Abstract:
A  Pseudomonas  sp. strain TKU015 is deposited under DSMZ GmbH (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) Number DSM 21747). The  Pseudomonas  sp. strain TKU015 can be used to produce chitinase, chitosanase and nattokinase. A method of producing chitinase, chitosanase and nattokinases can use the  Pseudomonas  sp. strain TKU015.

Description:
REFERENCE TO SEQUENCE LISTING 
       [0001]    A sequence listing is enclosed as an attachment, and the content of the sequence listing information recorded in computer readable form is identical to the written sequence listing. 
       BACKGROUND 
       [0002]    Soybeans don&#39;t contain plasmin. Generally, natto is produced by inoculating Bacillus natto or Bacillus subtilis into braised soybeans and then fermenting the soybeans. A kinase, which names as nattokinase, exists in viscousness distributed on surface of produced natto. Nattokinase acts as a fibrinolytic enzyme of thrombus; therefore, nattokinase is very useful in prevention and curing of brain stroke and myocardial infarction, as well as Alzheimer&#39;s disease caused by infarction in small vein. 
         [0003]    Plasmin is the only fibrinolytic enzyme contained in human body. Usually, blood contains a plasmin precursor called plasminogen. Fibrinolytic enzyme is a plasminogen activator that can activate plasminogen and thereby producing plasmin. Nattokinase has similar properties of fibrinolytic enzyme; therefore, nattokinase is capable of dissolving fibrin in blood serum. Furthermore, nattokinase reacts with pro-urokinase together with a pro-urokinase activator thereby obtaining urokinase. Urokinase reacts with plasminogen and fibrinolytic enzyme is obtained. The obtained fibrinolytic enzyme further dissolves thrombus and produces thrombus degradation products. In summary, nattokinase can decreases thrombus by increasing amount of fibrinolytic enzyme. 
         [0004]    Except in fermented soybean, nattokinase is also found in bacillus, actinomyces, epiphyte and alga. However, there isn&#39;t any prior art discloses that nattokinase can be produced from bacteria doesn&#39;t belong to pseudomonas sp. and bacillus sp. 
         [0005]    Shrimp and crab shell powder (SCSP) contains mass amount of protein and chitin, if SCSP can be fermented with a microorganism to produce valuable bioactive substance, pollution to the environment can be reduced and value in use of SCSP can be improved. 
       BRIEF SUMMARY 
       [0006]    In one exemplary embodiment, a  Pseudomonas  sp. strain TKU015 (gene pool number EU103629, DSMZ GmbH (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH) Number DSM 21747) is provided as attachment A. The  Pseudomonas  sp. strain TKU015 can be used to produce chitinase, chitosanase and nattokinases. 
         [0007]    In another exemplary embodiment, a method of producing nattokinase is provided. The method utilizes the  Pseudomonas  sp. strain TKU015 to ferment a solution of aquatic products castoff powder and nattokinases is thereby obtained. Pollution of the aquatic products to environment is reduced and value of the aquatic products is improved. 
         [0008]    The strain TKU015 is selected from soil in North Taiwan and cultured using shrimp shell powder as unique carbon/nitrogen source. Identification results show that TKU015 belongs to  Pseudomonas  sp. 
         [0009]    Nattokinase can be produced by fermenting a solution of aquatic products castoff powder using the  Pseudomonas  sp. strain TKU015. The aquatic products castoff comprises shrimp and crab shell, squid pen power, mushroom and rude chitin. A concentration range of the aquatic products castoff powder is i 0.1%-3%, preferably, the range is 0.2-1.5%. 
         [0010]    Generally, culture medium that is suitable for producing chitinase and chitosanase includes 0.5% by weight of shrimp shell powder (SSP), 0.1% by weight of K 2 HPO 4 , and 0.05% by weight of MgSO 4 .7H 2 O. The  Pseudomonas  sp. strain TKU015 is cultured in the medium (pH 8) for 3 days at 30° C. The obtained fermented supernatant is processed with Ammonium sulfate precipitation, DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography and Sephacryl S-100 chromatography, and then chitinase and chitosanase is separated. SDS-PAGE testing results show that molecular weight of chitinase and chitosanase is 68 kDa and 30 kDa respectively. Optimum reacting pH value, optimum reacting temperature, pH stable range, thermal stable range of chitinase are pH 5, 50° C., pH 5-7, and &lt;60° C.; optimum reacting pH value, optimum reacting temperature, pH stable range, thermal stable range of chitosanase are pH 4, 50° C., pH 3-9, and &lt;50° C. Activity of chitinase is inhibited by Mn 2+  and Fe 2+ , activity of chitosanase is inhibited by Mn 2+ , Cu 2+ and PMSF. 
         [0011]    Generally, culture medium that is suitable for producing chitinase and chitosanase includes 0.2-1.5% by weight of shrimp shell powder (SSP), 0.05-0.2% by weight of K 2 HPO 4 , and 0.02-0.1% by weight of MgSO 4 .7H 2 O. Preferably, the culture medium includes 1% by weight of shrimp shell powder (SSP), 0.1% by weight of K 2 HPO 4 , and 0.05% by weight of MgSO 4 .7H 2 O. The  Pseudomonas  sp. strain TKU015 is cultured in the medium (pH 7) for 2 days at 30° C. The obtained fermented supernatant is processed with Ammonium sulfate precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose chromatography, and then nattokinase is separated. SDS-PAGE testing results show that molecular weight of nattokinase is 21 kDa and 30 kDa respectively. Optimum reacting temperature, pH stable range, thermal stable range of nattokinase are pH 7, 50° C., pH 4-11, and &lt;37° C. Activity of nattokinase is completely inhibited by PMSF. Fe 2+  can improve activity of nattokinases. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]    These and other features and advantages of the various embodiments disclosed herein will be better understood with respect to the following description and drawings, in which like numbers refer to like parts throughout, and in which: 
           [0013]      FIG. 1  is a microscope photograph of a  Pseudomonas  sp. strain TKU015 provided in an embodiment of the present invention; 
           [0014]      FIG. 2  is a partial 16S rDNA nucleic acid sequence of  Pseudomonas  sp. strain TKU015; 
           [0015]      FIG. 3  is a graph showing influence of concentration of SSP to produced nattokinases activity; 
           [0016]      FIG. 4  is a graph showing influence of temperature to produced nattokinases activity; 
           [0017]      FIG. 5  is a graph showing influence of pH value to produced nattokinases activity; 
           [0018]      FIG. 6  is a graph showing influence of volume of culture medium to produced nattokinases activity; 
           [0019]      FIG. 7  is a graph showing growth curves of chitinase, chitosanase and nattokinases produced in a culture medium of  Pseudomonas  sp. strain TKU015; and 
           [0020]      FIG. 8  is SDS-PAGE analysis graph of chitosanase and nattokinases produced by  Pseudomonas  sp. strain TKU015. 
       
    
    
     DETAILED DESCRIPTION 
       [0021]    Culture of New Strain TKU015 
         [0022]    An amount of pre-gathered soil is dissolved in an appropriate amount of germfree water and the obtained solution is coated on a solid culture medium comprising 1% by weight of shrimp shell powder (SSP), 0.1% by weight of K 2 HPO 4 , 0.05% by weight of MgSO 4 .7H 2 0 and 1.5% by weight of agar. The medium is cultured for two days at 30° C., and the colony is observed. Produced single colony is inoculated into a nutrient agar (NA) medium and cultured for one day at 30° C. After that, the NA medium is stored at 4° C. Single colony filtrated from the NA medium is cultured in 100 mL liquid medium disposed in a 250 mL conical flask for two days at 30° C. using a shaking culture method. The liquid medium includes 1% by weight of SSP, 0.1% by weight of K 2 HPO 4 , and 0.05% by weight of MgSO 4 .7H 2 O disposed. Finally, the liquid medium is centrifugally purified, supernatant is separated, and activity of chitinase and chitosanase of the supernatant is tested. 
         [0023]    Characteristics of TKU015 Strain 
         [0024]    As shown in  FIG. 1 , TKU015 is one kind of Gram-negative, which has catalase, oxidase and motility, and can grow both in an aerobic and an anaerobic environment, but does not produce spores. 
         [0025]      FIG. 2  illustrates a portion of 16 S rDNA nucleotide sequence analysis result, which indicates that TKU015 is most close to Pseudomonas sp. and similarity of TKU015 and Pseudomonas sp. exceeds 97%. 
         [0026]    Testing result of glucose metabolism type indicates that TKU015 is oxidized type and is consistent with characteristics of Pseudomonas sp. 
         [0027]    Analysis of fatty acid composition indicates that main fatty acid composition of TKU015 includes C16:0 and H18:1ω7c, hydroxylated fatty acids C10:0 3OH, C12:0 2OH and C12:0 3OH can also be found in TKU015, but TKU015 doesn&#39;t contain C16:0 2OH and C16:0 3OH. The fatty acid composition is also consistent with Pseudomonas sp. 
         [0028]    Preparation of Chitin 
         [0029]    In step a, squid pen powder (SPP) is mixed with a 2N NaOH solution in a ratio of 3:40 (w/v), the obtained mixture is heated in a 100° C. hot water bath for 30 minutes, residual and filtrate are separated by filtrating, the residual is dried at 60° C. and then weighted. The dried residual is mixed with a 2N HCl solution in a ration of 1:8 (w/v). The residual is soaked fort two days at room temperature. Residual and filtrate are separated by filtrating, the residual is dried at 60° C. and then weighted. 
         [0030]    In step b, 5 g residual is added into 50 mL of 12N HCl solution and then stirred for 1 hour, the obtained mixture is poured into distilled water, supernatant liquor is removed precipitation, and the residual is washed with 50 mL H 3 PO 4  (pH 7), the washing step is repeated until pH value of supernatant equals to 7. After the supernatant is removed, the obtained sediment is chitin suspension, which can be stored at 4° C. for testing its activity of chitinase. 
         [0031]    In order to produce chitinase and chitosanase, generally, TKU015 is cultured in a liquid shaking culture medium (pH 8) including 0.5% by weight of SSP, 0.1% by weight of K 2 HPO 4  and 0.05% by weight of MgSO 4 .7H2O for 3 days at 30° C. The obtained fermented supernatant is purified with ammonia sulfate precipitation, DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography, and Sephacryl S-100 chromatography in sequence, and a chitinase and a chitosanase is obtained. Testing result of Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) indicates that the chitinase has a molecular weight of 68 kDa and the chitosanase has a molecular weight of 30 kDa. An optimum pH value and an optimum temperature for producing chitinase are pH 5 and 50° C., and chitinase is stable under pH 5-7 and a temperature less than 60° C. An optimum pH value and an optimum temperature for producing chitosanase are pH 4 and 50° C., and chitinase is stable under pH 3-9 and a temperature less than 50° C. Activity of chitinase can be prohibited by Mn 2+  and Fe 2+ , and activity of chitosanase can be inhibited by Mn 2+ , Cu 2+ , and phenylmethanesulfonyl fluoride (PMSF). 
         [0032]    Activity Test of Nattokinase 
         [0033]    Protease activity of the supernatant obtained from culture medium of chitinase (cultured in a liquid shaking culture medium (pH 8) including 0.5% by weight of SSP, 0.1% by weight of K 2 HPO 4  and 0.05% by weight of MgSO 4 .7H 2 O for 3 days at 30° C.) is respectively tested using casein and N-succinyl-Ala-Ala-Pro-Phe-pNa as substrate. The supernatant hasn&#39;t protease activity when casein is employed as substrate. In contrast, the supernatant has protease activity when N-succinyl-Ala-Ala-Pro-Phe-pNa is employed as substrate; this indicates that the supernatant has nattokinase activity (0.02 FU/mL). In summary, the supernatant contains chitinase, chitosanase and nattokinase. 
         [0034]    Selection of Carbon/Nitrogen Source 
         [0035]    Chitosanase and nattokinase also exist in supernatant obtained from culture medium that is cultured under an appropriate environment for producing chitinase. In order to maximize output of nattokinase, SSP is employed as main carbon source of nattokinase. In addition, other condition of producing nattokinase is also discussed. Preferably, a concentration of SSP is 1% by weigh (3.8 FU/mL) (referring to  FIG. 3 ). 
         [0036]    Appropriate Condition for Producing Nattokinase 
         [0037]    As described above, appropriate carbon source for producing nattokinase is 1% SSP. The carbon source is cultured under different temperature (25° C., 30° C., 37° C.) for 1-4 days. The nattokinase activity testing results are shown in  FIG. 4 , when cultured for 2 days at 30° C., a highest nattokinase activity (4 FU/mL) is obtained. 30° C. is most appropriate temperature for producing nattokinase. 
         [0038]    Culture mediums have different pH values (5-10) is cultured for 1-4 days at 30° C., and nattokinase activity testing results are shown in  FIG. 5 , when the pH value of culture medium is 7 a highest nattokinase activity (4 FU/mL) is obtained. 
         [0039]    Different volumes (50 mL, 100 mL, 150 mL, and 200 mL) of culture medium, which contains 1% SSP and has a pH value of 8, is disposed in a 250 conical flask and cultured for 1-4 days at 30° C. nattokinase activity testing results are shown in  FIG. 6 , when the volume of culture medium is 100 mL a highest nattokinase activity (5.5 FU/mL) is obtained. 
         [0040]    In summary, optimum conditions for producing nattokinase include: 100 mL shaking culture medium (pH 7) including 1% by weight of SSP, 0.1% by weight of K 2 PO 4  and 0.05% by weight of MgSO 4 .7H2O cultured for 2 days at 30° C., which can result in a highest nattokinase activity. 
         [0041]      FIG. 7  illustrates a growth curve of  Pseudomonas  sp. TKU015 cultured in a shaking liquid medium (pH 8) including 0.5% by weight of SSP, 0.1% by weight of K 2 HPO 4 , and 0.05% by weight of MgSO 4 .7H2O at 30° C. It is known that nattokinase activity is highest on the second day, and then nattokinase activity decreases with increasing of time. The chitinase and chitosanase activity is highest on the third day. 
         [0042]    Separating and Purifying of Nattokinase 
         [0043]    Crude enzyme extract preparation: TKU015 is cultured in an appropriate culture medium; the obtained fermentation broth is processed with ammonium sulfate precipitation and centrifugal separation; remained sediments are re-dissolved with a few 50 mM phosphate buffer solution (pH 7) and then dialyze at 4° C.; the resulted dialysis fluid is crude enzyme extract (50 mL). 
         [0044]    Anion exchange chromatography: the crude enzyme extract is injected into a column for performing DEAE-Sepharose CL-6B column chromatography, a flow rate of the crude enzyme extract is 4 mL/min, every 6 mL of resulted solution is collected in a cuvette, non-adsorbed protein is collected, and then the column is eluted with 50 mM phosphate buffer solution (pH 7) that contain NaCl at a gradient of 0-1 M such that adsorbed protein is eluted out; nattokinase is found in non-adsorbed area and is collected as nattokinase solution (100 mL). 
         [0045]    Hydrophobic chromatography: 80 mL of nattokinase solution (containing 1 M ammonium sulfate) is injected into a Phenyl Sepharose 6 Fast Flow column, a flow rate of the nattokinase solution is 4 mL/min, each 6 mL of resulted solution is collected in a cuvette; firstly, the column is eluted with 50 mM phosphate buffer solution (pH 7) that contain 1 M ammonium sulfate; and then the column is eluted with different 50 mM phosphate buffer solution (pH 7) that contain from 1 M to 0 M of NaCl to change hydrophobicity of the column, the nattokinase solution can be separated under at different hydrophobicity. Finally, the column is eluted with 50 mM phosphate buffer solution (pH 7) to wash hydrophobic protein out of the column. Area having nattokinase activity is collected (70 mL) and enzyme characteristics analysis and SDS-PAGE is performed. 
         [0046]    Comprehensive Results: during the purification process, nattokinase exists in non-adsorbed area of the DEAE-Sepharose column, after the followed hydrophobic Phenyl-Sepharose chromatography step, TKU015 nattokinase (45 mg) can be separated. DEAE-Sepharose separation results at pH 7 show that chitosan and nattokinase both exists in non-adsorbed area and isoelectric points of both should belong to alkaline pH. 
         [0047]    DEAE-Sepharose separation results show that chitosan appears after the solvent gradient is increased; therefore, chitosan should be a hydrophobic protein. In contrast, nattokinase appears prior to the solvent gradient is increased; therefore, nattokinase should be a hydrophilic protein. Purification ration of nattokinase is 5.1. 
         [0048]    The Molecular Weight Determination of Nattokinase 
         [0049]    SDS-PAGE: molecular weight of nattokinase is analyzed using 12.5% SDS-PAGE, and testing result is shown in  FIG. 8 , in which Lane 1 belongs to crude enzyme extract and Lane 4 belongs to TKU015 nattokinase. Molecular weight of nattokinase is about 21 kDa. 
         [0050]    Comprehensive Results: molecular weight of TKU015 nattokinase is about 21 kDa, as shown in Table 1, molecular weight of majority of nattokinase that are produced from microorganisms are in a range from 20 kDa to 45 kDa. Currently, it is not found that  Pseudomonas  is used in production of nattokinase. Molecular weight of TKU015 nattokinase is less than that of nattokinase produced by bacteria, and is similar to that of nattokinase produced by fungi such as  Armillaria mellea  AMMP (21 kDa) and  Rhizopus chinensis.  12(18 kDa). As shown in Table 1, majority of nattokinase are produced by bacteria and fungi. Molecular weight of nattokinase produced from bacteria is about 30 kDa, and nattokinase produced from the  Pseudomonas  sp. TKU015 is similar to that of fungi. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Properties comparison of nattokinase produced by different microorganisms 
               
             
          
           
               
                   
                 Molecular 
                   
                   
                   
                   
               
               
                   
                 mass 
                 Optimum 
                 Optimum 
                 Type of 
               
               
                 Source 
                 (kDa) 
                 pH 
                 temp (° C.) 
                 protease 
                 Reference 
               
               
                   
               
             
          
           
               
                   Pseudomonas  sp. TKU015 
                 21 
                 7 
                 50 
                 Serine 
                   
               
               
                   Streptomyces  spp. SW-1 
                 30 
                 8 
                   
                 Serine 
                 Wang et al., 
               
               
                   
                   
                   
                   
                 and 
                 1999 
               
               
                   
                   
                   
                   
                 Metallo 
               
               
                   Rhizopus chinensis . 12 
                 18 
                 10.5 
                 45 
                   
                 Xiao-Lan et al., 
               
               
                   
                   
                   
                   
                   
                 2005 
               
               
                   Bacillus natto  nattokinase 
                 27.7 
                   
                   
                 Serine 
                 Fujita et al., 
               
               
                   
                   
                   
                   
                   
                 1993 
               
               
                 
                   Bacillus amyloliquefaciens 
                 
                 28 
                 9 
                 48 
                 Serine 
                 Peng et al., 
               
               
                 DC-4 
                   
                   
                   
                   
                 2003 
               
               
                   Bacillus subtilis  CK 
                 28.2 
                 10 
                 70 
                 Serine 
                 Kim et al., 
               
               
                   
                   
                   
                   
                   
                 1996 
               
               
                   Bacillus  sp. subtilisin DJ-4 
                 29 
                 10 
                 40 
                 Serine 
                 Kim and Choi, 
               
               
                   
                   
                   
                   
                   
                 2000 
               
               
                   Bacillus  sp. subtilisin QK-2 
                 28 
                 8.5 
                 55 
                 Serine 
                 Ko et al., 2004 
               
               
                   Bacillus subtilis  BK-17 
                 31 
                   
                   
                 Metallo 
                 Jeong et al., 
               
               
                   
                   
                   
                   
                   
                 2001 
               
               
                   Bacillus subtilis  KCK-7 
                 44 
                 8 
                 50 
                 Serine 
                 Paik et al., 2004 
               
               
                   Bacillus  sp. KA38 
                 41 
                 7 
                 40 
                 Metallo 
                 Kim et al., 1997 
               
               
                   Bacillus subtilis  strain A1 
                 31.4 
                 7 
                 50 
                 Metallo 
                 Jeong et al., 
               
               
                   
                   
                   
                   
                   
                 2004 
               
               
                   Armillaria mellea  AMMP 
                 21 
                 6 
                 33 
                 Metallo 
                 Lee et al., 2005 
               
               
                   Bacillus  sp. KDO-13 
                 45 
                 7 
                 60 
                 Metallo 
                 Lee et al., 2001 
               
               
                   
               
               
                 The references cited in Table 1 are listed in detail: 
               
               
                 1. Wang et al., 1999: Wang J, Wang M, Wang Y (1999) Purification and characterization of a novel fibrinolytic enzyme from  Streptomyces  spp. Chin.  J. Biotechnol.  15: 83-89. 
               
               
                 2. Xiao-Lan et al., 2005: Liu X L, Du L X, Lu F P, Zheng X Q, Xiao J. (2005) Purification and characterization of a novel fibrinolytic enzyme from  Rhizopus chinensis  12,  Appl. Microbiol. Biotechnol.  67: 209-214. 
               
               
                 3. Fujita et al., 1993: Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S (1993) Purification and characterization of a strong fibrinolytic enzyme (Nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan.  Biochem Biophys. Res. Commun.  197: 1340-1347. 
               
               
                 4. Peng et al., 2003: Peng Y, Huang Q, Zhang R H, Zhang Y Z (2003) Purification and characterization of a fibrinolytic enzyme produced by  Bacillus amyloliquefaciens  DC-4 screened from douchi, a traditional Chinese soybean food. Comp. Biochem. Physiol.  Biochem. Mol. Biol.  134: 45-52. 
               
               
                 5. Kim et al., 1996: Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S (1996) Purification and characterization of a fibrinolytic enzyme produced from  Bacillus  sp. strain CK 11-4 screened from Chungkook-Jang.  Appl. Environ. Microbiol.  62: 1488-2482. 
               
               
                 6. Kim and Choi, 2000: Kim S H, Choi N S (2000) Purification and characterization of subtilisin DJ-4 secreted by  Bacillus  sp. strain DJ-4 screened from Doen-Jang.  Biosci. Biotechnol. Biochem.  64: 1722-1725. 
               
               
                 7. Ko et al., 2004: Ko J H, Yan J P, Zhu L, Qi Y P (2004) Identification of two novel fibrinolytic enzymes from  Bacillus subtilis  QK02. Comp.  Biochem. Physiol. C Toxicol.  Pharmacol. 137: 65-74. 
               
               
                 8. Jeong et al., 2001: Jeong Y K, Park J U, BaekH, Park S H, Kong I S, Kim D W, Joo W H (2001) Purification and biochemical characterization of a fibrinolytic enzyme from  Bacillus subtilis  BK-17.  World J. Microbiol Biotechnol.  17: 89-92. 
               
               
                 9. Paik et al., 2004: Paik H D, Lee S K, Heo S, Kim S Y, Lee H, Kwon T J (2004) Purification and characterization of the fibrinolytic enzyme produced by  Bacillus subtilis  KCK-7 from Chungkookjang.  J. Microbiol. Biotechnol.  14: 829-835. 
               
               
                 10. Kim et al., 1997: Kim H K, Kim G T, Kim D K, Choi W A, Park S H, Jeong Y K, Kong I S (1997) Purification and characterization of a novel fibrinolytic enzyme from  Bacillus  sp. KA38 originated from fermented fish.  J. Ferment. Bioeng.  84: 307-312. 
               
               
                 11. Jeong et al., 2004: Jeong Y K, Kim J H, Gal S W, Kim J E, Park S S, Chung K T, Kim Y H, Kim B W, Joo W H (2004) Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from  Bacillus subtilis  Strain A1.  World J. Microbiol. Biotechnol.  20: 711-717. 
               
               
                 12. Lee et al., 2005: Lee S Y, Kim J S, Kim J E, Sapkota K, Shen M H, Kim S, Chun H S, Yoo J C, Choi H S, Kim M K, Kim S J (2005) Purification and characterization of fibrinolytic enzyme from cultured mycelia of  Armillaria mellea . Protein  Expr. Purif.  43: 10-17. 
               
               
                 13. Lee et al., 2001: Lee S K, Bae D H, Kwon T J, Lee S B, Lee H H, Park J H, Heo S, Johnson M G (2001) Purification and characterization of a fibrinolytic enzyme from  Bacillus  sp. KDO-13 isolated from soybean paste.  J. Microbiol. Biotechnol.  11: 845-852. 
               
             
          
         
       
     
         [0051]    Analysis of the Characteristics of Nattokinase 
         [0052]    Optimum temperature and thermal stability range of TKU015 nattokinase are 50° C. and &lt;37° C. As shown in table 1, most optimum temperature and thermal stability range of nattokinase are 37-50° C. and &lt;50° C. With respect to optimum temperature, nattokinases produced by bacteria of  Bacillus subtilis  KCK-7 (50° C.,  Bacillus subtilis  Strain A1 (50° C.) and  Bacillus amyloliquefaciens  DC-4 (48° C.) are similar to TKU015 nattokinase. With respect to thermal stability range, TKU015 nattokinase is relatively non-stable to heat, nattokinase produced by fungi such as  Rhizopus chinensis.  12 (&lt;37° C.),  Fomitella fraxinea  FFP2 (&lt;40° C.), (Park et al, 2005) is similar to TKU015 nattokinase. Above results indicate that molecular weight and temperature characteristics of  Pseudomonas  sp. TKU015 nattokinase are similar to that of fungi nattokinase. 
         [0053]    Optimum pH value and pH stability range of TKU015 nattokinase are pH 7 and pH 4-11. As shown in Table 1, most optimum pH values of nattokinase are in a range from neutral to a little alkaline, and pH stability range are pH 7-10. With respect to optimum pH value, nattokinases produced by  Bacillus  sp. KA38 (pH 7),  Bacillus  sp. KDO-13 (pH 7), and  Bacillus subtilis  Strain A1 (pH 7) are similar to TKU015 nattokinase. With respect to pH stability range, TKU015 nattokinase is stable in a weakly acidic or a weakly alkaline environment, and can remain 80% of activity in a solution having a pH value of 11, nattokinases produced by  Bacillus amyloliquefaciens  DC-4 (pH 4-11) and  Bacillus  sp.  subtilisin  QK-2 (pH 3-12) are similar to TKU015 nattokinase. 
         [0054]    Table 2 shows that purified TKU015 nattokinase, which belongs to serine-type protease, is completely inhibited by PMSF, and Fe 2+  can increase its activity. As shown in table 1, nattokinase produced by  Bacillus amyloliquefaciens  DC-4,  Bacillus  sp. Strain DJ-4,  Bacillus subtilis  QK-2,  Bacillus subtilis  KCK-7,  Bacillus natto  NK (Fujita et al ., 1993) and  Bacillus subtilis  DC33 (Wang et al, 2006) are inhibited by PMSF. When the concentration of Fe 2+  is 5 mM, nattokinase activity is increased to 232% of the original activity, nattokinase shows increased activity when Ca 2+ , Cu 2+  or Mg 2+  is added (Peng et al, 2005); Fe 2+  can influence TKU015 nattokinase activity, however Ca 2+ , Cu 2+ or Mg 2+ does not. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Influence of metal ions on TKU015 nattokinase activity 
               
             
          
           
               
                   
                   
                 Concentration 
                 Relative activity (%) 
               
               
                   
                 Metal ions 
                 (mM) 
                 Nattokinase 
               
               
                   
                   
               
             
          
           
               
                   
                 None 
                 0 
                 100 
               
               
                   
                 Mg 2+   
                 5 
                 104 
               
               
                   
                 Cu 2+   
                 5 
                 95 
               
               
                   
                 Fe 2+   
                 5 
                 232 
               
               
                   
                   
                 10 
                 156 
               
               
                   
                 Mn 2+   
                 5 
                 102 
               
               
                   
                 Zn 2+   
                 5 
                 94 
               
               
                   
                 Ba 2+   
                 5 
                 109 
               
               
                   
                 Ca 2+   
                 5 
                 108 
               
               
                   
                 Al 3+   
                 5 
                 101 
               
               
                   
                 EDTA 
                 5 
                 117 
               
               
                   
                 PMSF 
                 5 
                 45 
               
               
                   
                   
                 10 
                 0 
               
               
                   
                   
               
               
                   
                 Relative activity 100% represents 19 FU/mL 
               
             
          
         
       
     
         [0055]    Table 3 shows influence of different surfactants at different concentration on TKU015 nattokinase activity. When concentration of SDS is 0.5, 1, or 2 mM, TKU015 nattokinase remains 50% activity; when concentration of Triton X-100 is 0.5%, 1%, 2% (v/v), TKU015 nattokinase activity is respectively increased to 160%, 206%, 344% of original activity. In comparison, when concentration of SDS is 0.1%,  Pseudomonas aeruginosa  PseA protease remains 61% activity; and when concentration of Triton X-100 is 0.1% or 0.5%,  Pseudomonas aeruginosa  PseA protease activity doesn&#39;t change (Khare et al., 2005). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Influence of surfactants on TKU015 nattokinase activity 
               
             
          
           
               
                   
                   
                   
                 Relative activity (%) 
               
               
                   
                 Surfactant 
                 Concentration 
                 Nattokinase 
               
               
                   
                   
               
             
          
           
               
                   
                 None 
                 0 
                 100 
               
               
                   
                 SDS 
                  0.5(mM) 
                 66 
               
               
                   
                   
                 1 
                 56 
               
               
                   
                   
                 2 
                 56 
               
               
                   
                 Tween 20 
                 0.5(%) 
                 76 
               
               
                   
                   
                 1 
                 70 
               
               
                   
                   
                 2 
                 85 
               
               
                   
                 Tween 40 
                 0.5(%) 
                 94 
               
               
                   
                   
                 1 
                 70 
               
               
                   
                   
                 2 
                 82 
               
               
                   
                 Triton X-100 
                 0.5(%) 
                 160 
               
               
                   
                   
                 1 
                 206 
               
               
                   
                   
                 2 
                 344 
               
               
                   
                   
               
               
                   
                 Relative activity 100% represents 18 FU/mL 
               
             
          
         
       
     
         [0056]    Table 4 shows influence of different organic solvents on TKU015 nattokinase activity. TKU015 nattokinase activity increase to 150% in the presence of acetone; methanol, toluene and xylene do not affect TKU015 nattokinase activity; ether inhibits 50% of TKU015 nattokinase activity; and TKU015 nattokinase remain about 70% of activity in the other solvents. With respect to  Pseudomonas  protease, activity of  Pseudomonas aeruginosa  PseA, (Khare et al., 2005) doesn&#39;t change in the presence of toluene and N,N-dimethylformamide (DMF); activity of  Pseudomonas aeruginosa  PST-01, (Ogino et al., 1999) doesn&#39;t change in the presence of toluene, DMF and actone; activity of  Pseudomonas aeruginosa  san-ai strain, (Lee et al., 2006) doesn&#39;t change in the presence of DMF. Similar to PseA protease, PST-01 protease and san-ai protease, TKU015 nattokinase remains 80% of activity in the presence of DMF. In addition, activity of TKU015 nattokinase and PST-01 protease respectively increased to 150% and 200% in the presence of acetone. With respect to thermal stability , TKU015 nattokinase is mixed with different solvents and then respectively placed for ten days at 25° C. and 4° C. As shown in Table 5, the solvents don&#39;t affect activity of nattokinase at 25° C. and 4° C. Similarly, the same effects are also found in  Pseudomonas  sp. PseA protease (Khare et al., 2005) and  Pseudomonas  san-ai protease (Lee et al., 2006). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Influence of organic solvents on activity of TKU015 nattokinase 
               
             
          
           
               
                   
                   
                 Relative activity(%) 
               
               
                   
                 Organic solvents 
                 TKU015 nattokinase 
               
               
                   
                   
               
             
          
           
               
                   
                 None 
                 100 
               
               
                   
                 methanol 
                 96 
               
               
                   
                 ethanol 
                 65 
               
               
                   
                 ethyl ether 
                 43 
               
               
                   
                 toluene 
                 106 
               
               
                   
                 ethyl acetate 
                 70 
               
               
                   
                 acetonitrile 
                 88 
               
               
                   
                 acetone 
                 150 
               
               
                   
                 butanol 
                 70 
               
               
                   
                 isoamylalcohol 
                 60 
               
               
                   
                 isopropylalcohol 
                 68 
               
               
                   
                 hexane 
                 66 
               
               
                   
                 xylene 
                 96 
               
               
                   
                 DMSO 
                 80 
               
               
                   
                 DMF 
                 64 
               
               
                   
                   
               
               
                   
                 Relative activity 100% represents 20 FU/mL 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Influence of organic solvents on TKU015 nattokinase 
               
               
                 activity at different temperature 
               
             
          
           
               
                   
                   
                 Temperature 
                 Relative activity(%) 
               
               
                   
                 Organic solvent 
                 (° C.) 
                 TKU015 nattokinase 
               
               
                   
                   
               
             
          
           
               
                   
                 None 
                 25 
                 100 
               
               
                   
                   
                 4 
                 100 
               
               
                   
                 methanol 
                 25 
                 90 
               
               
                   
                   
                 4 
                 116 
               
               
                   
                 ethanol 
                 25 
                 88 
               
               
                   
                   
                 4 
                 114 
               
               
                   
                 ethyl ether 
                 25 
                 70 
               
               
                   
                   
                 4 
                 98 
               
               
                   
                 toluene 
                 25 
                 100 
               
               
                   
                   
                 4 
                 120 
               
               
                   
                 ethyl acetate 
                 25 
                 92 
               
               
                   
                   
                 4 
                 118 
               
               
                   
                 acetonitrile 
                 25 
                 80 
               
               
                   
                   
                 4 
                 90 
               
               
                   
                 acetone 
                 25 
                 108 
               
               
                   
                   
                 4 
                 128 
               
               
                   
                 butanol 
                 25 
                 85 
               
               
                   
                   
                 4 
                 125 
               
               
                   
                 isoamylalcohol 
                 25 
                 100 
               
               
                   
                   
                 4 
                 88 
               
               
                   
                 isopropylalcohol 
                 25 
                 120 
               
               
                   
                   
                 4 
                 115 
               
               
                   
                 xylene 
                 25 
                 96 
               
               
                   
                   
                 4 
                 126 
               
               
                   
                 DMSO 
                 25 
                 94 
               
               
                   
                   
                 4 
                 90 
               
               
                   
                 DMF 
                 25 
                 98 
               
               
                   
                   
                 4 
                 120 
               
               
                   
                   
               
               
                   
                 Relative activity 100% represents 15 FU/mL 
               
             
          
         
       
     
         [0057]      Pseudomonas  sp. TKU015 is cultured in a liquid medium (100 mL/250 mL), which employ shrimp shell powder as carbon/nitrogen source, for 2-3 days at 30° C., and chitinase, chitosanase and nattokinase activity are tested in the medium. The fermented supernatant is processed by Ammonium sulfate precipitation, DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography and Sephacryl S-100 chromatography, and then three pure enzymes (chitinase, chitosanase and nattokinase) are obtained. Test results of SDS-PAGE show molecular weight thereof are respectively 68 kDa, 30 kDa and 21 kDa. Respectively, optimum reacting pH value, optimum reacting temperature, pH stable range, thermal stable range of chitinase are pH 5, 50° C., pH 5-7, and &lt;60° C.; optimum reacting pH value, optimum reacting temperature, pH stable range, thermal stable range of chitosanase are pH 4, 50° C., pH 3-9, and &lt;50° C.; and optimum reacting pH value, optimum reacting temperature, pH stable range, thermal stable range of nattokinase are pH 7, 50° C., pH 4-11, and &lt;37° C. Activity of chitinase is inhibited by Mn 2+  and Fe 2+ , activity of chitosanase is inhibited by Mn 2+ , Cu 2+  and PMSF, and activity of nattokinase is completely inhibited by PMSF. Nattokinase belongs to serine-type protease, and Fe 2+  can increase its activity. Acetone can affect activity of chitinase, acetonitrile can affect activity of chitosanase, and ether can affect activity of nattokinase. With respect to organic solvent resistance, after placed for 10 days at 25° C. and 4° C., test results show that toluene, xylene and DMF don&#39;t affect activity of chitinase while the other organic solvents decrease activity of chitinase at 25° C., ether, acetonitrile and acetone can affect activity of chitinase; toluene, xylene and DMF don&#39;t affect activity of chitosanase while the other organic solvents decrease activity of chitosanase at 25° C., and ether and acetone can affect activity of chitosanase at 4° C.; and all the organic solvents don&#39;t affect activity of nattokinase. 
         [0058]    Generally, shrimp and crab shell are useless and often thrown away. However shrimp and crab shell contains mass amount of protein and chitin, the present invention utilize a new strain TKU015 to ferment the shrimp and crab shell, and finally, valuable bioactive substance nattokinase can be produced, pollution to environment is reduced and value in use of shrimp shell castoff is improved. 
         [0059]    The above description is given by way of example, and not limitation. Given the above disclosure, one skilled in the art could devise variations that are within the scope and spirit of the invention disclosed herein, including configurations ways of the recessed portions and materials and/or designs of the attaching structures. Further, the various features of the embodiments disclosed herein can be used alone, or in varying combinations with each other and are not intended to be limited to the specific combination described herein. Thus, the scope of the claims is not to be limited by the illustrated embodiments.