Purification and characterization of two novel extra cellular proteases from Serratia rubidaea

A protease, producing bacterial culture (isolate ‘C’) was obtained from slaughterhouse waste samples, Hyderabad, India. It was related to Serratia rubidaea on the basis of 16S r RNA gene sequencing and biochemical properties. Cultural characters of S. rubidaea identified it as a psychrophile secreting protease at 10–30 °C. Single step purification of culture supernatant on sephacryl S-100 column revealed two proteases CP-1 and CP-2. The molecular masses of the enzymes determined by SDS-PAGE and zymography were approximately 97 and 45 kDa, respectively. N-terminal sequencing of CP-1 revealed a novel surface protein of S. rubidaea and CP-2 protease has shown 100% homology with protease of Serratia sp. A fold purification of 1.5 with 54% recovery was achieved in CP1 and purification of CP-2 resulted in 88% yield with a fold purification of 2. The optimum pH values of CP-1 and CP-2 were shown to be 10 and 8, respectively. The maximum activities for the enzymes were at 40 °C and 30 °C. Both the proteases are inhibited by EDTA indicating that they are metallo proteases. The activity of CP-1 was enhanced with Cu²⁺ that of CP-2 was enhanced with Zn²⁺ and Ca²⁺. These proteases have stability in presence of detergents, surfactants and solvents. These properties make these proteases an ideal choice for application in detergent formulations, food, leather industries, vaccine and enzyme peptide synthesis.     

ADAMTS proteases in vascular biology

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteases comprise the most recently discovered branch of the extracellular metalloenzymes. Research during the last 15 years, uncovered their association with a variety of physiological and pathological processes including blood coagulation, tissue repair, fertility, arthritis and cancer. Importantly, a frequent feature of ADAMTS enzymes relates to their effects on vascular-related phenomena, including angiogenesis. Their specific roles in vascular biology have been clarified by information on their expression profiles and substrate specificity. Through their catalytic activity, ADAMTS proteases modify rather than degrade extracellular proteins. They predominantly target proteoglycans and glycoproteins abundant in the basement membrane, therefore their broad contributions to the vasculature should not come as a surprise. Furthermore, in addition to their proteolytic functions, non-enzymatic roles for ADAMTS have also been identified expanding our understanding on the multiple activities of these enzymes in vascular-related processes.     

Bacillus subtillis RTSBA6 6.00, a new strain isolated from gut of Helicoverpa armigera (Lepidoptera: Noctuidae) produces chymotrypsin-like proteases

Exploring bacterial communities with proteolytic activity from the gut of the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) insect pests was the purpose of this study. As initial efforts to achieve this goal here we report the isolation of new Bacillus subtillis RTSBA6 6.00 strain from the gut of H. armigera and demonstrated as proteases producer. Zymographic analysis revealed 12 proteolytic bands with apparent molecular weights ranging from 20 to 185 kDa. Although some activity was detected at acidic pH, the major activity was observed at slight alkaline pH (7.8). The optimum temperature was found to be 35 °C with complete loss of activity at 70 °C. All proteases were completely inactivated by PMSF (phenylmethylsulfonyl fluoride) and TPCK (N-tosyl-l-phenylalanine chloromethyl ketone), suggesting that proteases secreted by B. subtillis RTSBA6 6.00 belong to serine proteases class with chymotrypsin-like activity. The occurrence of protease producing bacterial community in the gut of the H. armigera advocates its probable assistance to insect in proteinaceous food digestion and adaptation to protease inhibitors of host plants.     

Digestive proteolytic activity in larvae and adults of Bactrocera oleae Gmelin (Diptera: Tephritidae)

Digestive proteolytic activity in larvae and adults of Bactrocera oleae was studied using specific substrates and inhibitors. The optimal pH for general proteolytic activity was 4 and 10 for soluble and membrane-bound fractions of larvae, and 9 for the soluble fraction of adults. The highest activities of general proteases were revealed at temperatures of 25 °C and 45 °C for both the soluble and membrane-bound fractions of larvae as well as the soluble fraction of adults. Determination of the specific protease activities demonstrated the presence of serine and cysteine proteases in addition to two exopeptidases in the larvae and adults. However, trypsin-like protease, chymotrypsin-like protease, and two exopeptidases of larvae, and chymotrypsin-like protease as well as cathepsin L of adults had no activity in the soluble fraction. The presence of specific proteases was verified by using specific inhibitors such as PMSF, TLCK, TPCK, E-64, EDTA, phenanthroline, and DTT. Finally, feeding of B. oleae larvae on different olive varieties revealed the highest trypsin-like protease, chymotrypsin-like protease, elastase, cathepsin B, cathepsin L, and cathepsin D on Amigdalifolia, Coratina, Baladi, Mari, Conservalia, Baladi, and Arbequina, respectively. These results showed digestive proteolytic activities in B. oleae for the first time, and could be the basic knowledge required for finding a control procedure to decrease the damage of this destructive pest around the world.     

Hypervalent organotellurium compounds as inhibitors of P. falciparum calcium-dependent cysteine proteases

Hypervalent organotellurium compounds (organotelluranes) have shown several promising applications, including their use as potent and selective cysteine protease inhibitors and antiprotozoal agents. Here, we report the antimalarial activities of three organotellurane derivatives (RF05, RF07 and RF19) in two Plasmodium falciparum strains (CQ_S 3D7 and CQ_R W2), which demonstrated significant decreases in parasitemia in vitro. The inhibition of intracellular P. falciparum proteases by RF05, RF07 and RF19 was determined and the IC₅₀ values were 3.7 ± 1.0 μM, 1.1 ± 0.2 μM and 0.2 ± 0.01 μM, respectively. Using an assay performed in the presence of the ER Ca^2 +-ATPase inhibitor we showed that the main enzymatic targets were cysteine proteases stimulated by calcium (calpains). None of the compounds tested caused haemolysis or a significant decrease in endothelial cell viability in the concentration range used for the inhibition assay. Taken together, the results suggest promising compounds for the development of antimalarial drugs.     

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis,genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of ～ 1.7 kb located in 2 chromosomal bands of 600–720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes.     

PEG/NaPA aqueous two-phase systems for the purification of proteases expressed by Penicillium restrictum from Brazilian Savanna

The partitioning of proteases expressed by Penicillium restrictum from Brazilian Savanna in an inexpensive aqueous two-phase system composed of poly (ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The effects of PEG molecular weight and concentration, as well as NaPA concentration and the concentration of fermented broth on protease partitioning were studied. Partitioning into the top PEG-rich phase was increased in systems with smaller PEG-molecular weight, higher NaPA concentration and lower PEG concentration. For most systems studied, purification has been achieved by directing the biomolecule partition to the opposite phase of the other proteins, providing the enzyme purification. The highest partition coefficient was obtained using 20 wt% NaPA, 4 wt% PEG 2000 g mol⁻¹ and 45 wt% fermented broth, leading to a purification factor of 1.98 and partition coefficient of 37.73. The system showed high mass balances and yield, indicating enzyme stability and applicability for industrial processes. The partitioning results using the PEG/NaPA/NaCl system show that this method could be used to purify or concentrate protease from fermented broth.     

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC₅₀ values of 12 nM and 42 nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.     

Synthesis of dammarane-type triterpene derivatives and their ability to inhibit HIV and HCV proteases

We synthesized dammarane-type triterpene derivatives and evaluated their ability to inhibit HIV-1 and HCV proteases to understand their structure–activity relationships. All of the mono- and di-succinyl derivatives (5a–5f) were powerful inhibitors of HIV-1 protease (IC₅₀ < 10 μM). However, only di-succinyl (5e) and 2,3-seco-2,3-dioic acid (3b) derivatives similarly inhibited HCV protease (IC₅₀ < 10 μM). A-nor dammarane-type triterpenes (4a and 4b, IC₅₀ 10.0 and 29.9 μM, respectively) inhibited HIV-1 protease moderately or strongly, but were inactive against HCV protease. All compounds that powerfully inhibited HIV-1 or HCV protease did not appreciably inhibit the general human proteases, renin and trypsin (IC₅₀ > 1000 μM). These findings indicated that the mono-succinyl dammarane type derivatives (5a–5d) selectively inhibited HIV-1 protease and that the di-succinyl (5e, 5f) as well as 2,3-seco-2,3-dioic acid (3b) derivatives preferably inhibited both viral proteases.     

Nutritional value and proteases of Lentinus citrinus produced by solid state fermentation of lignocellulosic waste from tropical region

This paper examined the growth and yield performance of Lentinus citrinus on cupuaçu exocarp (Theobroma grandiflorum) mixed with litter (CE + LI) or rice bran (Oryza sativa) (CE + RB) in the ratio of 2:1 (800 g:200 g) to investigate the nutritional composition and proteolytic potential of the fruiting body produced. Significance values of yield were determined on substrate combinations. In CE + LI the biological efficiency of the mushrooms was 93.5% and the content of fat (4.5%), fiber (11.0%), protein (27.0%) and amino acids were higher when compared with CE + RB. Among the amino acids, the amount of glutamic acid, aspartic acid, alanine, arginine and leucine was high. The biological efficiency on CE + RB reduced to 84.2% and based on the nutritional value, carbohydrates (53.59%), energy (324.33 kcal) and minerals such as zinc, iron, copper, potassium and phosphorus were higher in this substrate combination. Protease activity from fruiting body was significant in CE + LI (463.55 U/mL). This protease showed an optimal activity at 50 °C in neutral and alkaline pH with maximum stability at 30 °C at alkaline pH. This is the first report of L. citrinus fruiting body nutritional composition with potential for human food and application in industrial processes.     

The PARL family of mitochondrial rhomboid proteases

Rhomboids are an ancient and conserved family of intramembrane-cleaving proteases, a small group of proteolytic enzymes capable of hydrolyzing a peptide bond within a transmembrane helix that anchors a substrate protein to the membrane. Mitochondrial rhomboids evolved in eukaryotes to coordinate a critical aspect of cell biology, the regulation of mitochondrial membranes dynamics. This function appears to have required the emergence of a structural feature that is unique among all other rhomboids: an additional transmembrane helix (TMH) positioned at the N-terminus of six TMHs that form the core proteolytic domain of all prokaryotic and eukaryotic rhomboids. This “1 + 6” structure, which is shared only among mitochondrial rhomboids, defines a subfamily of rhomboids with the prototypical family member being mammalian Parl. Here, we present the findings that in 11 years have elevated mitochondrial rhomboids as the gatekeepers of mitochondrial dynamics and apoptosis; further, we discuss the aspects of their biology that are bound to introduce new paradigm shifts in our understanding of how the organelle uses this unique type of protease to govern stress, signaling to the nucleus, and other key mitochondrial activities in health and disease.     

An inhibitor from Lupinus bogotensis seeds effective against aspartic proteases from Hypothenemus hampei

The coffee berry borer, Hypothenemus hampei (Ferrari), is one of the most devastating coffee pests (Coffea arabica L.) worldwide. Digestion in the midgut of H. hampei is facilitated by aspartic proteases. This is the first report of an aspartic protease inhibitor from Lupinus bogotensis. The L. bogotensis aspartic protease inhibitor (LbAPI) exhibited a molecular mass of 12.84 kDa, as determined by MALDI-TOF, and consists of a single polypeptide chain with an isoelectric point of 4.5. In thermal activity experiments, stability was retained at pH 2.5 after heating the protein at 70 °C for 30 min, but was unstable at 100 °C. The protein was also stable over a broad range of pH, from 2 to 11, at 30 °C. In in vitro assays, LbAPI was highly effective against aspartic proteases from H. hampei guts with a half maximal inhibitory concentration (IC₅₀) of 2.9 μg. LbAPI inhibits pepsin in a stoichiometric ratio of 1:1. LbAPI inhibition of pepsin was competitive, with a K_i of 3.1 μM, using hemoglobin as substrate. Its amino-terminal sequence had 76% homology with the seed storage proteins vicilin and β-conglutin. The homology of LbAPI to vicilins from Lupinus albus L. suggests that they may also serve as storage proteins in the seed. LbAPI could be a promising tool to make genetically modified coffee with resistance to H. hampei.     

Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Three distinct alkaline serine proteases (named CTSP-1, -2, and -3) were purified from the polychaete Cirriformia tentaculata and characterized in terms of their enzymatic properties and kinetics. The estimated molecular masses of CTSP-1, -2, and -3 enzymes were found to be 28.8, 30.9, and 28.4 kDa, respectively. The enzymes were active at the temperature range of 50–60 °C under pH 8.5–9.0 and completely inactivated by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, but not by 1,10-phenanthroline and bestatin, suggesting that they are all typical serine proteases and not metalloproteases or cysteine proteases. CTSP-1 and -2 cleaved arginine, whereas CTSP-3 digested tyrosine residue at the carboxyl sides in their peptide substrates. A typical hepta-sequence (I-X-X-G-X-X-A) conserved in serine proteases from annelid species was found in N-termini of all CTSPs. CTSP-2 was the most active enzyme among the proteases purified as shown by kinetic values. The enzymes cleaved all chains of fibrinogen within 20 min and also hydrolyzed actively fibrin polymer as well as cross-linked fibrin. In addition, the enzymes could actively digest the fibrin clot in blood plasma milieu. Taken together, the results obtained demonstrate that CTSP enzymes have a potential of becoming therapeutic agents for thrombus dissolution.     

Antioxidant,Fusarium growth inhibition and Nasutitermes corniger repellent activities of secondary metabolites from Myracrodruon urundeuva heartwood

Myracrodruon urundeuva heartwood is resistant to biodeterioration and lectin purified from heartwood showed antifungal and termiticidal activities. This report deals with antioxidant, antifungal and termite repellent activities of secondary metabolites from M. urundeuva heartwood. Saline (SE, active hemagglutinin preparation) and methanolic (ME, without hemagglutinating activity) extracts contain phenolic compounds, gallic acid, flavonoids, luteolin, cinamic derivatives, proanthocyanidins, hydrolysable tannins, and leucoanthocyanidins. Both SE and ME showed antioxidant activity and were effective in Fusarium growth inhibition. SE was efficient against Fusarium decemcellulare, Fusarium moniliforme, Fusarium oxysporum, and Fusarium solani but it had little effect against Fusarium lateritium. ME practically had no effect on F. decemcellulare and was more active than SE against F. lateritium. SE induced mortality of Nasutitermes corniger (LC₅₀ of 1.81 mg ml^?1 for soldiers and 2.59 mg ml^?1 for workers) and had no repellent activity. ME had no termiticidal activity but was a good repellent. The detected bioactivities point out the possibility of participation of secondary metabolites in the resistance of M. urundeuva heartwood to biodeterioration. Additionally, the results indicate the use of wood residues, as extracts, a source of natural bioactive agents.     

Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model

Background

Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host–parasite interaction.

Tanshinones as selective and slow-binding inhibitors for SARS-CoV cysteine proteases

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CL^pro and PL^pro, viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (1–7) act as time dependent inhibitors of PL^pro, but no improved inhibition was observed following preincubation with the 3CL^pro. In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC₅₀ = 0.7 μM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.     

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom,Chaetoceros gracilis

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.     

Peptide synthesis in neat organic solvents with novel thermostable proteases

Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80 °C and 60 °C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40–50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Design,synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: Potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one—1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC₅₀ values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure–activity relationship studies, in vitro screening and X-ray crystallography.