RESUMO
Microplastics (MPs) and associated contaminants have become a major environmental concern. From available literature, their ubiquitous presence is now well established. However, the kind and level of toxicological impacts these MPs accomplish on various life forms are not well understood. Nevertheless, the environmental toxicity of MP is now being revealed gradually with supporting studies involving groups of lower organisms. Additionally, the presence of microplastics also disturbs the functions of ecosystem through affecting the vulnerable life forms, thus ecological manifestations of MPs also need to be analyzed. The present review encompasses an overview of toxicological effects mediated by various types of MPs present in the environment; it covers the types of toxicity they may cause and other effects on humans and other species. In this review, aquatic systems are used as primary models to describe various eco-toxicological effects of MPs. Various research gaps as well as methods to alleviate the level of MPs, and future strategies are also comprehensively highlighted in the review.
Assuntos
Monitoramento Ambiental , Microplásticos/toxicidade , Poluentes Químicos da Água/toxicidade , EcossistemaRESUMO
Enzymes are biocatalysts that have found profound applications in the current biotherapeutic industry and play a crucial role in diagnosis, prevention, and biochemical analysis of major diseases. However, stability, protein degradation and immunogenicity in the body present unique challenges that are faced upon sustained use of such enzymes. The present chapter is an attempt to dissect the state-of-the-art in relation to the challenges of development of therapeutic enzymes and the recent advances to address them. At the very outset, diseases where enzymes have found effective applications and the various causes of enzyme instability have been discussed. In recent times, polymer or nano- conjugated resistant delivery methods, as well as mutagenesis have led to manifold increase in enzyme stability against thermal denaturation, acidic gut environment, proteolysis and immunogenicity. Further, methods of analytical characterization of proteins have been highlighted and explored to shape future research directions.
Assuntos
Estabilidade Enzimática , Enzimas/química , Enzimas/farmacologia , ProteóliseRESUMO
Proteins are prone to unfolding and subsequent denaturation by changes in temperature, pH and other harsh conditions. Nanoparticles act as artificial 'chaperones' due to favourable orientation of the proteins on their scaffold which prevents aggregation and reconfigures denatured proteins into their native functional state. In the present study, thermal denaturation of Cholesterol oxidases from Pseudomonas aeruginosa PseA, Rhodococcus erythropolis MTCC 3951 and Streptomyces sp. were studied at temperatures 50-70⯰C. Further, these thermally denatured proteins were refolded using functionalized Magnetic Iron (II, III) oxide nanoparticles which was confirmed using DLS, Zeta Potential Measurements, fluorescence and CD spectroscopy. The refolded proteins were found to regain their secondary structure and activity to a great extent.
Assuntos
Colesterol Oxidase/química , Nanopartículas de Magnetita/química , Desnaturação Proteica , Redobramento de Proteína , Ativação Enzimática , Compostos Férricos/química , Tamanho da Partícula , Análise Espectral , Temperatura , TermodinâmicaRESUMO
In this study, urease mediated calcite precipitation technique was used for remediation of Zn (II). A urease positive Enterobacter cloacae EMB19 was used to produce calcite impregnated with Zn ions. In co-presence of Ca (II), Zn (II) concentrations of 10 and 20 mg L-1 were completely remediated by the bacterium from the media at 72 and 96 h of incubation, respectively. Co-precipitation of Ca (II) and Zn (II) to form calcite-Zn precipitate is one of the major mechanisms of Zn remediation in the present study. Role of urease in calcite-Zn precipitation was substantiated by using urease/carbonate and ammonium enriched cell free culture supernatant (CFS) obtained after sufficient microbial growth. Using CFS, 68% removal of initial 50 mg L-1 Zn (II) was detected. Energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction pattern (XRD) of the precipitate supports calcite mediated remediation of Zn. Remediation of multiple metals viz. Cd, Zn, Cu was also analyzed using CFS laden with urease. The preparation showed 40% Cd, 23% Zn, and 8% Cu reduction from the solution containing initial 25 mg L-1 of each metal. Overall, it can be concluded that, the E. cloacae mediated calcite precipitation technique could effectively be used for alleviation of Zn (II) and other heavy metals from the contaminated environment.
Assuntos
Carbonato de Cálcio/química , Enterobacter cloacae/enzimologia , Urease/química , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Zinco/análise , Amônia/química , Carbonatos/química , Precipitação Química , Recuperação e Remediação AmbientalRESUMO
In the present study, urease positive Serratia marcescens (NCIM2919) and Enterobacter cloacae EMB19 (MTCC10649) were individually evaluated for remediation of cadmium (II) using ureolysis-induced calcium carbonate precipitation. Both the cultures were observed to efficiently remove cadmium from the media through co-precipitation of Cd (II) and Ca (II). S. marcescens and E. cloacae EMB19, respectively showed 96 and 98% removal of initial 5.0â¯mgâ¯L-1 soluble Cd (II) from the urea and CaCl2 laden media at 96â¯h of incubation period. At higher Cd (II) concentrations of 10 and 15â¯mgâ¯L-1, cadmium removal efficiency was much higher in case of E. cloacae EMB19 compared to S. marcescens. In-vitro cadmium (II) remediation study using urease containing cell-free culture supernatant of S. marcescens and E. cloacae EMB19 showed respective 98 and 53% removal of initial 50â¯mgâ¯L-1â¯Cd (II) from the reaction mixtures in co-presence of Ca (II). While in sole presence of Cd (II), only 16 and 8% removal of Cd (II) were detected for S. marcescens and E. cloacae EMB19, respectively. The elemental analysis of the co-precipitated mineral products using Energy Dispersive X-ray spectroscopy (EDX) clearly showed the prevalence of Ca and Cd ions. The morphology Cd-Ca composites formed with respect to both the cultures were observed to be of different shape and size as revealed through Scanning Electron Microscopy (SEM). Entire study hence comes out with a sustainable bioremediation option which could be effectively used to tackle Cd (II) or other heavy metal pollution.
Assuntos
Cádmio/isolamento & purificação , Enterobacter cloacae/enzimologia , Serratia marcescens/enzimologia , Urease/metabolismo , Cádmio/química , Poluentes Ambientais/química , Poluentes Ambientais/isolamento & purificação , Metais PesadosRESUMO
The rampant use and misuse of antibiotics in human medicine, agriculture and veterinary have become the key contributors to global antimicrobial resistance. One of the significant resistance mechanisms that inactivates antibiotics and impedes treatment of bacterial infections is the expression of ß-lactamases. Rising evidence of newer variants of ß-lactamases in the environment is therefore a serious threat to the presently available antibiotic armoury. The present work describes the purification of a variant ß-lactamase isolated from a soil strain EMB20 of Bacillus cereus. The lactamase was purified using three-phase partitioning and gel filtration chromatography to a 30-fold purification and 15% recovery yield. Contrary to the general trend, the lactamase was not a metalloenzyme, but its activity was enhanced in the presence of Mg2+ and Mn2+. The EMB20 lactamase exhibited improved stability against inhibitors and denaturing agents such as urea and GdmCl as compared to its commercial analogue. The improved stability of EMB20 lactamase was further validated by circular dichroism and fluorescence spectroscopy. This study reemphasizes the rising prevalence of environmental lactamase variants. Decoding the structure-function correlation of such lactamases in the presence of inhibitors will provide insights into the response of this enzyme towards inhibitors as well as its substrates.
Assuntos
Bacillus cereus/enzimologia , beta-Lactamases/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Guanidina/química , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Especificidade por Substrato , Ultrafiltração , Ureia/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , beta-Lactamases/isolamento & purificaçãoRESUMO
Present work reports the inhibition of Bacillus cereus EMB20 ß-lactamase by a deep eutectic solvent, maline in an uncompetitive manner. Far-UV CD and intrinsic fluorescence spectroscopy revealed a disrupted secondary as well as tertiary structure as a function of maline concentration. The effect of individual components of maline on ß-lactamase inhibition showed that malonic acid was mainly responsible for inhibiting the ß-lactamase. Structural and docking studies found that malonic acid led to major perturbations in the secondary and tertiary structure of the enzyme while H-bonding with the active site residues. Further the antibacterial and cytotoxic studies also confirmed the potential of maline as a potent growth inhibitor of ß-lactamase producing B. cereus EMB20.
Assuntos
Bacillus cereus/enzimologia , Bacillus cereus/crescimento & desenvolvimento , Líquidos Iônicos/farmacologia , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Bacillus cereus/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Dicroísmo Circular , Concentração Inibidora 50 , Cinética , Malonatos/farmacologia , Simulação de Acoplamento Molecular , Solventes , Espectrometria de Fluorescência , beta-Lactamases/químicaRESUMO
The lignocellulosic agro-wastes are an attractive renewable resource in biorefinery for production of value-added platform chemicals and biofuels. The study describes use of different agro-wastes as substrate for production of lactic acid, a C3-platform chemical and high demand industrial product by Lactobacillus brevis in a one-pot bioprocess. The simultaneous saccharification and co-fermentation (SSCF) process was achieved by L. brevis governed fermentation of sugars, derived from saccharification of ionic liquid pretreated feedstocks by nanoimmobilized cellulase, which was further recovered and used for consecutive cycle. The lactic acid yields of 0.22, 0.49, 0.52â¯g/g were obtained from cottonseed cake, wheat straw and sugarcane bagasse, respectively. The ionic liquid-tolerant L. brevis, cellulolytic reusable nanoimmobilized enzyme coupled with valorization of renewable feedstocks points towards a holistic approach for future biorefineries with sustainable production of bioproducts.
Assuntos
Ácido Láctico , Levilactobacillus brevis , Biomassa , Fermentação , Líquidos IônicosRESUMO
The oxidation of cholesterol results in the formation of oxysterols such as 7-ketocholesterol (7KC), which are implicated in a number of age-related disorders such as atherosclerosis, Alzheimers' disease and macular degeneration. Current modalities use antioxidants and other natural or synthetic molecules to reduce 7KC-induced cytotoxity. The alternative application of enzymes from microbial sources to degrade oxysterols in vitro and in vivo is an innovative approach. The present study aims to assess the potential of the bacteria Rhodococcus erythropolis MTCC 3951 in degrading 7KC and mining relevant enzymes involved. This strain has been previously reported to be a degrader of xenobiotics such as polyphenols, toluene and catechol. Under optimized conditions, Rhodococcus erythropolis MTCC 3951 is found to degrade 93% of 1g/l concentration of 7KC within 15days of incubation. The extra- and intra-cellular extracts were also able to hydrolyse the compound indicating the involvement of enzymatic systems in the process. The strain produced cholesterol oxidase, lipase, dehydrogenase and reductase in the presence of 7KC. We have also identified a few intermediate products to predict the degradation pathway.
Assuntos
Colesterol Oxidase/metabolismo , Cetocolesteróis/metabolismo , Lipase/metabolismo , Oxirredutases/metabolismo , Rhodococcus/metabolismo , Rhodococcus/enzimologiaRESUMO
The present work aimed to improve catalytic efficiency of Trichoderma reesei cellulase for enhanced saccharification. The cellulase was immobilized on two nanomatrices i.e. magnetic and silica nanoparticles with immobilization efficiency of 85% and 76% respectively. The nanobioconjugates exhibited increase in Vmax, temperature optimum, pH and thermal stability as compared with free enzyme. These could be efficiently reused for five repeated cycles and were stable in 1-ethyl-3-methylimidazoliumacetate [EMIM][Ac], an ionic liquid. Ionic liquids (IL) are used as green solvents to dissolve lignocellulosic biomass and facilitate better saccharification. The cellulase immobilized on magnetic nanoparticles was used for in situ saccharification of [EMIM][Ac] pretreated sugarcane bagasse and wheat straw for two cycles. The structural deconstruction and decrease in biomass crystallinity was confirmed by SEM, XRD and FTIR. The high hydrolysis yields (â¼89%) obtained in this one-pot process coupled with IL stability and recycled use of immobilized cellulase, potentiates its usefulness in biorefineries.
Assuntos
Celulase , Lignina , Biomassa , Hidrólise , Líquidos Iônicos , NanoconjugadosRESUMO
There is an increasing demand of γ-aminobutyric acid (GABA) as drug and food additive, as well as feedstock to produce 2-pyrrolidone, a precursor for the synthesis of nylon 4. 2-Pyrrolidone is a petrochemical and depleting reserve which raises concern for its bio-based production. The study herein describes bio-based economical GABA production from Lactobacillus brevis by solid-state fermentation (SSF) using toxic deoiled cottonseed cake (CSC) as substrate. In general, the use of cottonseed cake remains restricted due to the presence of toxic gossypols. Thus, simultaneous detoxification observed during fermentation also widens the scope of utilization of this residual seedcake for feed use vis-a-vis production of other value added chemicals. The SSF conditions were optimized for maximum GABA production, viz., 19.7 mg/g, CSC of GABA was obtained at 6th day of fermentation with 70 % degradation of gossypols simultaneously. The potential of this bio-based GABA as a platform chemical is demonstrated in the synthesis of 2-pyrrolidone. Thus, a simple and cost-effective strategy for utilizing toxic biomass has been developed as an alternate to chemical synthetic route.
Assuntos
Óleo de Sementes de Algodão/química , Óleo de Sementes de Algodão/metabolismo , Gossypium/química , Levilactobacillus brevis/crescimento & desenvolvimento , Pirrolidinonas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/químicaRESUMO
The present study aims to degrade 7-Ketocholesterol (7KC), a major oxysterol implicated in many age-related disorders, through microbial means and find candidate enzymes involved for further application in food systems and as a therapeutic. During initial screening of previously isolated bacteria from our laboratory, Pseudomonas aeruginosa PseA was found to be a potential degrader strain using 7KC as a sole carbon source. Under optimized conditions, it is able to degrade 88% of an initial concentration of 1000ppm (1g/l) 7KC. Preliminary in vitro studies with extra-cellular extract has shown degradation of the compound, thus reinforcing the occurrence of suitable enzymatic systems involved in the process. We have been able to identify cholesterol oxidase as one such potential enzyme. Some intermediate products of degradation have also been identified. This is the first detailed report of 7KC degradation by a P. aeruginosa strain.
Assuntos
Cetocolesteróis/metabolismo , Pseudomonas aeruginosa/metabolismo , Biodegradação Ambiental , Colesterol Oxidase/biossínteseRESUMO
Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. "FVHLFEW" was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.
Assuntos
Marinobacter/enzimologia , alfa-Amilases/química , Sequência de Aminoácidos , Dicroísmo Circular , Guanidina/química , Concentração de Íons de Hidrogênio , Filogenia , Estrutura Secundária de Proteína , Cloreto de Sódio/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , alfa-Amilases/classificaçãoRESUMO
Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Present work encompasses production optimization and nanoimmobilization of an α-amylase from moderately halophilic Marinobacter sp. EMB8. Media ingredients and culture conditions were optimized by "one-at-a-time approach." Starch was found to be the best carbon source at 5% (w/v) concentration. Glucose acted as catabolic repressor for amylase production. Salt proved critical for amylase production and maximum production was attained at 5% (w/v) NaCl. Optimization of various culture parameters resulted in 48.0 IU/mL amylase production, a 12-fold increase over that of unoptimized condition (4.0 IU/mL). α-Amylase was immobilized on 3-aminopropyl functionalized silica nanoparticles using glutaraldehyde as cross-linking agent. Optimization of various parameters resulted in 96% immobilization efficiency. Starch hydrolyzing efficiency of immobilized enzyme was comparatively better. Immobilized α-amylase retained 75% of its activity after 5th cycle of repeated use.
RESUMO
The present work targets the fabrication of an active, stable, reusable enzyme preparation using functionalized silica nanoparticles as an effective enzyme support for crude halophilic Bacillus sp. EMB9 protease. The immobilization efficiency under optimized conditions was 60%. Characterization of the immobilized preparation revealed marked increase in pH and thermal stability. It retained 80% of its original activity at 70 °C while t 1/2 at 50 °C showed a five-fold enhancement over that for the free protease. Kinetic constants K m and V max were indicative of a higher reaction velocity along with decreased affinity for substrate. The preparation could be efficiently reused up to 6 times and successfully hydrolysed whey proteins with high degree of hydrolysis. Immobilization of a crude halophilic protease on a nanobased scaffold makes the process cost effective and simple.
Assuntos
Bacillus/enzimologia , Enzimas Imobilizadas/química , Peptídeo Hidrolases/química , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida , Endopeptidases , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Microbiologia Industrial , Cinética , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Dióxido de Silício/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Soro do Leite , Proteínas do Soro do Leite/químicaRESUMO
Halophilic enzymes have been manifested for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation in presence of high temperature, pH, presence of organic solvents and chaotropic agents. The present study aims at understanding the stability and activity of a halophilic Bacillus sp. EMB9 protease in organic solvents. The protease was uniquely stable in polar solvents. A clear correlation was evident between the protease function and conformational transitions, validated by CD and fluorescence spectral studies. The study affirms that preservation of protein structure, possibly due to charge screening of the protein surface by Ca(2+) and Na(+) ions provides stability against organic solvents and averts denaturation. Salt was also found to exert a protective effect on dialyzed protease against chaotropism of solvents. Presence of 1 % (w/v) NaCl restored the activity in the dialyzed protease and prevented denaturation in methanol, toluene and n-decane. The work will have further implication on discerning protein folding in saline as well as non-aqueous environments.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Peptídeo Hidrolases/química , Desnaturação Proteica , Álcoois/química , Dimetil Sulfóxido/química , Estabilidade Enzimática , Cloreto de Sódio/químicaRESUMO
Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein-urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca(2+) and Na(+) ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.
Assuntos
Proteínas de Bactérias/química , Estabilidade Enzimática , Guanidina/química , Peptídeo Hidrolases/química , Cloreto de Sódio/química , Bacillus/enzimologia , Desnaturação ProteicaRESUMO
A salt-stable alkaline protease from moderately halophilic Bacillus sp. EMB9, isolated from the western coast of India, is described. This protease was capable of efficiently removing silver from used/waste X-Ray films, as well as hydrolyzing defatted soy flour with 31% degree of hydrolysis (DH). Production of the protease was optimized by using response surface methodology. Ca(2+) and NaCl were the most critical factors in enhancing the yield. Under optimized culture conditions, a maximum of 369 U protease/mL was obtained, which is quite comparable to the yields of commercial proteases. The elevated production level coupled with ability to efficiently hydrolyze protein-laden soy flour and complete recovery of silver from used X-Ray films makes it a prospective industrial enzyme.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Endopeptidases/metabolismo , Prata/isolamento & purificação , Alimentos de Soja , Filme para Raios X , Bacillus/metabolismo , Farinha , Hidrólise , Índia , Modelos Estatísticos , Hidrolisados de Proteína , Prata/metabolismoRESUMO
De-oiled Jatropha curcas seed cake, a plentiful by-product of biodiesel industry was used as substrate for the production of a useful xylanase from Sporotrichum thermophile in solid state fermentation. Under the optimized conditions, 1025U xylanase/g (deoiled seed cake) was produced. The xylanase exhibited half life of 4h at 45°C and 71.44min at 50°C respectively. It was stable in a broad pH range of 7.0-11.0. Km and Vmax were 12.54mg/ml and 454.5U/ml/min respectively. S. thermophile xylanase is an endoxylanase free of exoxylanase activity, hence advantageous for xylan hydrolysis to produce xylooligosachharides. Hydrolysis of oat spelt xylan by S. thermophile xylanase yielded 73% xylotetraose, 15.4% xylotriose and 10% xylobiose. The S. thermophile endoxylanase thus seem potentially useful in the food industries.
Assuntos
Biotecnologia/métodos , Endo-1,4-beta-Xilanases/biossíntese , Fermentação , Glucuronatos/biossíntese , Jatropha/química , Oligossacarídeos/biossíntese , Óleos de Plantas/isolamento & purificação , Sementes/química , Sporothrix/enzimologia , Metabolismo dos Carboidratos/efeitos dos fármacos , Carbono/farmacologia , Fermentação/efeitos dos fármacos , Umidade , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Cinética , Sporothrix/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Resíduos , Xilanos/metabolismoRESUMO
A moderately halophilic protease producer, Bacillus sp. strain isolated from sea water is described. The protease is purified to homogeneity by ammonium sulphate precipitation and CM cellulose chromatography. The serine protease has a molecular mass of 29 kDa. Enzymatic characterization of protease revealed K(m) 2.22 mg mL(-1), Vmax 1111.11 U mL(-1), pH optimum 9.0, t1/2 190 min at 60°C and salt optima 1% (w/v) NaCl. The protease is remarkably stable in hydrophilic and hydrophobic solvents at high concentrations. The purified preparation is unstable at room temperature. Ca(2+) ions are required for preventing this loss of activity. Interestingly, the activity and stability are modulated differentially. Whereas, divalent cation Ca(2+) are involved in maintaining stability in solution at room temperature by preventing unfolding, monovalent Na(+) and K(+) ions participate in regulating the activity and assist in refolding of the enzyme. Application of the protease is shown in efficient removal of blood stain.