Crude Protease Research Articles

Characterization of protease activity from hepatopancreas of blue crab

Proteolytic enzymes play an important role in determining the quality of blue crab during postmortem storage. Activity of endogenous proteases is involved in the texture softening and autolysis of blue crab, which limits the customer acceptance and marketing price. This research aimed to characterize the protease activity of crude enzyme extract from the hepatopancreas of blue crab. The optimum activity of crude protease extract was found at pH 7.0 and 50°C. The crude protease enzyme was highly stable over a wide pH range of 4.0-11.0 and showed high stability at temperatures below 40°C. In addition, the protease activity continuously decreased with an increasing concentration of NaCl (0-15% w/v). Therefore, an understanding of the endogenous proteases in the blue crab could be used to develop appropriate storage methods during its distribution process.

Extraction of Protease Produced by Sea Mud Bacteria and Evaluation of Antifouling Performance

Enzyme-based antifouling coatings are potential alternative to traditional tributyltin-based coatings in the marine bio-fouling control depended on its environmental friendliness. Proteases are usually the key antifouling active components in enzyme based antifouling coatings. In this work, based on biological antagonism effect, a marine proteolytic bacterial strain of Bacillus velezensis was isolated from the sea mud, and denoted as SM-1. The scanning electron microscope (SEM) revealed that the bacteria are rod-shaped with length 1-1.3 μm. The protease-producing conditions of SM-1 were investigated, and it was found that the culture solution displays higher proteolytic activity under the culture conditions of 35°C, 10‰-20‰ salinity, pH 6–9 and more than 7d culture time. Moreover, the 25kDa protein was confirmed to be the main active component in the crude protease, which was revealed via the experiment of SDS-PAGE. The antifouling assay indicated that the protease SM-1 has remarkable effect on the decomposition of barnacle cement and diatom secretion, and also can effectively inhibit the attachment of barnacle cyprids, diatom and mussel byssus. Therefore, this protease potentially can be used as environmental-friendly antifoulant of enzyme-based marine antifouling coatings, and this work also provides a new approach to obtain antifouling protease via isolating proteolytic bacteria from the sea mud surrounding representative fouling organisms.

The Application of Crude Ginger Protease as an Inhibitive Assay for Heavy Metals

In this work, a novel source of protease for the bioassay of heavy metals has been developed using proteases extracted from ginger. The result shows that the optimum protease activity was reached at 1 mg/mL protein concentration of the ginger protease. The optimum casein concentration toward crude ginger protease activity was 1.75 mg/mL. The most suitable pH for protease from crude ginger protease was within the range from pH 5.0 to 7.0. The proteases exhibited high protease activity in a broad range of temperature from 20 to 60 oC. The optimum incubation time for the enzyme occurred at minute 30. Among the six heavy metals tested, only three heavy metals inhibited proteolytic activity of ginger crude with an inhibition more than 30% at 1 mg/L. The calculated LC50 for mercury, copper and silver were, 0.182, (95%, confidence interval (C.I.) 0.134 to 0.283), 0.071, (95% C.I. of 0.056 to 0.096), 0.054, (95% C.I. of 0.039 to 0.085), respectively. Data on the sensitivity of various proteases to heavy metals shows that the crude ginger protease is comparable in sensitivity for mercury to the achromopeptidase, bromelain, papain assays. The crude ginger protease assay for copper is comparable in sensitivity to the papain assay and appears to be more sensitive than the rest of the assays. For silver, the ginger protease was the most sensitive while other assay methods are either unable or be able to detect higher concentration of silver. The crude proteases extracted from ginger showed a good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of mercury, copper, and silver in the environment.

Degumming raw silk by a halotolerant metalloprotease isolated from metabolites of Vibrio sp. LA-05

The crude sample of a halotolerant metalloprotease produced by Vibrio sp. LA-05 was systematically investigated as a silk degumming agent. The experimental results showed that the required time, when the sericin was completely eliminated, varied with the incubation temperatures. The sericin was almost completely removed when raw silk was co-incubated with the crude protease solution (490 U/mL) at 45 °C for 3 h. The results of SEM, thermal analyses, tensile properties and regenerated silk fibroin indicated that the properties of silk fibre degummed with crude protease were superior to those of the traditional Na2CO3 method. Furthermore, SDS-PAGE analyses revealed that the sericin could be hydrolyzed into small peptides by protease during the degumming process. Sericin proteolysis product (SPP) recovered from the protease degumming solution displayed dose-dependent ABTS˙+ scavenging activity and ferric ion reducing capacity. The antioxidant activity of SPP was significantly better than that of sericin recovered from the Na2CO3 degumming solution. The findings in this study suggest that this halotolerant metalloprotease may be developed to an eco-friendly, energy- and resource-saving silk degumming agent.

Antioxidant and antifungal activities of collagen hydrolysates from skin of milkfish (Chanos chanos) hydrolyzed using various bacillus proteases

The skin of fish contains collagen which can be used as a material for food, pharmacy or cosmetics. Collagen from fish is a good source of bioactive peptides which are produce through enzymatic hydrolysis and may be beneficial for human health. This research was conducted to investigate antioxidant and antifungal activities of milkfish (Chanos chanos) collagen which was hydrolyzed by Bacillus licheniformis and Bacillus thuringiensis proteases. The collagen was isolated from milkfish skin and the hydrolysis was done using B. licheniformis protease and crude protease from B. thuringiensis at alkaline condition, 55°C for 30 and 60 minutes. The hydrolysates obtained were assayed for antioxidant activities using ABTS and DPPH methods and antifungal activities against Candida albicans. The results showed that generally, collagen hydrolyzed using B. thuringiensis protease both 30 and 60 minutes showed higher antioxidant activities than of that hydrolyzed using B. licheniformis protease. The hydrolysates also reduced C. albicans at least for 5 log cycles. The results indicated that collagen hydrolyzed using B. thuringiensis protease is very potent as antioxidant and antifungal against C. albicans in future pharmaceutical product.

The application of a halotolerant metalloprotease from marine bacterium Vibrio sp. LA-05 in liquid detergent formulations

The application of a halotolerant metalloprotease from marine bacterium Vibrio sp. LA-05 in liquid detergent formulations was investigated in detail. The washing simulation experiment revealed that blood stains were successfully removed from cotton cloths by washing them with three commercial liquid detergents (Liby, Bluemoon and Walch) supplemented with the crude protease at 15–45 °C for 30 min. However, macroscopic residues of stains were remained on the cloths treated with tap water, liquid detergent alone and liquid detergent supplemented with the commercial protease subtilisin A. The analyses of microstructure, chemical elements and tensile properties showed that the washing treatments with crude protease as a bio-additive almost did not affect the texture, elemental composition or tensile properties of cloths. Sheep blood agar plate and SDS-PAGE analyses indicated that its hydrolysis activity to blood proteins was not significantly influenced even in the presence of liquid detergents. The hydrolysis mechanism was further studied and results demonstrated that this protease could specifically hydrolyzed six proteins in Sheep Red Blood Cells (SRBCs) and four proteins in sheep plasma. These findings suggest that this crude halotolerant metalloprotease as a bio-additive may have considerable potentials in the application of liquid detergent formulations.

Comparative Evaluation of Protease Extraction from Leaves of <i>Syzygium polyanthum</i>

In this study, the comparison of four extraction methods for protease from the leaves of Syzygium polyanthum was evaluated. The methods include sodium phosphate buffer, 1-methyl-8-oxyquinolinium betaine (QB) solvent based extraction, hydrogen-bond acceptor (HBA) solvent based extraction and Trichloroacetic-acetone (TCA) precipitation. The effectiveness of these extraction methods was measured based on the specific activity demonstrated by the crude protease extracted in this experiment. The results indicated that QB solvent-based extraction method could extract the crude protease from the leaves of S. polyanthum with the highest specific activity of 10.204 ± 0.329 U/mg compared to other extraction methods.

Effective biotransformation of Reactive Black 5 Dye Using Crude Protease from Bacillus Cereus Strain KM201428

Effective effluent treatment is a paramount step towards conserving the dwindling clean water resources. The present study describes the use of crude protease extract from Bacillus Cereus Strain KM201428 biotransformation of azo dye Reactive Black 5 (RB5). Batch experimental results displayed over 97% decolorization efficiency with initial dye concentration of 1.0 x 10-4M. The decolorization process was highly dependent on contact time, dye concentration and pH. The optimum contact time and pH for decolorization were 120 hours and pH 9 respectively at 25˚C. Biotransformation of RB5 dye was monitored using UV-Vis spectrophotometer and formed metabolites characterized by LC–QTOF-MS. Comparison of resultant LC–QTOF-MS chromatograms after decolorization confirmed complete cleavage of RB5 dye. First order kinetic fitted well with experimental data for different RB5 dye concentrations. Lineweaver-Burk plot was used to describe the apparent relationship between the decolorization rate and the dye concentration at optimized condition with the coefficient of determination R2 =1. This study comprehensively illustrates the potential of crude protease from Bacillus Cereus Strain KM201428 as an effective and environmentally friendly bacterial isolate in biodegradation of RB5 and opens a new approach in the treatment of wastewaters contaminated with high load of azo dyes.

Vallaris solanacea: A rich source for biopharmaceuticals

Plant based medications had served from the earliest period of the human civilization as the most important therapeutic weapon available to man to fight various human and animal diseases. Vallaris solanacea (Roth) Kuntze (Family: Apocyanacea) locally named as Agarmoni, bread flower is a tall climbing shrub. It is distributed throughout India and cultivated as an ornamental plant for its white fragrant flowers traditionally used against ring worms and skin infections. The plant extract contains reducing sugars, tannins, saponins, gums, steroids, alkaloids and glycosides and is medicinally important with its cytotoxic, antioxidant, antinociceptive, antimicrobial, analgesic, anti-inflammatory and antidiarrhoel activities. As an accumulators of pollutants it has an an important role in phytoremediation. Fascinated by the fact that this plant is a natural source of pharmaceuticals, in the present study a proteolytic enzyme has been extracted from the latex of the plant Vallaris solanacea. Preliminary investigations were carried out on this protease which included the effect of time, enzyme concentration, pH, temperature, activators and inhibitors on the caseinolytic activity of crude protease. Stability towards temperature and pH were also checked. Further a cysteine protease was purified from crude latex of Vallaris solanacea by fractionation with ammonium sulphate, ion exchange and gel chromatography. The enzyme was purified to a state of near homogeneity as checked by polyacylamide gel electrophoresis at pH 8.3. The proteolytic enzyme present in the latex of Vallaris solanacea is referred to as solanain. Its antimicrobial properties were studied. Thus this information emphasizes that Vallaris can be explored for new drugs.

Immobilization of Enzymes on Flexible Tubing Surfaces for Continuous Bioassays.

Immobilized enzymes can be used to catalyze biochemical reactions in a batch process, however, it is more difficult to use them in a continuous process. Herein, we develop an enzyme immobilization technique for flexible tubing surfaces, which can be used to catalyze biochemical reactions in a continuous process. In this technique, the tubing is first treated with (3-aminopropyl)triethoxysilane at 50 °C and baked at 100 °C in vacuum to form a network of reactive amine functional group on the inner tubing surface. Subsequently, dextran polyaldehyde, a polymeric cross-linker, is used to immobilize crude protease extract and catalase for hydrolyzing casein and degrading H2O2, respectively, in a continuous process. The immobilized proteases are highly stable even after a long-term storage at 4 °C. After 12 weeks of storage, 90% of the original protease activity can be preserved. Meanwhile, the immobilized catalase is able to degrade 0.1% H2O2 solution flowing at 5 μL/min. The immobilization technique is potentially useful for bioassays and industrial wastewater treatments when continuous processes are preferred.

Biocontrol of Meloidogyne incognita by Metarhizium guizhouense and its protease

Crude extracts of the fungus Metarhizium guizhouense PSUM02 grown on protein- enriched medium showed high proteolytic activity and produced one main protein indicated as fungal protease with 33 kDa molecular mass. The protease activity had high stability when incubated at 10 or −20 °C for 6 months. The use of Metarhizium spores (1 × 104 spore mL−1) in combination with its protease (354.1 ± 2.2 U mL−1) completely inhibited hatching of the root-knot nematode Meloidogyne incognita within 24 h. Moreover, the mortality of juveniles increased to 100% at 48 h post treatment. The results were significantly different (p < 0.05) from Metarhizium spores or protease individually, as well as from extraction buffer and distilled water controls. Metarhizium spores suspended in crude protease rapidly germinated in 24 h and then mycelia invaded the immature eggs of M. incognita, while the eggs containing juveniles and the hatched juveniles were resistant. SEM observations showed extensive damage to eggshell and cuticle when treated with Metarhizium spore and protease, either individually or in combination. This is the first report on biocontrol potential of M. guizhouense against M. incognita. The combination of Metarhizium spores and protease has enhanced potential to develop into a safe and reliable method for plant parasitic nematode control.

Preliminary Screening of Plant Proteases as a Potential Source for the Development of an Inhibitive Assay for Heavy Metals

Heavy metals pollution has become a great threat to the world. Since instrumental methods are expensive and need skilled technician, a simple and fast method is needed to determine the presence of heavy metals in the environment. In this work, a preliminary study was carried out on the applicability of various local plants as a source of protease for the future development of the inhibitive enzyme assay for heavy-metals. The crude proteases preparation was assayed using casein as a substrate in conjunction with the Coomassie dye-binding assay. The crude protease from the kesinai plant was found to be the most potent plant protease. The crude enzyme exhibited broad temperature and pH ranges for activity and will be developed in the future as a potential inhibitive assay for heavy metals.

PRODUCCIÓN DE PROTEASAS A PARTIR DE Bacillus safensis EN FERMENTACIÓN SUMERGIDA USANDO METODOLOGÍA DE SUPERFICIES DE RESPUESTA

In present study, various cultural parameters for Bacillus safensis were optimized in submerged fermentation for alkaline protease production using response surface methodology. Nutritional components were screened through Placket-Burman design. The optimized cultural parameters were, 1% inoculum, initial medium pH 9.0, incubation temperature 30OC and fermentation time 72h. Among nutritional parameters, casein, KH2PO4 and peptone were found significant which were further optimized through Box-Bhenken Design with three levels. The experimental results showed that 4.0% casein, 0.05% KH2PO4 and 0.575% peptone yielded highest protease production. The proposed model was significant which clearly explained the response. The crude protease produced by Bacillus safensis showed optimum activity at pH 8.0 and at 40oC. Kinetics revealed that the Km and Vmax values of 1.027 mg/ml and 67.57 mg/ml/min using casein as substrate, respectively

Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata

Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest – Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ± 11 nM) and chymotrypsin (Ki of 446 ± 9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50 = 8 μg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.

Effect of temperature and pH on the properties of skim milk gels made from a tamarillo (Cyphomandra betacea) coagulant and rennet

Reconstituted skim milk was gelled with a crude protease extract from tamarillo [Cyphomandra betacea or Solanum betacea (syn.)] fruit and compared with gels prepared with calf rennet. The effects of temperature and pH on the gelation of skim milk were investigated by small deformation oscillatory rheology. The tamarillo extract-induced gels had a faster rate of increase in the elastic modulus (G') at the early stage of gelation than rennet-induced milk gels. This was probably due to the broader proteolytic activity of tamarillo protease extracts as shown by sodium dodecyl sulfate-PAGE analysis. Confocal microscopy also showed that the milk gels resulting from the addition of tamarillo extracts had larger voids than rennet-induced milk gels. The proteolytic activity of tamarillo extracts was found to be optimal at pH 11. For both rennet and tamarillo extracts, the aggregation time was similar between pH 6.7 and 6.5, but the aggregation time of rennet-induced milk gels was lower than that of milk gels obtained by the addition of tamarillo extracts at pH lower than 6.5. An increase in temperature was found to have a significant effect on aggregation time, particularly at 20°C, where rennet did not coagulate milk in 3 h but the tamarillo extracts coagulated milk within 2 h. The results of this study suggest that extracts from tamarillo fruit could be used for milk gelation, particularly under lower temperature or high pH conditions.

Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases

The herbaceous plant Cynanchum otophyllum Schneid. is widely used as a milk coagulant to make a Chinese traditional milk product, milk cake. However, the milk-clotting compounds and their mechanism remain unclear. In this study, crude proteases were extracted from the dried leaves of Cynanchum otophyllum Schneid. using citric acid-phosphate buffer and then partially purified by weak anion exchange chromatography. Two proteases, QA and QC, with molecular weights of 14 and 27 kDa, respectively, were shown to exhibit milk-clotting activity. A study of the effects of pH and temperature on the milk-clotting activity and proteolytic activity of the proteases showed that they exhibited good pH stability from pH 5.5 to 7.5 and good thermal stability at temperatures from 50 to 70°C. The QA and QC were the cysteine proteases, able to hydrolyze β-casein and κ-casein completely, and α-casein partially. The cleavage site on κ-casein determined by Orbitrap (Thermo Fisher Scientific, San Jose, CA) analysis showed that QA and QC could cleave κ-casein at Ser132-Thr133. Overall, the results suggest that the Cynanchum otophyllum Schneid. proteases are a promising milk-clotting enzyme that could be used for manufacturing milk cake and cheese.

STATISTICAL AND KINETIC STUDIES OF ACID PROTEASE BY ASPERGILLUS SPP. ISOLATED FROM SOIL CONTAMINATED WITH ABATTOIR WASTE

Objective: Aim of the present investigation was to optimize the acid protease production from Aspergillus spp. through statistical method in solid state fermentation and to study the inhibitory enzyme kinetics.Methods: To fulfill above mentioned aim, seven solid substrates were screened though using PBD (Plackett-Burman Design) and concentrations of three significant were determined by using one of the Response surface methodologies (RSM), Box-Behnken design (BBD). Inhibitory enzymatic effects were carried by using previously developed models.Results: From PBD, wheat bran, soybean meal, and dried potato peel (DPP) were screened as major influencing nutritional factors for enzyme production. Better optimal values were determined by BBD as wheat bran: 8.841 g, soybean meal: 4.557 g, and DPP: 0.661 g with predicted protease activity as 817.83 U/g (±44.047 U/g). Linear, interactive, and quadratic effects of aforesaid substrates on enzyme activity were formulated by quadratic model through multiple regression model (R2Adj:Adjusted R square = 94.78%; R2Pre:Predicted R square = 98.13%). Partial substrate inhibition to crude acid protease activity was notified with casein concentration higher than 0.4 mmol and inhibitory constant, KN, was computed with previous developed mathematical models. Ratio of reaction rate constants, k4/k2, was found to be 0.233 that had confirmed partial casein inhibition to enzyme velocity. Improved activity and kinetics of caseinolysis make amicable for industrial applications.Conclusion: Quick optimization was performed with statistical methodology over conventional approach. Inhibitory enzyme kinetic studies were important for industrial applications of acid protease.

Characterization of detergent compatible protease from halophilic Virgibacillus sp. CD6.

A halophilic bacterium, Virgibacillus sp. strain CD6, was isolated from salted fish and its extracellular protease was characterized. Protease production was found to be highest when yeast extract was used as nitrogen source for growth. The protease exhibited stability at wide range of salt concentration (0-12.5%, w/v), temperatures (20-60°C), and pH (4-10) with maximum activity at 10.0% (w/v) NaCl, 60°C, pH 7 and 10, indicating its polyextremophilicity. The protease activity was enhanced in the presence of Mg2+, Mn2+, Cd2+, and Al3+ (107-122% relative activity), and with retention of activity>80% for all of other metal ions examined (K+, Ca2+, Cu2+, Co2+, Ni2+, Zn2+, and Fe3+). Both PMSF and EDTA inhibited protease activity, denoting serine protease and metalloprotease properties, respectively. High stability (>70%) was demonstrated in the presence of organic solvents and detergent constituents, and the extracellular protease from strain CD6 was also found to be compatible in commercial detergents. Proteinaceous stain removal efficacy revealed that crude protease of strain CD6 could significantly enhance the performance of commercial detergent. The protease from Virgibacillus sp. strain CD6 could serve as a promising alternative for various applications, especially in detergent industry.

Characterization of a double WAP domain-containing protein from the red swamp crayfish Procambarus clarkii

Crustaceans express multiple whey acidic protein (WAP) domain containing proteins which are components of host immunity. In the present study, a new double WAP domain containing protein was identified from red swamp crayfish Procambarus clarkii, designated Pc-DWD. The ORF is 387 bp, encoding 128 amino acids consisting of signal peptide of 18 residues, and two tandem WAP domains of 38 and 44 residues. Multiple alignment indicates the presence of conserved motifs in both WAP domains, and phylogenetic analysis shows that Pc-DWD is a new member of the type-IV crustin family. Pc-DWD transcripts were found most abundantly in hemocytes, gills, intestine and heart, and induced by Vibrio anguillarum, Staphylococcus aureus and white spot syndrome virus challenge. RNAi knockdown of Pc-DWD expression led to increased expression of white spot syndrome virus genes and increased crayfish mortality after virus infection. Recombinant Pc-DWD exhibited strong protease inhibitory activity towards commercial subtilicin A and protease K. Pc-DWD inhibited the crude proteases from V. anguillarum and S. aureus cultures and from the crayfish tissue extracts. We infer that Pc-DWD acts in crayfish bacterial and viral immunity.

Extraction, purification and characterization of a novel cysteine protease from the latex of plant Vallaris solanacea

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.