INTRODUCTION Enzymes?

Enzymes are the biological substances (proteins) that act as catalysts and help complex reactions occur everywhere in life. These are central to every biological stepwise reaction which act in a sequential manner catalyzing hundreds of by which nutrient molecules are degraded, chemical energy is conserved and transformed, and biological macro-molecules are made from simpler precurpors. Most of the enzymes are highly specialized proteins (except for a few RNA molecules possessing catalytic power) wi8th high substrate specificity and whose catalytic activity depends on the integrity of its native protein conformation. Enzymes have proved themselves to be important practical tools in medical purposes, dishwashing, textile, silver recovery, pulp industries, food processing, feeds and chemical industrial as well as waste treatment. They have been isolated from microorganisms like bacteria, yeast and mould and even plants and animals. Enzymes have replaced many chemical used in detergents, food processing AND IN LEATHER INDUSTRIES AS THESE IS JMORE EXPLAINS CURRENTLY ON THE DEVELOPMENT OF ENVIRONMENT FRIENDLY TECHNOLOGY. For example, leather industry wastes leads to environmental pollutions and health hazards (Malathai and chakraborty, 1001). Major pollutants from the leather industry that may have significant environmental impact include lime, sulphide & chromium (Alessandro et al, 2003). Enzymatic dehairing is being increasingly looked upon as a reliable alternative to the conventional lime culphide process as it completely eliminates lime & sulphide from

the effulent, helps recover hair as a byproduct and eliminates the sating process required during eleliming (Zambare et al., 2007. The 1999 world market for industrial enzymes was estimated at more than $1.6 billion.yhe market for detergent enzymes, including protease, celluase, -amylose, & lipase, occupies approximately 40% of the world market (Horikosh & susumu). Additionally, microbial proteases are among the most important hydrolytic enzymes. They account for 40% of the total enzyme sales in food, leather, Detergent, pharmaceutical industries and in eliagnostics, waste management and silver recovery (Gupta et al., 2002). A protease is an enzyme that undergoes proteolysis. Proteases differ in their ability to hydrolyze various peptide bonds. Examples of proteases include: fungal proteases, pepsin, trypsin, chymotrypsin, papain, bromelain & subtitsin. Protease digest various proteins found in gelatin, meat, grains, vegetables extracts and release amino acids and small peptides. In food industry, the proteases ar used for making bread, beer, cheese, lookies & cakes and meat tenderizing. In leather industries, these enzymes are employed for removing wool, hair & pigments from animal skin and to soften it. Application of proteases in laundry detergents is for removing protein stams such as blood, chocolate, milk, keratin, gelatin, grains and vegetables. Owing to digest to the better cleansing properties of enzymes based detergents at lower washing temperature and pollution alleviating capacity overt conventional Synthetic detergents (Mukherjee et al., 2008; Krik et al., 2002). In pharmaceutical industry, proteases have been used in debridement of wounds, in skin ulceration and as

contact lens enzyme cleaners (Anwar & Saleemudin., 2002). Additionally, proteolytic enzymes have been used for a long time in various forms of therapy. Their use in medicine is indicating their benefits in oncology, inflammatory conditions, blood rheology control and immune regulation. Oral proteases have been shown to be absorbed and carried into the blood stream where they are bound to alphs2-macroglobulin and hydrolyze immune complexes, proteinacious debris, damaged proteins, and acute phase plasma proteins in the blood stream. Commercially, proteases are produced in highly controlled aseptic conditions for food supplementation and systemic enzyme therapy against fatal diseases like cancer, malaria, controlling vasoconstrictor activity, neuroplasticty, haptitis C, thrombolytic therapy and AIDS. It has been found that HIV, the essential for the retroviral life cycle and it has been good target for chemo therapy with specific inlibitors. Orally ingested protease can bypass the conditions of the GI tract and be absorbed into the blood stream while still maintaining their enzymatic activity. The organism most often used are Aspergillus niger and oryzae. The world market for enzymes is growing at the rate of 7.6% per year and expected to reach $7.3 billion by 2013 as predicted in the Fredonia Group Inc. business report (WWW.freedoniagroup.com). http://www. Freedmoniagroup.com/world-enzymes. Html The market for detergent enzyme including protease, cellulose, - amylase & lipase is given below: -

Fig- overview of contribution of different enzyme to total rate of enzyme (http////mbr.asm.org/cgi/content/62/3/5977#secdo). Microbial proteases account for approximately 60% of the total world wide enz. Sales. Among the various proteases produced, the alkaline proteases particularly those isolated from Bacillus species have found major application in detergent industry as they hjave high temp. and ph optima and are able to withstand harsh conditions that prevail during wash cycle. Majority of proteases are known to work near neutral or nearly alkaline ph and temp. much less than 100 C (arulmani et al., 2007; Kumar and Bhalla., 2004). Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures. The present investigation is based on a protease which shows fibrinolytic activity. According to data of world Health Organization (WHO) in 2000, 29% of the total mortality rate in the world is caused by cordiouascular diseases (Mine Yetal, 2005). When fibrin clots are not lysed, they accumulate in bold vessels and cause thrombosis leading to cardiovascular diseases (Kim et al., 1996; Kim et al 2000). These diseases, including acute myocardial infarction, ischemic heart disease, volucular heart disease, peripheral vascular disease, arrhythmias, high blood pressure, and stroke, are the leading causes of death throughout the world (Mine et al, 2005). The major protein component of blood clots, fibrin, is formed from fibrinogen via the proteolysis by thrombin. Meanwhile, the fibrin clots can be hydrolyzed by plasmin to avoid thrombosis in blood vessels. Under an unbalanced situation due to some disorders, the

clots are not hydrolyzed, & thus thrombosis occurs. Over the years, thrombolytic therapies via injecting or orally administrating thrombolytic agents to lyse thrombi in blood vessels have been extensively investigated. (Goldhaber & bounameause, 2001; Tough 2005). Based on working mechanisms, thrombolytic agents are of two types; one is plasminogen activators and urokinase which activates plasminogen into active plasmin to degrade fibrin & the other is plasmin like proteins which directly degrade fibrin (Peng et al, 2005). Moreover, on the basis of catalytic mechanisms, microbial fibrinolytic enzymes are classified into three types: - Serine protease (eg. Alottokinase), metalloprotease (eg. Armillariamella ea metalloproteae and mixture of both serine and metalloprotease (eg. Proteaswe from streptomyces sp. Y405) (Peng et al, 2005). Despite widespread uses, thrombolytic agents such as t-99 and urokinase are expensive, exhibit low fibrin specificity, and have undesired sideeffects such as gastrointestinal bleeding, resistance to repercussion and allergic reactions (Hua et al, 2008. Therefore, continuous efforts have been focused in the search of sager and less expensive thrombolytic agents from diverse sources. Until recently, fibrinolytic enzyme with potential thrombolytic application have been purified from various forces such as fermented food, earthworms, snake alenoms and microbialo sources (chag et al, 2005). from mixeobial soucces, bacteria, actinomycetes, fungi & algae are reported to produce fibrinolytic enzymes. For the treatment of thrombotic diseases, at present, fibrinolytic therapy has been often employed mainly with the use of the plasminogen activators such as streptokinase,

urokinase, and the genetically engineered tissue plasmino9gen activator. Intravenous administration of urokinase & streptokinase has been widely used but these enzymes have low specificity to fibrin and are expensive. Although t-PA and urokinase are still widely used in thrombolytic therapy today, their expensive prices & undesireable sideeffects, such as the risk for internal hemorrhage within the intestinal tract when orally administrated, prompt researchers to search for cheaper and safer resources. For thrombolytiuc therapy, microbial fibrinholytic enzymes have now attracted much more attention than typical thrombolytic agents because of the expensive prices and the undesireable side effects if the latter. The fbrinolytic enzymes were successively discovered from different micro-organisms, the most important among which is the genus Bacillus from traditional fermented food. The physiochemical properties of these enzymes have been characterized, and their effectiveness in thrombolysis in aiiuo has been further identified. Therefore, microbial fibrinolytic enzymes, especially those from food grade micro-organisms have the potential to be developed as fuctional fpood additives and drugs to present or cure thrombosis and other related diseases. Keeping in view the seneficial application of fibrinolytic protease, the present study was planned with the following objectives: (1) Isolation of fibrinolytic protease producing micro organisms from natural habitates. (2) Medium optimization for maximum production. (3) Partial purification and biochemical characterization of the fibrinolytice protease.

REVIEW OF LITERATURE ISOLATION AND SCREENING Micro-organisms are isolated from various natural sources where they are present in abundance for used at large scale production for enzyme. Bacillys subtilis natto, producing a fotent fibrinolytic enzyme, natto kinase, which was isolated in the extract of natto produced via fermentation from boiled soyabean. (Merualle et al., 2011). Bacillus amyloliquefaciens Am6 was isolated from the soil of detergent industry in Tunisia and the use of powder from Mirabilis frlaps tubers acted as a new microbial growth substrate for the production of fibrinolytic protease by this strain (agrebi et al., 2010). Streptomyces megaspores SD5, isolated from the water of a hot spring, can produce a strong thermostable fibrinolytic enzyme (Chitte and Day., 2000). The fibrinolytic enbzymes were isolated from different fermented foods by various bacillus species such as B.amyloliquefaciens DC-4 from the Chinese souabean-fermented food named douche (feng and Zhang., 2002), Bacillis species CK from the Korean doen-jang (choi et al., 2005; Kim & choi 2000), and Cacillus sp. KA38 from the Korean salty fermented fish called joet gal (Kim et al., 1997). Bacillus subtilis producing Nottokinase, B Haemolytic streptolocci with heamolytic activity & Preudomenas sp. Producing urokinase activity were isolated from soil sample collected from various regions in Kolkata, India identified by colony morphology, Grams staining, biochemical tests and selective media;

different samples of blood and biomass from infected throat. The bacteria was identified by blood agar selective media, Grams staining & biochemical tests, and from human urine sample and identified by cetrimide agar selective media, grams staining and biochemical tests. These three fibrinolytic proteases were screened using nutrient agar medium containing 2% casin & 2ml human serum (Dubey et al, 2011). Bacillus sphaericus of H5a5b serotype was obtained from the culture collection of vector control research centre (VCRC),Puducherry code named as VCRC B42 for isolating novel fibrinolytic enzyme named as thrombinase (Nalaraman & Prabakaran, 2007). Bacillus subtilis dc 33 producing a strong fibrin-specific enzyme was newly isolated from ba-boo douche, a typical and flavor-rich fermented soyabean food in China (Wang et al, 2006). Bacillus subtitis CD-8547, a mutant capable of high fibrinolytic enzyme production by mutagenesis was isolated from douche, a chinese soyabean fermented food (Wang et al., 2008). Stroptromyces omiyaensis containing serine protease was isolated from soil samples showed highly potent fibrinolytic activity (Vesugi et al, 2011). Bacillus polymaxa was isolated from soil MRC-1A code named as NRC-A producing fibrinolytic enz maintained on nutrient agar medium (Mahmoud et al, 2011).

Bacterium a26 was isolated from seawater in sfax city (Timisia) which were plated on to skim-milk agar plates to show for protease activity and then screened for fibrino activity through fibrin agar plate method (Agrebi et al, 2009). Bacillus subtilis ICTF-1was isolated from marinemiche of the western seacost of Maharashtra (India) using spread plate & streaking method which were screened for fibrinolytic enzyme production (mahajan et al, 2011). Paenibacillus polynyxa EJS-3producing fibrinolytic enzyme ws isolated from tissues of stemona japonica mig a Chinese traditional medicine which were screed by fibrin plate method Lu et al, 2010). Streptomyces so, C9684 was isolated from Korean soil with profound prote0lytic enzyme with fibrinolytic activity and were screened for above activity by and were screened for above activity by fibrin plate method (Simkhada et al, 2010). The samples of bionectria sp., an unconventional enzyme-producing fungas was isolated from las vung as pedemontana forest (tueman, argentina), b/w 400 & 700 metres above sea level which were directly ddetected by the naked eye (mushrooms, cap & shelf fungi) as well as mycelia mats growing on soils, logs, twigs, leaves, roots, stones etc. where aseptically transferred to sterile flasks which were further screened for proteolytic activity and fibrinolytic activity through 1% skimmed milk Agar plate as well as fibrin plate method respectively (Rovati et al, 2010). Ganoderma luciduim UK12. Was collected from different ecological miches of southern India and the pure cultures were prepared and maintained in pltato

dextrose Agar medium which were screened for the production of fibrinolyutic protease by growing them on fibrin sett basal medium (Kumaran et al, 2011). Paecilonyces tancipes, a famous Chinese medicinal entomopathogenic fungus used in Japan, Korea & China, was obtained from the culture collection of DNA Bank of Mushrooms (CCDMB, IVM01135), Inchoon, Republic of Korea having novel, highly potent fibrinolytic proteins and were screened for fibrinolytic activity by fibrin Agarose plate method of Astrup & Mullertz with slight modifications (Kim et al, 2011). The earthworm lumbricus subellus was isolated from a local farm containing an anticoagulant / fibrinolytic protease which were scrneened through clotting time delay method of Baugham and fibrin plate method (Jeon et al, 1995). The in vitro fibrinolytic activity on plasma clot is the inherent property of fibrinolytic protease present in latex of Synadenium Grantii Hook f which was isolated from outskirts of R.L.Jalappa hospital and Research Centre, Kolar and were screened for fibrinolytic protease by plasma clot method described by Simon & Michalle (Daganend & Murthy, 2010). TIME PROFILE Protease Production in solid-state fermentation was studied over a 10-day period. It is notable, enzyme production was studied over and incubation time of 48 hr. for bacteria and 8-9 days for fungi. In Bacillus subtilis DC 33, highest protease activity for fibrinolytic enzyme was achieved after 72 hrs. of incubation period (Hang et al., 2006).

Fibronolytic serine protease isolated from Bacillus amylolique faciens An6 showed maximum activity after 24hr. of incubation (Agrebi et al., 2010). A low molecular weight chymotrypsin-like novel fibronolytic enzyme from stroptomyces sp. CS624 shoed maximum activity after 72 hrs. of incubation (Mander et al, 2011). The enzyme production was maximum at 120h of fermentation by alkaline protease from Streptomycecs gulbaragensis which showed profound application in removal of blood stains (Vishalakshi et al., 2009). OPTIMIZATION OF CARBON SOURCES Bacillus subtilis DC33 showed increase in fibrinolytic protease activity with galactose (Wang et al., 2006). Enterococcus faecalis with strong fibrinolytic activity was optimized by 1% Glucose which supported the highest growth (OD 600, 3.521) as well as relative fibrinolytic activity of 100% (Voon et al., 2002). A fungal species, Rhizomucor miehei Schipper used Glucose (3%) for the maximum fibrinolytic protease production (Ali & Ibrahim., 2008). H&W as a carbon source was found to be the most suitable substrates for the production of fibronolytic activity from a newly isolated marine bacterium Bacillus subtilis A26 (Agrebi et al., 2009). Bacillus subtilis ICTF-1 showed maltose to be the best source for fibrinbolytic enzyme production (Mahajan et al., 2011). In case of bacillus subtilis CD-8547, rice power (5%) supported the maximum production of fibrinolytic enzyme (Wang et al., 2008). OPTIMIZATION OF NITROGEN SOURCES

Streptomyces sp. SD5 showed peptone to be the best source for fibrinolytic protease production (Chitte & Dey, 2000). Soyabean power (4%). Was found to be the best nitrogen source for the production of fibrinolytic enzyme in case of Bacillus sp. i.e. bacillus subtilis CD-8547 (Wang et al., 2008). A marine bacterium Bacillus subtilis ICTF-1 showed soy peptone and yeast extract to be the best source for fibrinolytic protease production (Mahajan et al., 2011). Casein peptone was found to be the most suitable substrates for the production of fibrinolytic activity fro in a marine bacterium Bacillus subtilis A26 (Agrebi et al., 2009). Bacillus subtilis DC33 used tryptone for the maximum fibrinolytic protease production (Wang et al., 2006). ENZYME PURIFICATION A marine bacterium strain Bacillus subtilis ICTF-1 supernatent was purified by in three steps, i.e., ammonium sulfate fractionation followed by Unoq Sepharose strong onion exchange and butyl sephaarose FF hydrophobic interaction chromatography. Molecular weight of purified enzyme was estimated by SDSPAGE (Mahajan et al., 2011). An anticoagulant / fibrinolytic protease from lumbricus rubellus was purified by a combination of ammonium sulfate fractionation and two chromatographic procedures i.e. Benzamicline-Sepharose Colum & DEAE-Sephacel column. Molecular weight of purified enzyme was estimated by SDS-PAGE and gel filteration and fibrinolytic activity was analyzed by Zymography (Jeon et al., 1994).

Streptomyces sp. CS624 was concentrated by ammonium sulfate precipitation and purified using Gel filteration with Sepharose CL-68 column and hydrophobic interaction with phenyl Sepharose CL-4B column. Molecular weight of purified enzyme was determined by SDS-PAGE. (Mander et al., 2011). Aeromonas sp. JH1 sample was purified by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, Sephadex G-75 gel filteration and (FPLC) Fast performance liquid chromatography using linear Superdex 75 Hr 10/31 gel filteration and finally lyophilized. The molecular weight was determined by FPLC and SDS-PAGE as well as fibrinolytic activity was checked by Zymography (Cho et al., 2011). Paenibacillus polymyxa EJS-3proteins were purified by ammonium sulfate precipitation and a combination of chromatographic steps having Hiprep Phenyl FF column, RESOURCEM & column and Sephacryl S-300 HR column as well as further purification was carried out by SDS-PAGE and fibrinolytic activity was analyzed by fibrin Zymography. The molecular weight of purified enz obtained by MALDI-TOF-MS (Lu et al., 2010). Bacillus amyloliquefaciens An6 fibrinolytic protease was purified to homogeneity using acetone precipitation, and cation exchange chromatography. Molecular weight of purified enzyme was estimated by SDS-PAGE and activity was analysed by fibrin Zymography (Agrebi et al., 2010). Bacillus subtilis, -haemolytic streptococci and recombinant E.coli containing short fragment genomic DNA of Pseudomenas sp. Showed fibrinolytic activity was

were subjected to purification by ammonium sulfate precipitation, onion exchange column chromatography (DESE Cellulose and Gel filteration with Sephadex G200. The molecular weights were determined by SDS-PAGE (Dubey et al., 2011). Ganoderma lucidum YK12 was concentrated by ammonium sulphate precipitation and purified by DEAE-Cellulose, Anion Exchange chromatography as well as sephadex G-150 column chromatography. Molecular weight of purified enzyme was estimated by SDS-PAGE (Kumaran et al., 2011). Bacillus polymaxa NRC-a sample was purified by using different standard techeniques, i.e., ammonium sulphate precipitation, DEAE-Sepharose ion exchanges chromatography and Sephacryl S-200 Gel filteration chromatography. Molecular weight of purified enzyme was obtained by SDS-PAGE (Mohamoud et al., 2011) Paecilomyces tencipes, a fungal species, was purified to homogeneity using ethanol precipitation cation exchange chromatography (CM-cellulose), Anion Exchange chromatography (CL6B fast flow), Gel filteration chromatography (Sephadex G-75) and Fast protein liquid chromatography (porous 20 HQ). Molecular weight of purified enzyme was determined by SDS-PAGE and fibrinolytic activity was analyzed by fibrin Zymography (Kim et al., 2011). TEMPERATURE AND PH OPTIMIZATION S.No NAME REFERE Proe Opt.te Ppt. Temp.sta PH

(Spacies/E nz.)

NCES

ln

mp.

PH For

bility

stabil ity

Enz. For

proeln prod n 1. KCK-7 Paik et al., 2004 500 C 8 Stable Over

upto 600 Ph 7C 10

2. Bacillus Sp. KDO-13 3. R.Chinensi s 12 4. B.subtilis A36

Lee et al., 2001

600 C

Upto 500 7-9 C

Xiao-lan Et al., 2005

450 C

10.5 370 C

6.88.8

Agrebi et 24L al., 2009

600 C

50-700 C

7-12

5. B.polyma MRC-A

Mahmoud 72lr et 2011 al.,

400 C

8.5

Upto 400 7-9 C

6. B.amyloliq uefaceins An6 7. Paenibacill us Polynyxa EJS-3 8. Bacillus subtilis CD-8547 9. B.subtilis DC33

Agrebi et 24L al., 2010 Lu et al., 2010 72L

600 C

40-700 C

7-10

370 C

7.5

30 -400 C 7-8

Wang al., 2008 Wang al., 2006

et 72L

500 C

35-600 C

7-10

et 72L

550 C

Upto Below 600 C

5-12

10. Oidiosendr on flavum

Tharwat, 2006

24l 21d ays

450 & 8 550 C

Upto 700 6-11 C

11. Perennipori Kim a fraxinea 12. Rhizonucor Ali miechei Ibrahim, al.,2008

et 10

B/W

Upto 550 2-9 C

days 350400 c & 6 days 350 C 8

30-400 C

6-9

2008 13. Stre[tp,uces Chitte Megasporu s SD5 Dey., 2000 5 450 C 9 0-450 C 5-10 & 18L 550 C 8 37-600 C 6-9

14. Stretpomyo Vishalaks s

hi et al., days

Gulbargens 2009 is 15. Subtilisin DFE 16. Jeot-gal enzyme 17. SW-1 Peng al., 2003 Kim et al; 1997 Wang al; 1999 18. Streptomyc Mander et 72L es Sp. CS624 al; 2011 600 C 7 Stable at 6.5or below 7.5 500 C et 370 C 8 400 C 7 et 480 C 9 Less than 6-10 500 C Upto 400 7-9 C 4-370 C 4-9

CLONING OF PROTEASE GENE A fibrinolytic enzyme producing strain was isolated from Chinese soyabean paste and identified B.amylolique-faicens LSSE-62 , which is a close relative to

B.subtilis as a food grade micro organism. The solid state fermentation of chick feas with B. amyloliquefaciens was investigated for the first time. This FF (fibrinolytic Protease) was expressed in E.coli DH5 resulting in the production of active fibrinolytic protease (Wei et al., 2011). The cloning & sequencing of the gene encoding a novel protease (FP) from fusarium sp. BLB showed higher hydrolytic activity towards synthetic peptides than that of any other protease tested, including natokinase which suggest that FP from this filamentous fungus is superior to Nattokinase in dissolving fission when absorbed into the blood. To maximize industrial enzyme production, the host organism is generally manipulated to carry multiple copies of the gene (sugimoto et al., 2007). Nattokinase, an oral health product for the prevention of otherosclerosis, was expressed from Bacillus subtilis to a live delivery vehicle, lactococcus lactis using promoter pnisz & signal peptide SPUSP for inducke and secretary expression of nattokinase in L.Tactis. Western blotting analysis demonstrated that nattokinase was successfully expressed. The recombinant nattokinase showed potent fibrinolytic activity. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle of l.lactis would facilitate the wide-spread application of mottokinase in the control & prevention of thrombosis diseases (Lian et al., 2007).