.18 meprin A, EC 3.4.24.19 procollagen C-endopeptidase, EC 3.4.24.20 peptidyl-Lys metalloendopeptidase, EC 3.4.24.21 astacin, EC 3.4.24.22 stromelysin 2, EC 3.4.24.23 matrilysin, EC 3.4.24.24 gelatinase A, EC 3.4.24.25 vibriolysin, EC 3.4.24.26 pseudolysin, EC 3.4.24.27 thermolysin, EC 3.4.24.28 bacillolysin, EC 3.4.24.29 aureolysin, EC 3.4.24.30 coccolysin, EC 3.4.24.31 mycolysin, EC 3.4.24.32 b-lytic metalloendopeptidase, EC 3.4.24.33 peptidyl-Asp metalloendopeptidase, EC 3.4.24.34 neutrophil collagenase, EC 3.4.24.35 gelatinase B, EC 3.4.24.36 leishmanolysin, EC 3.4.24.37 saccharolysin, EC 3.4.24.38 gametolysin, EC 3.4.24.39 deuterol ysin, EC 3.4.24.40 serralysin, EC 3.4.24.41 atrolysin B, EC 3.4.24.42 atrolysin C, EC 3.4.24.43 atroxase, EC 3.4.24.44 atrolysin E, EC 3.4.24.45 atrolysin F, EC 3.4.24.46 adamalysin, EC 3.4 .24.47 horrilysin, EC 3.4.24.48 ruberlysin, EC 3.4.24.49 bothropasin, EC 3.4.24.50 bothrolysin, EC 3.4.24.51 ophiolysin, EC 3.4.24.52 trimerelysin I, EC 3.4.24.53 trimerelysin II, EC 3.4.24.54 mucrolysin, EC 3.4 .24.55 pitrilysin, EC 3.4.24.56 insulysin, EC 3.4.24.57 O-sialoglycoprotein endopeptidase, EC 3.4.24.58 russellysin, EC 3.4.24.59 mitochondrial intermediate peptidase, EC 3.4.24.60 dactylysin, EC 3.4.24.61 nardilysin, EC 3.4.24.62 magnolysin , EC 3.4.24.63 meprin B, EC 3.4.24.64 mitochondrial processing peptidase, EC 3.4.24.65 macrophage elastase, EC 3.4.24.66 choriolysin L, EC 3.4.24.67 choriolysin H, EC 3.4.24.68 tentoxilysin, EC 3.4.24.69 bontoxilysin, EC 3.4.24.70 oligopeptidase A, EC 3.4.24.71 endothelin-converting enzyme, EC 3.4.24.72 fibrolase, EC 3.4.24 .73 jararhagin, EC 3.4.24.74 fragilysin, EC 3.4.24.75 lysostaphin, EC 3.4.24.76 flavastacin, EC 3.4.24.77 snapalysin, EC 3.4.24.78 gpr endopeptidase, EC 3.4.24.79 pappalysin-1, EC 3.4.24.80 membrane-type matrix metalloproteinase-1, EC 3.4.24.81 ADAM10 endopeptidase, EC 3.4.24.82 ADAMTS-4 endopeptidase, EC 3.4.24.83 anthrax lethal factor endopeptidase, EC 3.4.24.84 Ste24 endopeptidase, EC 3.4.24.85 S2P endopeptidase, EC 3.4.24.86 ADAM 17 endopeptidase and the like. Among these, EC 3.4.21.62 subtilisin is preferable. The EC 3.4.21.62 includes alcalase, alcalase 0.6L, alcalase 2.5L, ALK-enzyme, bacillopeptidase A, bacillopeptidase B, Bacillus subtilis alkaline proteinase, Bacillus subtilis alkaline proteinase bioprase, Bacillus subtilis carlsberg, bioprase, bioprase AL15, bioprase APL 30, colistinase, esperase, genenase I, kazusase, maxatase, nagarase, nagarse, opticlean, orientase 10B, protease S, protease VIII, protease XXVII, protin A 3L, savinase, savinase 16.0L, savinase 32.0 L EX, savinase 4.0T savinase 8.0L, SP 266, subtilisin, subtilisin A, subtilisin BL, subtilisin bpn ', subtilisin carlsberg, subtilisin DY, subtilisin E, subtilisin GX, subtilisin J, subtilisin NOVO, subtilisin S41, subtilisin Sendai, subtilopeptidid, subtilopeptidid, subtilopeptopidid , Subtilopeptidase bpn ', subtilopeptidase C, superase, thermoase, thermoase PC-10, peptidase produced by Bacillus sp. 97001 strain, and the like.

  The microorganism that produces the peptidase of the present invention is only required to have the ability to produce a peptidase that degrades or reduces jellyfish, and is preferably Bacillus sp. 97001, which is an independent administrative agency, National Institute of Advanced Industrial Science and Technology. Deposited at the deposit center as FERM P-20726. The results of morphological observation and physiological property test of this strain are shown below.

Results in Table 1 and literature ("Bergey's Manual of Systematic Bacteriology" Vol.2 (1986) Williams & Wilkins and "The Genus Bacillus" (1973) US Department of Agriculture ", this strain was named Bacillus sp. 97001 strain.
The microorganism of the present invention may be a microbial strain obtained by breeding such as a natural or artificial mutation method.

  In the method for producing the peptidase of the present invention, the above-described microorganism may be inoculated into a medium and cultured according to a conventional method. The medium used for the culture preferably contains appropriate amounts of carbon source and nitrogen source that can be assimilated by microorganisms. The carbon source and nitrogen source are not particularly limited, and examples of the carbon source include soluble starch, galactose, glucose, sucrose, fructose, maltose, mannitol, mannose, raffinose, and organic acids that can be assimilated, such as citric acid. Nitrogen sources include organic nitrogen sources such as casamino acids, corn gluten meal, corn steep liquor, soyton, soy bean meal, tryptone, huma media, polypeptone, yeast extract, soy flour, meat extract, cottonseed meal and cultivator. It is valid. Furthermore, inorganic salts such as potassium salt, calcium salt, cobalt salt, sodium salt, magnesium salt, manganese salt, zinc salt, phosphate, etc., and if necessary, inorganic or organic micronutrients and vitamins are appropriately added to the medium. Can be added. The culture temperature is preferably 10 to 45 ° C., particularly around 30 ° C., and the pH is preferably 6 to 8, particularly around 7. The culture is usually completed in 1 to 5 days under these conditions.

The peptidase of the present invention may be at least an enzyme capable of degrading or reducing jellyfish, and is a peptidase classified as EC 3.4. The peptidase of the present invention may be a culture solution containing bacterial cells, or may be a concentrate of the culture solution. Moreover, the crushed material of a microbial cell may be sufficient, and the microbial cell extract obtained by the solubilization by self-digestion or surfactant may be sufficient. The peptidase of the present invention may be purified from a culture solution and / or a bacterial cell extract and purified. The degree of purification is not particularly limited, and may be appropriately selected and used. In order to purify the peptidase of the present invention, it may be carried out according to a general means for collecting enzymes. For example, after the culture of the microorganism of the present invention is completed, the microorganism is removed from the culture solution by a normal separation means such as centrifugation or filtration to obtain a crude enzyme solution. Furthermore, the crude enzyme solution may be recovered by means such as ultrafiltration, salting out or solvent precipitation as necessary to obtain the peptidase of the present invention. The peptidase of the present invention may be used after powdered. Further, it may be purified by appropriately combining general enzyme purification means, for example, anion exchange resin, cation exchange resin, affinity chromatography, hydrophobic chromatography, chromatography with hydroxyapatite and gel filtration. .
Peptidases derived from the Bacillus sp. 97001 strain of the present invention are classified as endopeptidases, the active center is a serine residue, the molecular weight is about 26,000, the optimum temperature is 55 to 70 degrees, and the optimum temperature is around 60 ° C. Maximum activity was shown. The optimum pH is pH 7-9 and is stable in the range of pH 6-9. Inhibitory action is diisopropylfluorophosphate (DEP), phenylmethanesulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), soybean trypsin inhibitor.

  The preparation for decomposing jellyfish of the present invention only needs to contain at least the peptidase of the present invention. For example, the crude enzyme solution obtained during the production process of the peptidase of the present invention may be contained as it is. A culture solution containing a microorganism that produces the peptidase of the invention may be contained, or a microorganism producing the peptidase of the invention may be contained. The preparation for degrading jellyfish of the present invention contains at least the peptidase of the present invention, and the other formulation and shape are not particularly limited. For example, a polyhydric alcohol such as glycerol can be contained for the purpose of stabilization, or sodium benzoate can be blended in any proportion for the purpose of preventing spoilage. The shape may be liquid or powder, and the powder may be granulated, or may be pasted using another substrate.

  In the method for decomposing jellyfish of the present invention, the jellyfish may be decomposed or reduced in volume by acting on the jellyfish using the peptidase of the present invention. In addition, the jellyfish decomposition method of the present invention was obtained by purifying the jellyfish to an appropriate degree from the above-mentioned culture solution, culture supernatant, culture supernatant concentrate, and culture supernatant. It is preferable to use any one of an enzyme, a microbial cell of the microorganism of the present invention obtained by culturing, a treated product of the microbial cell obtained by culturing, conditions for decomposing or reducing the jellyfish, etc. Can be selected and used as appropriate. For example, it is performed by executing the following steps (1) to (3).

(1) The jellyfish that invaded the intake of the seawater utilization plant or the fishery facility and flowed into the intake channel were separated and collected by appropriate means.
(2) If necessary, the jellyfish is subjected to pretreatment such as cutting and / or crushing by an appropriate means,
(3) The pretreated jellyfish is reacted with a peptidase produced by the microorganism of the present invention or a similar enzyme in a suitable container for a predetermined time to decompose or reduce the volume of the jellyfish.

  In step (1) of the decomposition method of the present invention, the jellyfish that has entered the intake or fishery facility of the seawater utilization plant and has flowed into the intake channel is collected by appropriate means. The jellyfish can be separated and collected by a conventional method, such as trapping the attacking jellyfish with a net, a metal mesh or a screen. In addition, in order to prevent the jellyfish itself collected separately from being degraded by microorganisms, it is preferable that the steps up to step (3) are stored and operated at a low temperature. Furthermore, it is preferable to shorten the elapsed time until step (3) as much as possible. In step (2), the jellyfish separated and collected in step (1) of the method of the present invention is subjected to pretreatment such as cutting and / or crushing by appropriate means in step (2). This is intended to increase the efficiency of the reaction in step (3). The apparatus used for cutting and / or crushing of the jellyfish can achieve the above-mentioned object, and an apparatus that can decompose the jellyfish according to the present invention may be used. Etc. may be appropriately selected and used. In step (2) of the method of the present invention, a jellyfish that has been subjected to treatment such as cutting and / or crushing by means of appropriate cutting and / or crushing, etc. It reacts with a peptidase classified as EC 3.4 of the present invention and capable of decomposing jellyfish, preferably a peptidase produced by Bacillus sp. In step (3), in order to keep the efficiency of the reaction high, it is preferable to appropriately control the pH and / or temperature of the reaction system, and further to perform stirring and mixing. The apparatus used for the stirring and mixing may be an apparatus that can decompose the jellyfish of the present invention, and may be used by appropriately selecting an industrially used stirring apparatus, reaction tank, kneader or the like. . The order of performing step (2) and step (3) of the decomposition method of the present invention is not particularly limited. For example, step (2) and step (3) may be performed simultaneously. By combining the steps (1) to (3) described above, the decomposed or reduced volume jellyfish has a solid content that is greatly reduced and has the same form as general wastewater. The decomposed or reduced volume jellyfish can be processed by conventional biological and / or chemical sewage treatment methods to reduce BOD (biochemical oxygen demand) and / or COD (chemical oxygen demand). Can be disposed of efficiently. The jellyfish that attacked the intakes of seawater use plants such as power plants and fishing facilities and became a large amount of difficult-to-treat waste did not have an appropriate method, and the final disposal of the jellyfish was difficult. This problem is facilitated by the decomposition method of the invention.

  In the method of decomposing jellyfish of the present invention, the peptidase of the present invention is preferably 0.3 to 72 AU, more preferably 10 to 55 AU, and most preferably 25 to 40 AU in treating 1 kg of live jellyfish. If it is. The AU as a unit in the present invention is an Anson unit, and indicates a unit of proteolytic activity measured using a dimethyl casein method.

  The jellyfish decomposing apparatus of the present invention is related to the jellyfish decomposing method of the present invention, and is a container according to the amount of jellyfish to be processed, preferably controlling pH and / or temperature, more preferably. In order to increase the contact efficiency and keep the decomposition rate high, a stirring device may be installed. Appropriate control of the pH and / or temperature may be automatic control means or manual control means, and the control device can be appropriately selected and installed according to the control means. For example, various reaction tanks and kneaders that are used industrially may be used.

  Moreover, the above-described decomposition method of the present invention can be applied as it is as a method for producing the jellyfish decomposition product of the present invention. Since the solid content of jellyfish contains collagen, by applying the present invention, a jellyfish degradation product containing jellyfish-derived collagen and / or a degradation product thereof can be efficiently produced. The jellyfish degradation product containing the jellyfish-derived collagen and / or degradation product thereof can be applied to food materials, cosmetic materials and the like.

  The jellyfish extermination method of the present invention is a peptidase classified as EC 3.4 of the present invention and capable of decomposing jellyfish, preferably Bacillus sp., For jellyfish floating in the sea and / or adhering to an object in the sea. The peptidase produced by strain 97001 or a similar enzyme may be sprayed into the sea and brought into contact with the jellyfish. The jellyfish extermination method of the present invention is effective not only for jellyfish corpses but also for living jellyfish by using the peptidase of the present invention. Is possible. The amount and form of the peptidase of the present invention may be appropriately selected and used according to the spraying range, the amount of jellyfish to be exterminated, the spraying method, the inflow of seawater, the seawater temperature, the salinity concentration, etc., and is not particularly limited. It is possible to break down and even disinfect live jellyfish.

  In the method of the present invention, the facility targeted for jellyfish decomposition and volume reduction is a seawater utilization plant. The seawater utilization plant is not particularly limited as long as it takes seawater from a water intake and uses it, for example, a thermal power plant, a nuclear power plant, a liquefied natural gas re-vaporization facility, a fishery facility, etc. .

  EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[Example 1. Culture of Bacillus sp. 97001 strain]
Dispense Bacillus medium (yeast extract (0.4%), polypeptone (0.8%), sodium chloride (0.2%): adjusted to pH 7.2 with sodium hydroxide solution) into 4 test tubes each. Then, after sterilizing at 121 ° C. for 20 minutes and cooling, Bacillus sp. 97001 strain was inoculated one by one, and cultured at 30 ° C. with shaking for 18 hours to prepare a seed culture solution. Dispense 400 ml of the Bacillus medium into four 2 L culture flasks, disinfect and cool them at 121 ° C. for 20 minutes, and then add 16 ml of the seed culture solution (for four test tubes) to each flask. Each 4 ml was inoculated and cultured with shaking at 30 ° C. for 18 hours to prepare a preculture solution. Next, 160 L of a medium having the same composition as above with an antifoaming agent 0.01% (W / V) added thereto was placed in a 200 L capacity jar fermenter, sterilized at 121 ° C. for 20 minutes, cooled, 1.6 L of the above preculture solution was inoculated and cultured with aeration and stirring at 30 ° C. for 18 hours. After completion of the culture, the cells and the culture supernatant were separated.

[Example 2. Preparation of peptidase from Bacillus sp. 97001]
The culture supernatant obtained in Example 1 was concentrated at 4 ° C. using a membrane filtration device (fractionated molecular weight 10,000), and the liquid composition was then replaced with potassium phosphate buffer (50 mM, pH 7) using the same device. As a result, 4 L of peptidase solution was obtained. The molecular weight of the obtained peptidase was about 26,000. When the optimum temperature was measured according to a conventional method, the maximum activity was observed at around 60 ° C. Moreover, it was stable in the pH range of 6-9.

[Example 3. Degradation of moon jellyfish by peptidase from Bacillus sp. 97001]
100 mL of the peptidase solution obtained in Example 2 and 100 mL of potassium phosphate buffer (50 mM, pH 7) are mixed, and one living moon jellyfish (umbrella diameter: about 4.5 cm) is added thereto, and the mixture is allowed to stand at 30 ° C. And visually observed. As a result, the moon jellyfish was completely decomposed in about 6 hours, and the form disappeared and the volume was reduced.
In addition, instead of using the jellyfish body of the above method, an experiment was performed under the same conditions as the above method except that the jellyfish was cut into about 2 cm square, and the jellyfish cut in about 2 hours was completely decomposed, Volume decreased as a result of disappearance of form.

  As a control group, a mixed solution obtained by mixing the peptidase solution obtained in Example 2 with potassium phosphate buffer (50 mM, pH 7) and deactivating the enzyme by autoclaving at 121 ° C. for 30 minutes was used. The other conditions were the same. In the control group, moon jellyfish was not decomposed.

[Example 4. Degradation of Echizen jellyfish by peptidase from Bacillus sp. 97001]
100 mL of the peptidase solution obtained in Example 2 and 100 mL of potassium phosphate buffer (50 mM, pH 7) were mixed, and this was applied to a slice of the Echizen jellyfish organism (20 cm × 15 cm × 3 cm) and allowed to stand at 45 ° C., Visual observation was performed. As a result, Echizen jellyfish was completely decomposed in about 5 hours, and the volume was reduced as a result of the disappearance of the form.
In addition, when the experiment was carried out under the same conditions as in the above method except that the Echizen jellyfish cut into about 2 cm square instead of the Echizen jellyfish fillet of the above method, the Echizen jellyfish was completely decomposed in about 0.5 hours. The volume was reduced as a result of the disappearance of the form.

  In the control group, a mixed solution in which the enzyme was inactivated by autoclaving at 121 ° C. for 30 minutes was used in a solution obtained by mixing the peptidase solution obtained in Example 2 and potassium phosphate buffer (50 mM, pH 7). Except for the above, the conditions were the same as those described above. In the control group, Echizen jellyfish was not decomposed.

[Example 5. Comparison of peptidase from Bacillus sp. 97001 and other enzymes]
Using the peptidase solution obtained in Example 2 and 10 commercially available enzymes (commercial products A, B, C, D, E, F, G, H, I and J) as jellyfish degrading enzymes, This was performed under the same conditions as in Example 4 except that each of the cut pieces was cut into 2 cm squares. At this time, the amount of each enzyme used was adjusted to 50 mg based on the protein amount of the enzyme. The table method is shown in Table 3, and the result is shown in Table 2.

  As a result, the reaction results of the moon jellyfish and Echizen jellyfish with the peptidase derived from the Bacillus sp. 97001 strain of the present invention obtained in Example 2 were completely degraded in about 2 hours and Echizen jellyfish in about 0.5 hours. When other commercially available enzymes were used, they were not degraded at all or were degraded over 12 hours. It has been shown that by using the peptidase of the present invention, jellyfish can be decomposed and reduced in volume more efficiently.

Claims (10)

  A peptidase classified as EC 3.4 and capable of decomposing jellyfish.   The peptidase according to claim 1, which is derived from Bacillus sp. 97001 strain.   A protein having a peptidase property and / or a substantially equivalent property or a salt thereof includes an amino acid sequence encoding the protein or a mutation caused by deletion, substitution or addition of one or more amino acid residues in the sequence. The peptidase according to claim 1 or 2, which has an amino acid sequence and is a biologically active and stable protein.   A microorganism having the ability to produce the peptidase according to any one of claims 1 to 3.   A method for producing a peptidase obtained by culturing the microorganism according to claim 4 to produce a peptidase, and collecting the peptidase.   A method for decomposing jellyfish using the peptidase according to any one of claims 1 to 3.   A jellyfish decomposing apparatus using the peptidase according to any one of claims 1 to 3.   A preparation for jellyfish degradation comprising the peptidase according to any one of claims 1 to 3.   A jellyfish degradation product obtained by using the peptidase according to any one of claims 1 to 3. A jellyfish extermination method using the peptidase according to any one of claims 1 to 3.