Immobilization of fibrinolytic protease from Mucor subtilissimus UCP 1262 in magnetic nanoparticles.

This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.

Extractive fermentation for process integration of protease production by Aspergillus tamarii Kita UCP1279 and purification by PEG-Citrate Aqueous Two-Phase System.

The present study evaluated the influence of the variables polyethylene glycol (PEG) molar mass, pH, PEG concentration and sodium citrate concentration in the integrated production of the protease from Aspergillus tamarii Kita UCP1279 by extractive fermentation, obtaining as a response the partition coefficient (K), activity yield (Y) and concentration factor (CF). The enzyme preferably partitioned to the top phase and obtained in the system formed by variables MPEG = 400 g mol-1, CPEG = 20% (w w-1), and CCIT = 20% (w w-1) and pH 6, in this condition were obtained CF = 1.90 and Y = 79.90%. The protease showed stability at a temperature of 60 °C for 180 min, with optimum temperature 40 °C and pH 8.0. For the ions and inhibitors effects, the protease activity increased when exposed to Fe2+, Ca2+ and Zn2 + and inhibited by EDTA, being classified as metalloprotease. The kinetic parameters Km (35.63 mg mL-1) and Vmax (1.205 mg mL-1 min-1) were also estimated. Thus, the protease showed desirable characteristics that enable future industrial applications, especially, for beer industry.

Isolation and production of organic solvent tolerant protease from bacterial burn infection, Staphylococcus aureus KP091274.

Organic solvent-resistant proteases are used to synthesize valuable pharmaceutical and industrial compounds. Using an available and inexpensive source can be very effective in producing this enzyme. For this purpose, Staphylococcus aureus KP091274 was isolated from burn infection and a medium optimization procedure in the presence of organic solvents was considered for four factors of incubation time, the concentration of Mg2+, glycerol and sorbitol using the response surface methodology. The results of this statistical method showed that incubation time has the most effect and glycerol concentration has the least positive effect on enzyme secretion. As a result of applying the optimized conditions in the bacterial culture medium (3mM of Mg2+, 1.5% W/V of glycerol, 0.4% W/V of sorbitol and 72 hours of incubation), the enzyme secretion reaches its maximum.

Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization.

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine-metallo enzyme type.

Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil.

As an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4-30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5-35 °C and pH 6.0-12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low Km value of 1.0 mg/ml and Vmax 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.

Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties.

Peptidomics allows the identification of peptides that are derived from proteins. Urinary peptidomics has revolutionized the field of diagnostics as the samples represent complete systemic changes happening in the body. Moreover, it can be collected in a non-invasive manner. We profiled the peptides in urine collected from different physiological states (heifer, pregnancy, and lactation) of Sahiwal cows. Endogenous peptides were extracted from 30 individual cows belonging to three groups, each group comprising of ten animals (biological replicates n = 10). Nano Liquid chromatography Mass spectrometry (nLC-MS/MS) experiments revealed 5239, 4774, and 5466 peptides in the heifer, pregnant and lactating animals respectively. Urinary peptides of <10 kDa size were considered for the study. Peptides were extracted by 10 kDa MWCO filter. Sequences were identified by scanning the MS spectra ranging from 200 to 2200 m/z. The peptides exhibited diversity in sequences across different physiological states and in-silico experiments were conducted to classify the bioactive peptides into anti-microbial, anti-inflammatory, anti-hypertensive, and anti-cancerous groups. We have validated the antimicrobial effect of urinary peptides on Staphylococcus aureus and Escherichia coli under an in-vitro experimental set up. The origin of these peptides was traced back to certain proteases viz. MMPs, KLKs, CASPs, ADAMs etc. which were found responsible for the physiology-specific peptide signature of urine. Proteins involved in extracellular matrix structural constituent (GO:0005201) were found significant during pregnancy and lactation in which tissue remodeling is extensive. Collagen trimers were prominent molecules under cellular component category during lactation. Homophilic cell adhesion was found to be an important biological process involved in embryo attachment during pregnancy. The in-silico study also highlighted the enrichment of progenitor proteins on specific chromosomes and their relative expression in context to specific physiology. The urinary peptides, precursor proteins, and proteases identified in the study offers a base line information in healthy cows which can be utilized in biomarker discovery research for several pathophysiological studies.

Biochemical characterization of zebrafish Prss59.1.

Eleven genes, including prss59.1, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. To address the role of prss59.1, the prss59.1 gene knock-out zebrafish strain is currently being established by genome editing. In this study, for further phenotypic analysis of prss59.1, biochemical characterization of Prss59.1 was conducted using recombinant protein. A C-terminal histidine-tagged version of zebrafish Prss 59.1 was constructed. Although E. coli-produced recombinant Prss59.1 showed almost no activity, peptidase activities appeared after denaturation and renaturation. Zebrafish Prss59.1 showed the highest activity against Lys-MCA. The optimal temperature and pH of the activity toward Lys-MCA were 37 °C and pH 8.0, respectively. The Km value was 0.17 mM. Thus, zebrafish Prss59.1 possesses the closed character of trypsin, as expected from the DNA sequence.

Sodium caprylate wash during Protein A chromatography as an effective means for removing protease(s) responsible for target antibody fragmentation.

For recombinant proteins produced in Chinese hamster ovary (CHO) cells, fragmentation is a common phenomenon that results in generation of product-related low-molecular-weight (LMW) species. Recently while purifying a bispecific antibody (bsAb), we observed that the target protein experienced cleavage at a couple of potential sites, leading to truncated products. Further studies suggest that the cleavage can likely be attributed to residual CHO cell protease activity. In order to maximally remove potential protease(s) that contribute fragmentation, we optimized Protein A chromatography by adding sodium caprylate (SC) to the wash buffer. Upon optimization, fragmentation of Protein A eluate happened to a much lesser degree as compared to that of eluate from unoptimized process, and the increased sample stability is in accordance with significantly reduced host cell protein (HCP) level. Taken together, the data suggest that SC wash during Protein A chromatography is an effective means for removing HCPs including endogenous protease(s) that are responsible for target antibody fragmentation.

Biochemical properties of extracellular protease from Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins.

The characteristics of the extracellular protease, produced by Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins were investigated. The protease was purified by ammonium sulfate, ion exchange, and gel filtration chromatography to obtain a 20.0 kDa extracellular protease. The protease reached maximal activity at pH 9.0 and 50 °C and was stable at pH 7.0 to 11.0 and 20 to 40 °C. Its protease activity was easily inhibited in the presence of Zn2+ , Fe2+ , and Fe3+ . The enzymatic characterization of the protease revealed a Vmax 49.50 U/ml·min, Km 8.19 mg/ml, and the half-life = 28.06 min, ΔH* d  = 114.11 kJ/mol, ΔG* d  = 89.24 kJ/mol, and ΔS* d  = 77.00 J/mol·K at 50 °C. In addition, the protease hydrolyzed meat protein into small particles and produced soluble peptides. This study provides a basis for understanding the biochemical characteristics of the S. carnosus RT6 protease and its future application for fermented meat products.

The activity and stability of a cold-active acylaminoacyl peptidase rely on its dimerization by domain swapping.

The study of enzymes from extremophiles arouses interest in Protein Science because of the amazing solutions these proteins adopt to cope with extreme conditions. Recently solved, the structure of the psychrophilic acyl aminoacyl peptidase from Sporosarcina psychrophila (SpAAP) pinpoints a mechanism of dimerization unusual for this class of enzymes. The quaternary structure of SpAAP relies on a domain-swapping mechanism involving the N-terminal A1 helix. The A1 helix is conserved among homologous mesophilic and psychrophilic proteins and its deletion causes the formation of a monomeric enzyme, which is inactive and prone to aggregate. Here, we investigate the dimerization mechanism of SpAAP through the analysis of chimeric heterodimers where a protomer lacking the A1 helix combines with a protomer carrying the inactivated catalytic site. Our results indicate that the two active sites are independent, and that a single A1 helix is sufficient to partially recover the quaternary structure and the activity of chimeric heterodimers. Since catalytically competent protomers are unstable and inactive unless they dimerize, SpAAP reveals as an "obligomer" for both structural and functional reasons.

An integrated transcriptomic and proteomic approach to identify the main Torymus sinensis venom components.

During oviposition, ectoparasitoid wasps not only inject their eggs but also a complex mixture of proteins and peptides (venom) in order to regulate the host physiology to benefit their progeny. Although several endoparasitoid venom proteins have been identified, little is known about the components of ectoparasitoid venom. To characterize the protein composition of Torymus sinensis Kamijo (Hymenoptera: Torymidae) venom, we used an integrated transcriptomic and proteomic approach and identified 143 venom proteins. Moreover, focusing on venom gland transcriptome, we selected additional 52 transcripts encoding putative venom proteins. As in other parasitoid venoms, hydrolases, including proteases, phosphatases, esterases, and nucleases, constitute the most abundant families in T. sinensis venom, followed by protease inhibitors. These proteins are potentially involved in the complex parasitic syndrome, with different effects on the immune system, physiological processes and development of the host, and contribute to provide nutrients to the parasitoid progeny. Although additional in vivo studies are needed, initial findings offer important information about venom factors and their putative host effects, which are essential to ensure the success of parasitism.

Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

Perspectives for the use of latex peptidases from Calotropis procera for control of inflammation derived from Salmonella infections.

BACKGROUND: Anti-inflammatory properties have been attributed to latex proteins of the medicinal plant Calotropis procera. PURPOSE: A mixture of cysteine peptidases (LPp2) from C. procera latex was investigated for control of inflammatory mediators and inflammation in a mouse model of Salmonella infection. METHODS: LPp2 peptidase activity was confirmed by the BANA assay. Cytotoxicity assays were conducted with immortalized macrophages. Peritoneal macrophages (pMØ) from Swiss mice were stimulated with lipopolysaccharide (LPS) in 96-well plates and then cultured with nontoxic concentrations of LPp2. Swiss mice intravenously received LPp2 (10 mg/kg) and then were challenged intraperitoneally with virulent Salmonella enterica Ser. Typhimurium. RESULTS: LPp2 was not toxic at dosages lower than 62.2 µg/mL. LPp2 treatments of pMØ stimulated with LPS impaired mRNA expression of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6 and IL-10. LPp2 increased the intracellular bacterial killing in infected pMØ. Mice given LPp2 had a lower number of leukocytes in the peritoneal cavity in comparison to control groups 6 h after infection. The bacterial burden and histological damage were widespread in target organs of mice receiving LPp2. CONCLUSION: We conclude that LPp2 contains peptidases with strong anti-inflammatory properties, which may render mice more susceptible to early disseminated infection caused by Salmonella.

Protein function analysis of germinated Moringa oleifera seeds, and purification and characterization of their milk-clotting peptidase.

The present study aimed to investigate the biological functions of germinated M. oleifera seed proteins and to identify the identity of milk-clotting proteases. A total of 963 proteins were identified, and those with molecular weights between 10 and 30 kDa were most abundant. The identified proteins were mainly involved in energy-associated catalytic activity and metabolic processes, and carbohydrate and protein metabolisms. The numbers of proteins associated with the hydrolytic and catalytic activities were higher than the matured dry M. oleifera seeds reported previously. Of the identified proteins, proteases were mainly involved in the milk-clotting activity. Especially, a cysteine peptidase with a molecular mass of 17.727 kDa exhibiting hydrolase and peptidase activities was purified and identified. The identified cysteine peptidase was hydrophilic, and its secondary structure consisted of 27.60% alpha helix, 9.20% beta fold, and 63.20% irregular curl; its tertiary structure was also constructed using M. oleifera seed 2S protein as the protein template. The optimal pH and temperature of the purified protease were pH 4.0 and 60 °C, respectively. The protease had high acidic stability and good thermostability, thus could potentially be applied in the dairy industry.

Peptidases from Maclura Pomifera for Preparation of Food Protein Hydrolysates: Purification by Single-Step Chromatography and Characterization of Pomiferin I.

Our objective was to isolate peptidases from the latex of Maclura pomifera fruits and use them to hydrolyze food proteins, as well as to purify and characterize the main peptidase. Two partially purified proteolytic extracts were prepared by ethanol (EE) and acetone (AE) precipitation from an aqueous suspension of exuded fruit latex. EE was used to hydrolyze food proteins with a ratio of 0.19 caseinolytic units (Ucas) per mg of substrate. Different values of hydrolysis degree were observed for hydrolysates of egg white, soy protein isolate, and casein at 180 min (9.3%, 31.1%, and 29.1%, respectively). AE was employed to purify a peptidase which exhibited an isoelectric point (pI) of 8.70 and whose abundance in AE was 28.3%. This enzyme was purified to homogeneity using a single-step procedure by cation-exchange chromatography, achieving an 8.1-fold purification and a yield of 16.7%. The peptidase was named pomiferin I and showed a molecular mass of 63,177.77 Da. Kinetic constants (KM 0.84 mM, Vmax 27.50 uM s-1, kcat 72.37 s-1, and kcat/KM 86.15 mM-1 s-1) were determined employing N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester as substrate. Analysis by PMF showed only partial homology of pomiferin I with a serine peptidase from a species of the same family.

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates.

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 23 + 4 were performed to evaluate the following parameters: molar mass of PEG (MPEG ), concentration of PEG (CPEG ), concentration of citrate buffer (CCit ), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of MPEG  = 10,000 g/mol, CPEG  = 22 wt%, CCit  = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 µmol Trolox equivalent/mg protein, for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 µmol Trolox/mg protein). Nevertheless, the IC50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.

Proteomic analysis and optimized production of Alkalihalobacillus patagoniensis PAT 05T extracellular proteases.

Extracellular proteolytic extracts from the haloalkalitolerant strain Alkalihalobacillus patagoniensis PAT 05T have proved highly efficient to reduce wool felting, as part of an ecofriendly treatment suitable for organic wool. In the present study, we identified the extracellular proteases produced by PAT 05T and we optimized its growth conditions for protease production through statistical methods. A total of 191 proteins were identified in PAT 05T culture supernatants through mass spectrometry analysis. Three of the 6 detected extracellular proteases belonged to the serine-endopeptidase family S8 (EC 3.4.21); two of them showed 86.3 and 67.9% identity with an alkaline protease from Bacillus alcalophilus and another one showed 50.4% identity with Bacillopeptidase F. The other 3 proteases exhibited 55.3, 49.4 and 61.1% identity with D-alanyl-D-alanine carboxypeptidase DacF, D-alanyl-D-alanine carboxypeptidase DacC and endopeptidase LytE, respectively. Using a Fractional Factorial Design followed by a Central Composite Design optimization, a twofold increase in protease production was reached. NaCl concentration was the most influential factor on protease production. The usefulness of PAT 05T extracellular proteolytic extracts to reduce wool felting was possible associated with the activity of the serine-endopeptidases closely related to highly alkaline keratinolytic proteases. The other identified proteases could cooperate, improving protein hydrolysis. This study provided valuable information for the exploitation of PAT 05T proteases which have potential for the valorization of organic wool as well as for other industrial applications.

Extracellular neutral protease from Arthrospira platensis: Production, optimization and partial characterization.

Proteases are industrially important catalysts. They belong to a complex family of enzymes that perform highly focused proteolysis functions. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. In the present study, a novel extracellular neutral protease produced from Arthrospira platensis was detected and characterized. Its proteolytic activity was strongly activated by ß-mercaptoethanol, 5,5-dithio-bis-(2-nitrobenzoic acid) and highly inhibited by Hg2+ and Zn2+ metal ions which support the fact that the studied protease belongs to the cysteine protease family. Using statistical modelling methodology, the logistic model has been selected to predict A. platensis growth-kinetic values. The optimal culture conditions for neutral protease production were found using Box-Behnken Design. The maximum experimental protease activities (159.79 U/mL) was achieved after 13 days of culture in an optimized Zarrouk medium containing 0.625 g/L NaCl, 0.625 g/L K2HPO4 and set on 9.5 initial pH. The extracellular protease of A. platensis can easily be used in the food industry for its important activity at neutral pH and its low production cost since it is a valuation of the residual culture medium after biomass recovery.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl ß-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.

Production, properties and some applications of protease from alkaliphilic Bacillus sp. EBTA6.

Extracellular protease production by a novel strain, Bacillus sp. EBTA6, has been optimized by using central composite design of response surface methodology and properties and industrial applications of crude enzyme have been investigated. Three independent variables (temperature, pH and yeast extract concentration) chosen in the experimental design were significant terms and reduced cubic model fit with the design at p < 0.0001 level. The recommended temperature, pH and yeast extract concentration were 30 °C, 8, and 15 g/L, respectively. Crude enzyme displayed activity over a wide pH and temperature ranges having the optimum at 50-60 °C and pH 8. It was quite stable at high pH values and at 50 °C. Amongst the metal ions (Mg+, Cu2+, Ca2+, Zn2+, K2+, and Sn2+), Ca2+ enhanced the activity and the others either decreased or did not change it. The enzyme activity was reduced by phenyl-methyl-sulfonyl fluoride (PMSF), and ethylene diamine tetra acetic acid (EDTA). The results revealed that the protease was serine alkaline type. Tween 20 and Tween 80 did not inhibit the enzyme, however, sodium dodecyl sulfate (SDS), reduced it by 39%. It completely removed blood stain in 20 min and coagulated milk in the presence of CaCl2.