Alkaline Metalloprotease Research Articles

Characterisation of a novel metalloprotease produced by Bacillus subtilis JQ-2 and casein-derived bioactive peptides by the protease

A novel protease JQ-2 produced by Bacillus subtilis JQ-2, was purified and identified to be a peptidase M28 family alkaline metalloprotease with optimum pH and temperature of 11.0 and 52 °C, respectively. SDS-PAGE revealed high proteolytic activity of JQ-2 for κ-casein, and the cleavage site was identified to be at Lys111–Lys112. Ultrafiltration of the casein hydrolysate resulted in U1 fraction (>3 kDa) with good α-glucosidase inhibition ability and U2 fraction (<3 kDa) with good DPPH radical scavenging ability. Identification of the U2 fraction by mass spectrometry revealed three potential new bioactive peptides SLPQNIPP, GLPQEVLN and AVPYPQRDMPIQAF, which were verified by chemical synthesis. Among them, AVPYPQRDMPIQAF had the strongest DPPH radical scavenging ability and α-glucosidase inhibitory activity. The present study demonstrated a novel microbial protease capable of hydrolysing casein to produce new bioactive peptides with potential application in functional dairy products.

Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Soil Microbial Communities Involved in Proteolysis and Sulfate-Ester Hydrolysis Are More Influenced by Interannual Variability than by Crop Sequence

Proteases, catalysing protein hydrolysis, and arylsulfatases, catalysing sulfate-ester hydrolysis, are key microbial enzymes for N and S mineralization in soil. However, knowledge gaps remain regarding the effect of crop successions and seasonal and interannual meteorological variations on microbial communities responsible for those activities. Here, we compared the effect of six cropping sequences on the abundance and activity of microbial communities involved in proteolysis and sulfate-ester hydrolysis in northern France over four years, with two sampling dates per year. Crop sequences impacted soil microbial communities involved in proteolysis but not those involved in sulfate-ester hydrolysis. Oilseed rape following wheat presented a higher abundance of fungal 18S rDNA, culturable bacteria and alkaline metalloprotease genes and higher protease activity than other crop sequences (wheat following oilseed rape or pea, barley following wheat and pea following barley). Net N and S mineralization was not impacted by the cropping sequence. However, interannual variability of microbial parameters was large, and largely overcame the effect of crop sequences. Precipitation variability between years was the likely cause of this effect. In conclusion, the interaction between current crop, previous crops and yearly meteorology can strongly impact the soil microbial communities in agroecosystems.

Characterization of alkaline metalloprotease isolated from halophilic bacterium Bacillus cereus and its applications in various industrial processes.

Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.

Heterologous Expression of Alkaline Metalloproteinases in Bacillus Subtilis SCK6 for Eco-Friendly Enzymatic Unhairing of Goatskins

In this study, alkaline metalloprotease gene 1067 was cloned from Planococcus halotolerans SCU63T and heterologously expressed in Bacillus subtilis SCK6. Using Luria Bertani (LB) broth medium as the initial medium, the optimal medium was obtained through a series of fermentation and culture optimization (g/L): yeast extract (10), soybean powder (15), urea (20), potassium chloride (6.7), calcium chloride (13.3), NaCl (10). On the basis of the optimal medium, the highest enzymatic activity of 1259.21 U/mL could be obtained by culturing at 30°C for 40 h. with pH 8, inoculation amount of 4% and filling amount of 50 mL. EDTA inhibits protease activity and PMSF promotes it, indicating that it was a metalloprotease rather than a serine protease. The optimum reaction temperature of the protease is 70°C, and the optimum pH is 9. The metal ions Zn 2+, Co2+ and surfactant β-ME, Tween 80 can improve the activity of the protease. The results of unhairing, SEM and staining show that this metalloprotease can completely dehair goatskin. Compared with the conventional chemical method, the goatskin after enzymatic unhairing has softer texture and smoother surface, and there is no obvious damage to the goatskin.

Inhibition of quorum sensing-controlled virulence factors with natural substances and novel protease, obtained from Halobacillus karajensis

IntroductionIn recent years, a challenge in clinical treatment has developed due to bacterial resistance to antibiotics. One of the new mechanisms against infections is virulence factor inhibition. Many virulence factors are controlled by quorum sensing pathways such as biofilm formation and pyocyanin production. The goal of the present study was to investigate the effect of an obligate halophilic bacterial strain on Pseudomonas aeruginosa and Staphylococcus aureus, due to its halo-tolerant substances and enzymes. MethodsThe effect of Halobacillus karajensis on bacterial growth and production of virulence factors was studied in this work. The obligate halophile cells and supernatant fractions were extracted by the methanol/chloroform method and characterized by Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Gas Chromatography–Mass Spectrometry (GC-MS), and zymography. The effects of these fractions were studied on biofilm formation in P. aeruginosa and S. aureus as well as on pyocyanin production in P. aeruginosa. The effective protein in the fraction was analyzed by the SDS-PAGE method, and all protein fragments were studied for pyocyanin inhibition. ResultsThe crude supernatant extract, MMS fraction, from H. karajensis was effective for the biofilm reduction in S. aureus (74%) and P. aeruginosa (27%). Two proteases in this fraction, which were recognized by zymography on skim milk, were the probable causes for extracellular polymeric substances (EPS) hydrolysis in the biofilm matrix. Also, halide crystals and branched fatty acids, 12methyl-tetradecanoic acid, in the other fractions decreased the biofilm by 18% in S. aureus. The results showed that a new 25 kD protein, which was obtained from MMS fraction, inhibited pyocyanin production by 60% in P. aeruginosa. The zymogram and bioinformatics studies showed that this protein was a serine alkaline metalloprotease and had an interaction with AHL molecules. ConclusionThe inhibitory effects of the non-toxic natural substances and proteases on biofilm formation and pyocyanin production, specifically the 25 kD protease, are novel in this study and make them a good candidate for infected wound healing and inhibiting the virulence factors.

Soil available nitrogen and phosphorus affected by functional bacterial community composition and diversity as ecological restoration progressed

AbstractAvailable nitrogen (N) and phosphorus (P) are the most common elements limiting the success of ecosystem restoration. Soil microbial communities, harbouring the alkaline metalloprotease (apr) gene and alkaline phosphatase activity gene (phoD), play a crucial role in regulating and maintaining soil available N and P. However, the dynamics of apr‐ and phoD‐harbouring bacteria and their contributions to regulating soil available N and P balances remain largely unexplored as ecological restoration proceeded. In this study, we investigated the community dynamics of apr‐ and phoD‐harbouring bacteria during the plant growing season in association with the long‐term passive (40 years) and active (35 years) restoration of degraded ecosystems. The results showed that soil available N steadily increased while available P typically decreased as ecosystem restoration proceeded. The passive and active restoration efforts resulted in a higher available N balance than available P balance. Passive and active restoration significantly enhanced the abundances, diversities of OTUs, and beta diversities of apr‐ and phoD‐harbouring bacteria. Notably, the alpha diversity of apr‐harbouring bacteria did not keep pace with that of phoD‐harbouring bacteria with increased site age. Our results indicated that the distinct beta diversities of the apr‐ and phoD‐harbouring bacteria were the predominant cause of the variation in the available N and P balance. Our findings provide a foundation for a better understanding of the distinct responses of apr‐ and phoD‐harbouring bacteria to ecological restoration and how they ultimately regulate the available N and P balance in the developing soils of degraded ecosystems.

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential

ABSTRACT: The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study. Of total, 65.63% of the psychrotrophs that contained aprX gene were confirmed as Pseudomonas spp., using genus-specific PCR. Phylogenetic analysis of the 16S rRNA gene of the other psychrotrophs that were potential producers of AprX identified them as Serratia spp. (n=7), Raoultella ornithinolytica (n=1), and Acinetobacter schindleri (n=1) in the inspected samples and Psychrobacter sanguinis (n=1) and Leuconostoc mesenteroides (n=1) in the non-inspected samples. The production conditions of Brazilian MFC of these samples, while meeting the legal determinations, are not sufficient to control Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production of this type of cheese.

Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source

BackgroundSerratiopeptidase is an alkaline metalloendopeptidase, which acquired wide significance because of its therapeutic applications. The present study was undertaken for purification, characterization, and structural elucidation of serratiopeptidase produced from Streptomyces hydrogenans var. MGS13. ResultThe crude enzyme was purified by precipitating with ammonium sulfate, dialysis, and Sephadex gel filtration, resulting in 34% recovery with a 12% purification fold. The purified enzyme S.AMP13 was spotted as a single clear hydrolytic band on casein zymogram and whose molecular weight was found to be 32 kDa by SDS-PAGE. The inhibitor and stability studies revealed that this enzyme is metalloprotease, thermostable, and alkaline in nature. The maximum serratiopeptidase activity was observed at 37 °C and pH 9.0. The partial amino acid sequence of the purified enzyme S.AMP13 by LC-MS/MS analysis shows the closest sequence similarities with previously reported alkaline metalloendopeptidases. The amino acid sequence alignment of S.AMP13 shared a conserved C-terminus region with peptidase-M10 serralysin superfamily at amino acid positions 128–147, i.e., ANLSTRATDTVYGFNSTAGR revealed that this enzyme is a serralysin-like protease. The kinetic studies of the purified enzyme revealed a Km of 1 mg/mL for its substrate casein and Vmax of 319 U/mL/min. The 3D structure of the purified enzyme was modeled by using SWISS-MODEL, and the quality of the structure was authenticated by assessing the Ramachandran plot using PROCHECK server, which suggested that the enzyme was stable with good quality. ConclusionInhibitor, stability, electrophoretic, and bioinformatic studies suggested that the purified enzyme obtained from S. hydrogenans var. MGS13 is a serralysin-like protease.

Identificação de atividade deteriorante e do gene apr na microbiota isolada de leite cru em Caxias, MA

Dentre as bactérias contaminantes, as psicrotróficas destacam-se como as principais responsáveis pela deterioração do leite, pois são capazes de se multiplicar em temperaturas de refrigeração. O gene apr carreia a informação para a produção da metaloprotease alcalina, uma enzima termoresistente capaz de degradar as proteínas do leite. Objetivou-se investigar, por meio de métodos biomoleculares, a presença do gene apr e o potencial deteriorante de bactérias proteolíticas isoladas do leite cru em uma microrregião do Maranhão. Foram selecionadas 112 amostras de leite cru, coletadas de fevereiro a junho de 2018 em propriedades leiteiras das microrregiões de São João do Sóter e Caxias, MA. As análises microbiológicas foram realizadas no Instituto Federal do Maranhão (IFMA), Campus Caxias, e os ensaios biomoleculares no Laboratório de Inspeção de Produtos de Origem Animal (LIPOA) da Universidade Estadual de Londrina (UEL). Foram realizados testes para classificação de proteólise, lipólise e potencial acidificante em temperatura ótima (36 °C/2 dias) e atividade psicrotrófica (7 °C/10 dias). Foram achados 10 (9%) isolados positivos para o gene apr, todos pertencentes ao gênero Pseudomonas, corroborando com outras pesquisas no estado que apontam para o grande potencial deteriorante das bactérias encontradas na localidade. Desta forma, assim como em outras regiões do Brasil, o leite cru refrigerado produzido no estado do Maranhão precisa ser obtido de forma higiênica evitando contaminação com microrganismos deteriorantes que possam comprometer a integridade e a qualidade do leite fluido e derivados destinados ao comércio.

Production, Partial Purification, and Biochemical Characterization of a Thermotolerant Alkaline Metallo-protease from Staphylococcus sciuri

Protease-producing Staphylococcus sciuri was isolated from poultry soil samples and culture conditions for protease production were optimized. The isolated protease showed a maximum activity of 235.1U/ml. Enzyme purification procedure involved ammonium sulphate precipitation and Sephacryl S-200 HR gel filtration chromatography (GFC). The purification process resulted in the production of three protease fractions namely protease І (metallo-alkaline protease), II, and IІІ. The metallo-alkaline protease was purified to 25.49-fold with specific activity of 982.22U/mg and 3.76% recovery. The partially purified metallo-protease was optimally active at pH10.0 and 70°C and exhibited thermal stability up to 50°C. The protease activity was enhanced by Ca2+ and Mg2+, completely inhibited by Hg2+ and Cu2+, and significantly reduced by EDTA. The protease showed significant stability towards various surfactants, including SDS. The Km and Vmax values were 0.68mg/ml and 166.66nmol of azocasein/ml/h, respectively, while the activation energy (Ea) was 3.07Kcal/mol. Hence, it is evident that the produced protease possesses unique characteristics and could be a plausible candidate for various industrial and biotechnological applications.

Purification and Characterization of a Secretory Alkaline Metalloprotease with Highly Potent Antiviral Activity from Serratia marcescens Strain S3.

In this study we report a secretory protein that was purified from Serratia marcescens strain S3 isolated from soil from the tobacco rhizosphere. Subsequent mass spectrometry and annotation characterized the protein as secretory alkaline metalloprotease (SAMP). SAMP plays a crucial role in inhibiting Tobacco mosaic virus (TMV). Transmission electron microscopy (TEM), dynamic light scattering (DLS), confocal microscopy, and microscale thermophoresis (MST) were employed to investigate the anti-TMV mechanism of SAMP. Our results demonstrated that SAMP, as a hydrolytic metal protease, combined and hydrolyzed TMV coat proteins to destroy the virus particles. This study is the first to investigate the antiviral effects of a S. marcescens metalloprotease, and our finding suggests that S. marcescens-S3 may be agronomically useful as a disease-controlling factor active against Tobacco mosaic virus.

Proteolytic and lipolytic potential of Pseudomonas spp. from goat and bovine raw milk

ABSTRACT: Pseudomonas, the main genus of gram-negative microorganisms isolated from milk, is psychrotrophic, biofilm-forming, and thermo-resistant deteriorating enzyme producers. The aim of this study was to quantify Pseudomonas spp. in goat’s and cow’s milk produced in the Paraná state, Brazil, to evaluate the deteriorating activity of the isolates at mesophilic and psychrotrophic conditions and to identify, at the species level, the isolates with alkaline metalloprotease (aprX gene) production potential. Microbiological, biochemical and molecular methods were used for isolating, confirming and identifying of isolates. The mean counts were 1.6 (±6.3)x104 and 0.89(±3)x102 CFU/mL for goat and bovine milk samples, respectively, immediately after milking. Of the Pseudomonas colonies isolated from goat milk (n=60), 91.7% showed proteolytic potential when incubated at 35°C/48 h and 80% at 7°C/10 days, and lipolytic potential was observed in 95% of the isolates incubated in mesophilic and 78.3% at refrigeration conditions. From the isolates of bovine milk (n=20), 35% showed proteolytic activity only when incubated at 35°C/48 h, and lipolytic potential was observed in 25% of the isolates incubated at 7°C/10d and 35°C/48h. It was observed that 83.3% and 25% of the isolates genetically confirmed as Pseudomonas spp. of goat and bovine milk showed the potential for alkaline metalloprotease production, with the species P. azotoformans, P. koreensis, P. gessardii, P. monteilii and P. lurida being the most frequent in goat milk and P. aeruginosa the only species identified in cow milk.

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 104 cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.

Changes in proteolytic bacteria in paddy soils in response to organic management

ABSTRACTProteolytic bacterial communities, which mineralize organic nitrogen, play a key role in agricultural systems. In this study, alkaline metalloprotease (apr) gene fragments from proteolytic bacteria were investigated in bulk and rhizosphere paddy soil from four fields under organic management (for 2, 3, 5, and 9 years), and from one field under conventional management (for 2 years). We analyzed the abundance and structure of the proteolytic bacterial communities using real-time quantitative PCR and denaturing gradient gel electrophoresis. Our results showed that the abundance of proteolytic bacteria ranged from 1.57 × 108 to 8.02 × 108 copies/g of soil. In addition, the abundance of the proteolytic bacteria in the paddy soils under organic management was significantly higher than those in the paddy soil under conventional management. Moreover, the gene copy numbers in the rhizosphere soils were significantly higher than those in the bulk soils. The abundance of proteolytic bacteria tended to increase with the duration of organic management, with the highest abundance being found in the soil that had been under organic management for 5 years. However, the proteolytic bacteria communities in the paddy soils were not significantly affected by management practices. Phylogeny analysis showed that all gel bands obtained represented genes from Pseudomonas. Additionally, correlation analysis and canonical correspondence analysis showed that C/N, C, and N were important factors that influenced the abundance and community structure of the proteolytic bacteria. These results suggest that proteolytic bacteria are indicators in organic management systems, depolymerize organic N and hence maintain soil sustainability.Abbreviations: CM: conventional management; OM: organic management; DGGE: denaturing gradient gel electrophoresis; qPCR: real-time quantitative PCR detecting system; COFCC: China organic food certification center; CCA: canonical correspondence analysis

Purification and Characterization of Alkaline Metalloprotease from Bacillus sp. MTCC 9558 and its Application in Bioactive Peptide Production

Purification and Characterization of Alkaline Metalloprotease from Bacillus sp. MTCC 9558 and its Application in Bioactive Peptide Production

Agro-industrial wastes utilization for the generation of fibrinolytic metalloprotease by Serratia marcescens RSPB11

Abstract A previously isolated bacterial strain Serratia marcescens RSPB11, having the capacity to become an industrially important organism in thrombolytic therapy has been used for this study. Optimization of various fermentation process parameters for fibrinolytic alkaline metalloprotease production from mixed agro-industrial residues using statistical technique and Taguchi orthogonal array design under solid state fermentation (SSF) has been reported for the first time in this study. Before proceeding to Taguchi DOE, a one factor at a time approach has been employed for selection of suitable substrate and culture conditions. Five grams of mixed cake as sole nitrogen source containing green gram husk, groundnut oil cake, rice bran and wheat husk at 1.25 g each, moistened with 8.0 ml mineral salt solution followed by incubation with 3.0 ml inoculum at 30 °C, for 72 hours resulted in 25,385 ± 480 (U/g) serralysin. An overall yield of 148% increase in serralysin production was achieved by employing Taguchi design and one factor at a time approach when compared to control.

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.

Production, Purification, and Biochemical Characterization of Thermostable Metallo-Protease from Novel Bacillus alkalitelluris TWI3 Isolated from Tannery Waste.

Protease enzymes in tannery industries have enormous applications. Seeking a potential candidate for efficient protease production has emerged in recent years. In our study, we sought to isolate proteolytic bacteria from tannery waste dumping site in Tamilnadu, India. Novel proteolytic Bacillus alkalitelluris TWI3 was isolated and tested for protease production. Maximum protease production was achieved using lactose and skim milk as a carbon and nitrogen source, respectively, and optimum growth temperature was found to be 40°C at pH 8. Protease enzyme was purified using ammonium sulfate precipitation method and anion exchange chromatography. Diethylaminoethanol (DEAE) column chromatography and Sephadex G-100 chromatography yielded an overall 4.92-fold and 7.19-fold purification, respectively. The 42.6-kDa TWI3 protease was characterized as alkaline metallo-protease and stable up to 60°C and pH 10. Ca(2+), Mn(2+), and Mg(2+) ions activated the protease, while Hg(2+), Cu(2+), Zn(2+), and Fe(2+) greatly inhibited it. Ethylenediaminetetraacetic acid (EDTA) inhibited TWI3 protease and was activated by Ca(2+), which confirmed that TWI3 protease is a metallo-protease. Moreover, this protease is capable of dehairing goat skin and also removed several cloth stains, which makes it more suitable for various biotechnological applications.

Enhanced fibrinolytic protease production by Serratia marcescens RSPB11 through Plackett-Burman and response surface methodological approaches

A well characterized alkaline metalloprotease enzyme called serralysin with fibrinolytic activity has been reported in the newly isolated Serratia marcescens RSPB11. In view of its potential application in thrombolytic therapy this study has been made for understanding the nutritional parameters requirement needed for production. Therefore, medium components required for the production of serralysin were optimized using a two step statistical approach. Fermentation variables were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodological approach. A total of seven parameters viz., casein, dextrose, KH2PO4, MgSO4, NaCl, CaCl2 and inoculum have been considered for the optimization studies. The statistical model was constructed via central composite design (CCD) using five screened variables (casein, dextrose, KH2PO4, CaCl2 and inoculum size). An overall 51.8% increase in protease production was achieved in the optimized medium as compared with the unoptimized casein medium.With the application of statistical design methodology serralysin production increased significantly with optimized casein medium (23910 U/ml) when compared to yeast extract-peptone medium (5363 U/ml).