Research Article Annals of Genetics and Genetic Disorders Published: 27 Sep, 2018

Anticoagulant Property of Metalloprotease Produced by Pseudomonas fluorescens Migula B426

Usharani Brammacharry1 and Muthuraj Muthaiah2* 1Department of Biomedical Genetics, University of Madras, India

2State TB Training and Demonstration Centre, Intermediate Reference Laboratory, India

Abstract In this study we endeavoured for the production, purification and characterization of anticoagulant property of metalloprotease from Pseudomonas fluorescens Migula B426 strain. Shake flask fermentation was performed to produce the enzyme and the fibrinolytic activities of the enzyme of this isolate. These were estimated to range across 40 to 5000 IU/mL of urokinase through the standard curve using the thrombolytic area on the fibrin plate. Purification of crude enzyme was done through Sephadex S-300 gel filtration and its activity was 2474 IU/mL.Enzyme activity was enhanced bydivalent cations Mg2+ and Ca2+ in the presence of Ethylene Diamine Tetra Acetic acid (EDTA), a metal-chelating agent and two metalloprotease inhibitors, 2, 2′-bipyridine and o-phenanthroline, repressed the enzymatic activity significantly. Amino acids of N-terminal sequence have great similarity with those of metalloprotease from various Pseudomonas strains and have consensus sequence, HEXXH zinc binding motif. These results strongly suggest that the extracellular enzyme of P.fluorescens Migula B426 strain is a novel zinc metalloprotease and is based on its N-terminal amino acid sequence, effect of protease inhibitors and its fibrinolytic activity. It can be further developed as a potential candidate for thrombolytic therapy. Keywords: Fibrin; Metalloprotease; Pseudomonas fluorescens; Zinc binding motif

Introduction OPEN ACCESS Extracellular have great commercial value and find multiple applications in various *Correspondence: industrial sectors. Fibrinolytic enzyme is well known as a sub class of protease having competence Muthuraj Muthaiah, State TB to degrade fibrin. Deposition of fibrin in blood vessels normally increases thrombosis, resulting Training and Demonstration Centre, in myocardial infarction and other cardiovascular diseases [1]. Thrombosis can lead to cerebral Intermediate Reference Laboratory, and myocardial infarction due to unlysed blood clots. Approximately 31% of the total mortality Government Hospital for Chest rate in the world is due to cardiovascular diseases and another report of WHO shows about 18 Diseases, Puducherry, India, million people die every year from cardiovascular diseases [2]. Fundamental pathophysiological process related to this devastating problem is the formation of fibrin (blood clots) adhering to the E-mail: [email protected] unbroken wall of blood vessels. Fibrin can accumulate in the blood vessels resulting in interfering Received Date: 21 Aug 2018 blood flow leading to myocardial infarction and other serious cardiovascular diseases. Unless the Accepted Date: 20 Sep 2018 blockage is removed promptly, tissue that is normally supplied with oxygen by the vessel will be Published Date: 27 Sep 2018 severely damaged [3]. If damaged tissue or region is large, normal conduction of electrical signals Citation: through ventricle will be disturbed, leading to irregular heartbeat, cardiac arrest or even death [4]. Brammacharry U, Muthaiah A number of proteases which can interfere with blood clotting have been characterized from M. Anticoagulant Property of various sources including microorganisms and some of them are fibrinolytic capable of Metalloprotease Produced by digesting fibrin. Fibrinolytic enzymes from microbial sources have been reported from various Pseudomonas fluorescens Migula species of Bacillus, Pseudomonas, Staphylococcus, Alteromonas, Coryneform bacteria, Penicillium, B426. Ann Genet Genet Disord. 2018; Aspergillus, Fusarium, Trichotecium, Actinomyces, Streptomyces and Escherichia coli [5,6]. Bacillus 1(1): 1005. species produces a variety of fibrinolytic enzymes including nattokinase (NK) from Bacillus natto Copyright © 2018 Muthuraj [7], substilin DFE [8] and substilin DJ-4 from Bacillus amyloliquefaciens [9]. A fibrinolytic enzyme Muthaiah. This is an open access from Bacillus subtilis LD-8547 has also been reported by Wang et al. [3]. Almost all fibrinolytic article distributed under the Creative enzymes are serine proteases belonging to the subtilisin family of Bacillus origin. All these fibrinolytic Commons Attribution License, which enzymes have high substrate specificity to fibrin, different from other proteases with broad substrate permits unrestricted use, distribution, specificity. and reproduction in any medium, The Pseudomonas fluorescens Migula B426 strain produced a low molecular weight provided the original work is properly metalloprotease. A proteolytic enzyme with fibrinolytic activity was found in the culture medium; we cited.

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Fibrinogen solution (5ml 0.6%) in 50mM sodium phosphate buffer mAU 120 Active fraction (pH 7.4) was mixed with 5ml of 2% (wt/vol) agarose solution and poured into a petridish containing 0.1ml of thrombin solution 100 (10NIH unit/ml) and allowed to stand for 1h at room temperature in

80 order to form a fibrin clot layer. Sample solution (3mg in 25μl) was dropped into holes previously made on a fibrin plate using a capillary ° 60 glass tube (5mm diameter) and incubated at 37 C for 18h. An equal volume (3mg) of plasmin solution (1NIH unit/ml) was incubated in a 40 hole on the fibrin plate as a standard protease. The zone of fibrinolytic activity was observed as hydrolysis of fibrin [11]. 20 Quantitative assay of fibrinolytic activity 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 A standard curve of urokinase activity was plotted according to 0 50 100 150 200 min fibrin plate method [11] with slight modification for the estimation Figure1a: FPLC profile of purified fraction of extra cellular protein P. of fibrinolytic activity. In brief, 2ml of fibrinogen solution (50mg/ml) fluorescens. was mixed with 45ml of 0.8% agar solution (w/v) in 20 mmol/L Tris- HCl buffer (pH 8.0) at 50°C, and approximately 8ml of this solution

was poured into a petridish containing 40µl thrombin solution A B (80NIH/ml thrombin in 20mmol/L Tris-HCl, pH8.0). Petridish was allowed to stand for 35m to form fibrin clots, and five holes (3mm diameter) were made on the fibrin plate. Aliquots of 10μl of urokinase at 40, 80, 160, 320, 640, 1280, 2560 and 5000 IU/mL were separately added in each hole and incubated at 37°C for 24h, with duplicates. Diameter of the cleared zone was

Absorbance (280 nm) (280 nm) Absorbance measured and the thrombolytic activity was calculated. Standard curve showing the relationship between urokinase activity (IU/mL) and thrombolytic circle area (mm2) was established. Fibrinolytic activity of sample was determined using the curve after adding 15μl 5 10 15 20 5 10 15 20 Retention time (min) of sample on each hole of the fibrin plate, incubating at 37°C for 18h

Figure 1b: FPLC profile of crude extract (A) and purified fraction (B) of extra and measuring the lytic zone area. cellular protein of P. fluorescens. Microplate assay of fibrinolytic activity Fibrinolytic activity was assayed by measuring the decrease in therefore, are interested in its potential bio catalytic application and turbidity of fibrin polymer in a 96 well plate using a spectrophotometric describe in the following purification and anticoagulant properties of method. Ninety microliters of 1 mg/ml fibrinogen in 25mM phosphate metalloprotease enzyme. buffer (pH 7.5) was added to 10μl thrombin (10 U/ml), and the fibrin Materials and Methods polymer was incubated at room temperature for 1h. Thereafter, 10μl of plasmin (1μg) was added to the polymer and incubated for 30m at Production of extracellular enzyme from P. fluorescens room temperature; decrease in absorbance at 350nm was recorded Migula with a 96-well plate reader [11]. Seed culture of the bacterium, P. fluorescens Migula B426, was obtained from the culture collection of Vector Control Research Assays of elastase activity Centre that was inoculated in 600 ml of Glucose Peptone Salt (GPS) Radial diffusion in agarose gel containing was used for medium containing 1.0% (w/v) glucose and peptone, and 0.1M standard determination of elastinolytic activity. Agarose 1% was potassium dihydrogen phosphate (pH 7.0) and was incubated at 37°C dissolved in 50 ml of 50mM Tris-HCl buffer, pH 7.6 containing in a chamber rotating at 500rpm for 96h. Production of extra cellular 200mM NaCl and the mixture was cooled to 65°C. Elastin (0.8 mg/ protease enzyme in the culture supernatant was checked at different ml) was added to the agarose solution. Sodium dodecyl sulfate (SDS: stages from 24h to 96h for growth of the bacterium [10]. elastin ratio 1:4 by weight) was added to the mixture and sodium azide (0.05%) was added to prevent bacterial growth. Aliquots of 11ml each Precipitation and Purification of bacterial protein were poured into petridishes and the plates were kept overnight to Enzyme in the culture supernatant of P.fluorescens was precipitated allow the SDS to bind to the elastin [12]. Plates were stored in a moist with ammonium sulphate. Precipitated soluble protease enzyme was chamber at 4°C before use. For assay of elastase activity 5mm (20μl) collected after centrifugation at 10000 rpm for 20m. Precipitate was diameter wells were cut in the gel. Samples were placed in the wells dissolved in fresh PBS (50 mM, pH 7.0) buffer and assayed for the and allowed to diffuse into the gel and then incubated at 40°C in a total protease enzyme content. Culture supernatant precipitated with moist chamber up to two weeks. Zones of lysis were examined with ammonium sulphate was dialyzed and fractionated by gel filtration dark back ground illumination and measured in two perpendicular using sephacryl S300 columns (Amersham-Pharmacia, Sweden) in dimensions after 16, 24, 48, and 72h [13]. FPLC system. Microplate assay for elastase Qualitative assay of fibrinolytic activity on fibrin plate Elastolytic activity of enzyme was determined by using elastin Enzyme activity was determined by using fibrin plate method. (Sigma chemical Co) as substrate. Ezyme preparation (25μl) was

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Figure 2a: Native page of proteins from the culture filtrates ofP. fluorescens; Lane -2: Marker protein, Lane 4: FPLC Fractionated pure protein.

Figure 3a: Fibrinolytic activity assay on the fibrin plate (0.3%) with (a) 20 1 2 3 4 µl of extra cellular proteins and (b) 20 µl of plasmin, and (c) 20 µl thrombin. 203kDa

93kDa

66kDa

55kDa

43kDa

35kDa

Figure 2b: SDS-PAGE of proteins from the culture filtrates of P. fluorescens Lane1-Protein marker; Lane 2-Purified protein; Lane 3&4-Crude protein. incubated with 20mg of elastin in 2ml of 0.2mol/L boric acid buffer Figure 3b: Standard curve showing the relationship between urokinase activity (Y) and thrombolytic circle area (X). 2b. Established based on the (pH 7.4) containing an inhibitor, elastatinol (100μmol) and without data of 40, 80, 160, 320 and 640 IU/mL of urokinase. inhibitor, by constant shaking for 20 min at 37°C. Dilutions of sample were assayed for elastolytic activity to establish the linear range of the reactions. Reaction was stopped by adding 2ml of 0.7mol/l sodium phosphate buffer (pH 6.0) and immediately filtered. Absorbency of the filtrate was read at 540 nm against a control without the enzyme. One unit of elastase activity is defined as the amount of enzyme required to solubilize 20 mg elastin under the tested conditions [13]. Inhibitor sensitivity The sensitivity of protease inhibitor was tested by determining the hydrolysis of azocasein after preincubation with protease inhibitors. Inhibitors specific for metalloprotease ortho phenonthroline (1,10 Phe) 2mM; Ethylene Diamine Tetra Acetic acid (ETDA) 2mM, Figure 3c: Standard curve showing the relationship between urokinase (EGTA) 2mM and Elastatinol 2mM were used. 20μl of protease activity (Y) and thrombolytic circle area (X). 3c. Established based on the enzyme was pre incubated with 3μl of inhibitor in 100μl of buffer for data of 40, 80, 160, 320, 640,1280, 2560 and 5000 IU/mL of urokinase. 1h at room temperature to give the above concentrations of inhibitors and the enzyme activities were determined [14]. data obtained from the MALDI-TOF MS analysis. These results were also confirmed by sequence similarity searching with partial amino Phylogenetic analysis acid sequences obtained from ESI-QqTOF MS/MS Analysis. The The purified protein sample was subjected to Two-dimensional amino-acid sequences obtained from individual protease enzyme electrophoresis (2-DE) according to the method of Gorg et al. [15]. spots were subjected to homology analysis with the BLAST program 2-DE protein spots were excised and tryptically digested in modified (National Centre for Biotechnology Information NCBI-BLAST trypsin (sequencing grade, Roche, UK) as previously described by 2.2.6, [18]. Homology alignment was performed with the CLUSTAL Chemale et al. [16]. Peptides were eluted according to the method W program using CLC Genomics work bench 11 software [19]. A of Shevchenko et al. [17]. Protein spots of 47 different were excised phylogram was derived with the CLC Genomics work bench 11 from 2-DE gels and proteins were identified by peptide mass software and Clustal W alignments of the predicted amino acid fingerprinting using MALDI-TOF MS after in-gel tryptic digestion sequences with sequences of nine metalloproteases derived from of excised spots (Data is shown and yet to be published). Using the National Centre for Biotechnology Information. Phylogenetic specific ProtKpn database 47 protein spots from 2-DE gels can be trees demonstrated relatedness of 55 kDa and 35 kDa extra cellular successfully identified simply with their peptide mass fingerprinting proteases among metalloprotease [20].

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Estimation of Zn+ and Ca+ in purified protease enzyme The protease enzyme samples (100mg/ml) were pre-treated with nitric acid (HNO3) before treating it with perchloric acid (HCLO4) and avoid the repeated fuming with perchloric acid. Approximately 5-10 ml of pre-treated protease enzyme sample was taken in a clean beaker. A volume of 10ml of concentrated nitric acid and 10ml perchloric acid was added in a cooling condition. The beaker was placed on a hot plate for evaporation until dense white fumes of perchloric acid appear. The beaker was allowed to cool and diluted to about 50ml with Milli Q water. The solution was filtered through Whatmann paper and the filtrate was used for the Inductive Coupled Plasma (ICP) analysis. Standards of the target metals (Zn and Ca) were prepared by Figure 4: Effect of divalent cations on fibrinolytic protein activity. dissolution of the analytical-reagent grade nitrate salt in 45ml of Milli Q water with 0.1ml suprapur grade Nitric acid (Merk). To this, 50μl Yttrium ICP standard and 1ml of multi element standard (1 ppm concentration) was added and made up to 50ml with Milli Q water. Similarly, blank for the standards were also prepared. Sample of purified protease enzyme was dialyzed and dissolved in 45ml of Milli Q water with 0.1ml Suprapur grade Nitric acid (Merk). Yttrium ICP standard (50μl) was added and made up to 50ml with Milli Q water. The samples were loaded with an ultra-mass 700 Inductively Coupled Plasma mass spectrophotometer (Perkin Elmer, Optima 2100 spectrophoto meter). The emission was measured at its own specified wave length. The contents of zinc and calcium in the purified protease Figure 5a: Inhibition of protein activity by 1, 10-phenanthroline (mM). enzyme were calculated based on the standard curve prepared with standard calcium and zinc solutions and the data was retrieved using Data management software [21]. Results and Discussion Culture supernatant of P. fluorescens Migula B426was collected after 72h of growth in GPS medium and the enzymes inthe supernatant were precipitated using ammonium sulfate (50%) and then fractionated by gel-filtration using Sephacryl S300 (Amersham- Pharmacia, Sweden). The precipitated protein was dialyzed and fractionated by gel filtration using Sephacryl S300 columns (Amersham-Pharmacia, Sweden) in an FPLC system (Figure 1a and b). Pure protein was subjected to 10% Native page and single band Figure 5b: Inhibition of protein activity by inhibitors-1, 10- phenanthroline, was observed with a molecular weight of 90 kDa (Figure 2a and b). EGTA, elastatinol and EDTA (mM). Thus the FPLC fractionated pure protein was found to be a monomer. The protein fractions were eluted using Tris-HCl (0.05M), containing (mm2) were highly exponentially correlated (Figure 3c). Standard sodium chloride buffer (0.1M; pH 7.5) and monitored at 280nm. curve was used for the estimation of fibrinolytic activity of sample. The active fractions th (20 fraction showing activity on Effects of the divalent cations Mg2+, Ca2+ and the chelating zymography assay) were pooled, concentrated by lyophilisation and agent EDTA on the activity of the enzyme are presented in Figure stored at -20°C until further use. The purity of the pooled fractions 4. The divalent cations Mg2+ and Ca2+ enhanced the protease was checked by HPLC using a Dupont GF-250 column. enzyme activity. The EDTA, EGTA, and elastatinol decreased the The qualitative fibrin plate analysis revealed that the enzyme enzyme activity. Combination of divalent cations and chelating was produced from the P.fluorescens Migula B426 hydrolysis fibrin agent also decreased the enzyme activity. Since the average P value (Figure 3a). Sharper and contrasting clear zones observed around (<0.0001) of one way (ANOVA) analysis of variance is less than the well on the plates confirmed the fibrinolytic activity. Fibrinolytic <0.05 at 95% confidence intervals showing that the results of effect activity of the enzyme was further demonstrated by the cleavage of of divalent cations on enzyme is statistically significant. Inhibition fibrin polymer catalyzed by thrombin. There was transient increase of enzyme activity by EGTA, EDTA, 1, 10- phenanthroline and in the measurement of fibrinogen activity. Applications of 10μl of elastatinol is presented in Figure 5a and b. Since the average P value urokinase, at 40, 80, 160, 320 and 640 IU/mL, all produced clear (<0.0001) of one way (ANOVA) analysis of variance is less than 0.05 fibrinolytic zones in the fibrin plates. Urokinase activity (IU/mL) at 95% confidence intervals, the results of inhibition of enzyme by and the lytic circle area (mm2), averaged with two duplicates, were inhibitors test are statistically significant. The total contents of zinc highly linearly correlated in the range of 40 to 640 IU/mL (Figure 3b). and calcium in the purified protease enzyme were calculated based on However, in the range of 40 to 640 IU/mL, the activity and the area the standard curve prepared with standard calcium and zinc solutions

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Table 1: Estimation of Ca and Zn in purified protease enzyme. As Loc: Date: 2009/06/23 14:39:05 seq. No. Sample ID: Analyte 11 TP-13.6.09 time conc(calib) conc(sample) Net Intensity corr. Intensity 1.04 PPM

Mean 1.03 PPM

5,359,790.7 2009/06/23 5359790.7 14:39:01 Y 371.029 SD 1.03 PPM 5323900.2 2009/06/23 5341845.5 5,323900.2 14:39:05

%RSD: 0.005PPM

0.48 717.2 PPb 35860 PPb

Mean 728.3 PPb 36410 PPb

41053493.9 2009/06/23

39491662.3 14:39:09 Ca 396.847 SD 722.8 PPb 36410 PPb 40101440.9 39796551.6 2009/06/23

41407535.5 14:39:16

%RSD: 7.83 PPb 391.5 PPb

1.08 130.5 PPb 6524 PPb

Mean 130.7 PPb 6537 PPb

15749.2 2009/06/23 15206.8 14:39:25 Zn 206.200 SD 130.6 PPb 15238.1 6530 PPb 2009/06/23 15222.5 15676.0 14:39:25

%RSD: 0.19 PPb 9.5 PPb

0.15 and the data were retrieved using data management software (Figure thrombolytic agents are increasingly getting reported. In this study, 6a and b). The estimated amount of calcium and zinc was found to we described purification and characterization of a fibrinolytic be 36140 ppb (Ca), 6530 ppb (Zn) (Table 1) respectively. Culture enzyme from P. fluorescensMigula 426 strain. Until recently, only few supernatant of P. fluorescens was subjected to two-dimensional gel fibrinolytic enzymes were reported from microbes showing a large electrophoresis (Figure 7). Two prominent spots were identified group of organisms capable of producing several beneficial products based on zymography results and picked up by spot cutter for Nano including antibiotics. LC/MS analysis along with Edman sequencing to ascertain further In the previous study, the protease enzyme produced from P. the identity of the enzyme. 35 kDa and 55 kDa extracellular enzymes fluorescens Migula 426 was found to be a monomer with molecular of P. fluorescensMigula B426 were subjected to Nano LC/MS analysis weight of 90 kDa [22] and it exhibited a broad substrate activity and the sequence of enzyme were aligned using Bioinformatics tool towards various natural substrates such as azocasein, elastin, chitin available at National Centre for Biotechnology Information (NCBI) and fibrin [23]. In this study, we investigated that the metabolites of to identify the enzyme (Figure 8a and b). The phylogenetic tree was Pseudomonas fluorescens Migula strains (VCRC B426) hydrolysis constructed to find out the relationships among various biological the gelatin and fibrin rapidly. However, Fibrinogen and fibrin play metalloprotease enzymes among microbial strains. The amino essential roles in blood clotting, cellular and matrix interactions, acid sequences of 55 kDa and 35 kDa enzyme closely related with inflammation, wound healing, and neoplasia [24]. As there is metalloprotease of P. fluorescensand elastase of P. aeruginosa (Figure growing medical interest in thrombosis agents, great attention is 6a and b). Phylogenetic analysis was performed using 55 kDa and 35 being directed towards a search for thrombolytic agents of various kDa protease enzyme sequence and along with known (Gen Bank) organisms. The fibrinolytic agents, such as urokinase, tPA (tissue amino acid sequences of alkaline protease, , metalloprotease, type plasminogen activator) and streptokinase, have been used for serine, , , aminopeptidase and elastase. The thrombosis therapy. However, these agents have a low specificity analysis confirmed the 55 kDa showed more similarity with the to fibrin and are very expensive [25]. The availability of synthetic elastase of P. aeruginosa and 35 kDa showed more similarity with the substrates has made this determination an attractive adjuvant for metalloprotease of P. fluorescens. fibrinogen quantification. The result presented in this study inferred It becomes necessary to develop new thrombolytic agents as that the metabolite progressively cleaved the formation of the fibrin variety of cardiovascular diseases and drawbacks in the typical polymer, including the cross-linked fibrins. In this report, we found

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Figure 6a: ICP spectrum of calcium analysis in the purified protein of P. fluorescens.

Figure 7: Localisation of protein spots in2-D Gel Electrophoresis of culture supernatant proteins of P. fluorescens stained with the silver nitrate.

Figure 6b: ICP spectrum of zinc analysis in the purified protein of P. fluorescens. that the Pseudomonas fluorescens Migula strain (VCRC B426) secreted extra cellular metabolites showed fibrinolytic activity on fibrin plate. It has been reported that activity of the endogenous metalloprotease Figure 8a: Phylogenetic relationships of extra cellular enzyme (55kDa) of P. enzyme is inhibited by 1, 10-phenanthroline [26-28]. Results of this fluorescens Migula strain and other closely related bacterial sps. investigation confirmed that the enzyme was metalloprotease as it was strongly inhibited by 1, 10-phenanthroline. Effects of the divalent cations Mg2+ and Ca2+ and the chelating agent EDTA on the activity of the extra cellular protease were studied. Addition of either Mg2+ or Ca2+ increased protease activity, while removal of divalent cations by chelation with EDTA inhibited. Activity of the enzyme in the absence of added divalent cation (ca. 74% of maximal activity) suggested that the enzyme has divalent metal ions already bound to it. It showed that the extra cellular metabolite from P. fluorescens is a metalloprotease. Identical results were obtained when the native Proteus mirabilis enzyme was used and the results are in agreement with the results obtained by Loomes et al. [29], who have reported that numerous Proteus strains secrete an EDTA-sensitive metalloprotease [30]. Figure 8b: Phylogenetic relationships of extra cellular enzyme (35kDa) of P. fluorescens Migula strain and other closely related bacterial sps. The partial amino acid sequence analysis revealed that the 35kDa and 55kDa proteins are 91 % homology with Zinc of zinc metalloprotease [32]. The 55kDa amino acid sequence of metalloprotease () of Pseudomonas aeruginosa COG3227 Pseudomonas flourescens has high similarity to the sequences of (ref|ZP00973484.1) and Zinc metalloprotease (pseudolysin) of serralysin family including S. marcescens metalloprotease (serralysin) Pseudomonas aeruginosa COG3227 (ref|ZP 00967917.1) and 58 % ,50 and P. aeruginosa alkaline protease. Both metalloproteases have % homology with Zinc metalloprotease (serralysin) of Pseudomonas the extended zinc-binding motif HEXXHXXGXXH, and the third aeruginosa WH6 (ref|ZP00973484.1) and metalloprotease of histidine of the motif and a water molecule act as the third and fourth Pseudomonas fluorescens SBW25 (ref|YP_002872721.1) respectively, zinc ligands, respectively. The group possessing the XXGXXH zinc a group of zinc metalloprotease. The 35kDa zinc metallopeptidase binding motif is called the ‘metzincins family [33]. has consensus sequence, HEXXH Zinc binding motif [31]. An extra cellular metalloprotease was purified from the culture supernatant Metalloproteases, in which zinc is an essential metal ion for of Pseudomonas fluorescens strain KT1 to apparent homogeneity catalytic activity, are elaborated by various human pathogenic and the partial amino acid sequence revealed that the enzyme is bacteria, as well as by non-pathogenic ones [34]. Zinc in protease highly homologous with proteases of the serralysin family, a group enzyme s can either participate directly in chemical catalysis or be

Remedy Publications LLC. 6 2018 | Volume 1 | Issue 1 | Article 1005 Muthuraj Muthaiah, et al., Annals of Genetics and Genetic Disorders important for maintaining protease enzyme structure and stability. 14. Caldas C, Cherqui A, Pereira A, Simoes N. Purification and characterization Zinc ions serve as powerful electrophilic catalysts in many of an extra cellular protease from Xenorhabdus nematophila involved in and [35]. Zinc-binding stabilizes the folded conformations of insect immunosuppression. Appl Environ Microbiol. 2002;57:1297-304. domains so that the protease enzyme can function properly and an 15. Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, et al. example for this is zinc finger protease enzyme [36]. The N terminal The current state of 2-DE with immobilized pH gradients. Electrophoresis. amino acid residues of extracellular enzyme of P. fluorescens Migula 2000;21:1037-53. B426 revealed almost 91 % homology with Zinc metalloprotease 16. Chemale G, Morphew R, Moxon JV, Morassuti AL, Lacourse EJ, Barrett (pseudolysin 1EZM, EC 3.4.24.26) of P. aeruginosa strain and has J, et al. Proteomic analysis of glutathione from the liver fluke consensus sequence, HEXXH zinc binding motif. The enzyme might parasite, Fasciola hepatica. Proteomics. 2006;6(23):6263-73. be a novel type of Zinc metalloprotease based on its N-terminal 17. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric amino acid sequence, effect of protease inhibitors and its fibrinolytic sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. activity. This fibrinolytic enzyme is highly active and it can be further 1996;68(5):850-8. developed as a potential candidate for thrombolytic therapy. 18. Altschul SF, Madden TL, SchaVer AA, Zhang J, Zhang Z, Miller W. Acknowledgement Gapped BLAST and PSI-BLAST: a new generation of protease enzyme database search programs. Nucleic Acids Res. 1997;25(17):3389-402. Authors thank Dr. P. Jambulingam, Director, Vector Control 19. Pootanakit K, Chanan A, Sakol P. Identification of two isoforms of Research Centre, Puducherry, for his support and critical suggestions. aminopeptidase N in Aedes aegypti larval midgut. J Biochem Mole Biol. References 2003;36(5):508-13. 20. Swofford DL. Phylogenetic analysis using parsimony, version 3.0s. Illinois 1. Bode C, Runge MS, Smalling RW. The future of thrombolysis in the Natural History Survey, Champaign, Ill. 1993. treatment of acute myocardial infarction. Eur Heart J. 1996;17:55-60. 21. Carl EH, Jason FM, Gary DR. Atomic absorption measurements in 2. Kim JS, Kim JE, Choi BS, Park SE, Sapkota K, Kim S, et al. Purification inductively coupled plasma using spatial dispersion of the plasma image. and characterization of fibrinolytic metalloprotease from Perenniporia Analytical Commun. 1997;34:355-7. fraxinea mycelia. Mycol Res. 2008;112:990-8. 22. Usharani By, Paily K. Chitinase like activity of metabolites of Pseudomonas 3. Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, et al. Purification and fluorescens Migula on immature stages of the mosquito, Culex characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, quinquefasciatus (Diptera: Culicidae). Afr J Microbiol Res. 2012;6:2718- isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol. 26. 2006;33(9):750-8. 23. Usharani B, Paily K. Gelatinase activity of metabolites of Pseudomonas 4. Mihara H, Sumi H, Yoneta T, Mizumoto H, Ikeda R, Seiki M, et al. A novel fluorescens Migula on larvae and pupae of Culexquinquefasciatus fibrinolytic enzyme extracted from the earthworm, Lumbricus rubellus. J (DIPTERA: CULICIDAE). Int J Pharm Bio Sci. 2014;5 234-45. Physiol. 1991;41(3):461-72. 24. Mosesson MW. Fibrin polymerization and its regulatory role in hemostasis. 5. Balaraman K, Prabakaran G. Production & purification of a fibrinolytic J Lab Clin Med. 2006;116:8-17. enzyme (thrombinase) from Bacillus sphaericus. Indian J Med Res. 2007;126(5):459-64. 25. Nakajima N, Taya N, Sumi H. Potent fibrinolytic enzyme from the lysate of Katsuwonus pelamis digestive tract (Shiokara): purification and 6. Batomunkueva BP, Egorov NS. Isolation, purification and resolution of the characterization. Biosci Biotechnol Biochem. 1993;57:1604-5. extracellular protease enzyme ase complex of Aspergillus ochraceus 513 with fibrinolytic and anticoagulant activities. Microbiology. 2001;70:519- 26. Gearing AJH, Beckett P, Christodoulou M, Churchill M, Clements JM, 22. Crimmin, M, et al. Matrix metalloprotease enzymeases and processing of pro-TNF-alpha. J Leukocyte Biol. 1995;57:774-7. 7. Kim SH, Choi NS. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp strain DJ-4 screened from Doen-Jang. Biosci 27. McGeehan GM, Becherer JD, Blast RC, Boyer CM, Champion B, Biotechnol Biochem. 2000;64:1722-5. Conolly BM, et al. Regulation of tumor necrosis factor-a processing by a metalloprotease enzyme ase inhibitor. Nature (London). 8. Peng Y, Zhang YZ. Isolation and characterization of fibrinolytic enzyme 1994;370(6490):558-61. producing strain DC-4 from Chinese douche and primary analysis of the enzyme property. C H Tech Letters. 2002;12:30-4. 28. Mohler KM, Sleath PR, Fitzner JN, Cerretti DP, Alderson M, Kerwar SS, et al. Protection against a lethal dose of endotoxin by an inhibitor of tumor 9. Sumi H, Hamada H, Mihara H, Nakanishi K, Hiratani H. Fibrinolytic necrosis factor processing. Nature (London). 1994;370(6486):218-20. effect of the Japanese traditional food natto (nattokinase). Thrombosis & Haemostasis. 1989;62:549-56. 29. Christopher W, Diana C, Robert B. Molecular analysis of a metalloprotease from Proteus mirabilis. J Bacteriol. 1995;177(20):5790-8. 10. Prabakaran G, Paily KP, Padmanabhan V, Hoti SL, Balaraman K. Isolation of a Pseudomonas fluorescens metabolite/exotoxin active against both 30. Loomes LM, Senior BW, Kerr MA. A proteolytic enzyme secreted by larvae and pupae of vector mosquitoes. Pest Manag Sci. 2003;59:21-4. Proteus mirabilis degrades immunoglobulin of the immunoglobulin A1 (IgA1), IgA2, and IgG isotypes. Infect Immun. 1990;58(6):1979-85. 11. Alan KC, Hyo YK, Jung EP, Pankaj A, Il-Seon P, Seong MY, et al. Vibrio vulnificus secretes a broad-Specificity metalloprotease capable of 31. Hooper NM. Families of zinc metalloproteases. FEBS Lett. 1994;354(1):1- interfering with blood homeostasis through prothrombin activation and 6. fibrinolysis. J Bacteriol. 2005;187:6909-16. 32. Chen Qi-he HG, He GQ. Optimization of elastolysis conditions and 12. Abbott WS. A method of computing the effectiveness of an insecticide. J elastolytic kinetic analysis with elastase from Bacillus licheniformis. J Econ Entomol. 1995;18:265-7. Zhejiang Univ Sci B. 2006;7:482-90. 13. Kevin SM, Efrat K, Dennis EO. Identification of residues in the 33. Stocker W, Grams F, Baumann U, Reinemer P, Gomis Ruth FX, Mckay Pseudomonas aeruginosa elastase propeptide required for chaperone and DB, et al. The metzincins - Topological and sequential relations between secretion activities. Microbiol. 2004;150:3969-77. the , , , and matrixins ()

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astacins, adamalysins, serralysins, and matrixins (collagenases) define a 35. McCall KA. Function and mechanism of zinc metalloenzymes. J Nutr. superfamily of zinc-peptidases. Protein Sci. 1995;4(5):823-40. 2000;130:1437-46. 34. Miyoshi S, Wakae H, Tomochika K, Shinoda S. Functional domains of a zinc 36. Berg JM, Shi Y. The galvanization of biology: a growing appreciation for metalloprotease from Vibrio vulnificus. J Bacterial. 1997;179(23):7606-9. the roles of zinc. Science. 1996;271:1081-5.

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