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.     

Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Roles of adjuvants in aphicidal activity of enzymes from Beauveria bassiana (Ascomycota: Hypocreales) SFB-205 supernatant

Enzymes from Hypocrealean entomopathogenic fungi often encounter unfavorable abiotic and biotic factors during pathogenesis. The present work describes the roles of adjuvants, such as corn oil and polyoxyethylene-(3)-isotridecyl ether (TDE-3), in promoting aphicidal activity of enzyme precipitate from Beauveria bassiana SFB-205 supernatant. Supernatant enzymes including chitinase and proteases were lyophilized by attagel-mediated protein precipitation to produce attagel-mediated enzyme powder (AMEP). Corn oil-based AMEP + TDE-3 suspension showed 96.3% control efficacy against cotton aphids, Aphis gossypii Glover (Hemiptera: Aphididae), in glasshouse conditions at 2 days post-application, whereas water-based AMEP + TDE-3 suspension or TDE-3 alone showed < 20% efficacy. Corn oil-based AMEP + TDE-3 suspension was superior in degrading chitinase-specific substrate (p-nitrophenyl-β-d-acetylglucosaminide) under a drying condition compared to water-based AMEP + TDE-3 suspension. TDE-3 made supernatant, a source material of AMEP, degraded more cotton aphid proteins than supernatant alone in SDS-polyacrylamide gel electrophoresis; supernatant was used to clearly show the degradation without interfering with other proteins such as AMEP. These results suggest that 1) corn oil can slow down the evaporation of the diluted suspension drop and provide more time for enzymes from AMEP to degrade more cuticles at the time of application and 2) TDE-3 can disrupt chitin–protein matrixes within procuticle and facilitate enzymes from AMEP in degrading proteins which increases the exposure of chitin fibers to the attack of chitinases. This approach can provide another strategy in developing biopesticides using entomopathogenic fungi.     

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.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

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.     

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 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.     

Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses

4-α-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the α-1.4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1.4-α-d-glucan to an acceptor mono- or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. MeDPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while AtDPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4 kDa, with a pI of 5.13. The predicted structures of both enzymes showed the conserved 250's loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6–8 and 37 °C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16–50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP > 60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.     

Carbonic anhydrase activators. The first activation study of a coral secretory isoform with amino acids and amines

The activity of the coral Stylophora pystillata secretory carbonic anhydrase STPCA has been tested in presence of amino acids and amines. All the investigated compounds showed a positive, activating effect on k_cat and have been separated in weak (K_A in the range of 21–126 μM), medium (10.1–19 μM) and strong enzyme activators (K_A of 0.18–3.21 μM). D-DOPA was found to be the best coral enzyme activator, with an activation constant K_A of 0.18 μM. This enhancement of STPCA activity, as well as previous enzyme inhibition results, might now be tested on living organisms to better understand the role played by these enzymes in the coral calcification processes.     

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.     

Cloning,heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1

The keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 secretes two keratinolytic proteases, KerSMD and KerSMF. However, the genes encoding these proteases remain unknown. Here, we have isolated these two genes with a modified TAIL-PCR (thermal asymmetric interlaced PCR) method based on the N-terminal amino acid sequences of mature keratinases. These two keratinase genes encode serine proteases with PPC (bacterial pre-peptidase C-terminal) domain, which are successfully expressed with the help of pelB leader in Escherichia coli cells. Recombinant KerSMD (48 kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40 kDa). KerSMD has a t_1/2 of 90 min at 50 °C and 64 min at 60 °C, and a better tolerance to surfactants SDS and triton X-100. The predicted model of KerSMD helps to explain the phenomenon of auto-catalytic C-terminal propeptide truncation, the special function of PPC domain, and the molecular weight of the C-terminal-processed mature keratinase KerSMD. This work not only provides a new way to overproduce keratinases but also helps to explore keratinases folding mechanism.     

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.     

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.     

Effect of high salt concentrations on the stability of immobilized lipases: Dramatic deleterious effects of phosphate anions

We have analyzed the effects of the buffer nature on the stability of immobilized lipases. Commercial phospholipase Lecitase Ultra (LU), lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl-glyoxyl agarose beads. The enzymes were readily inactivated using 4 M sodium phosphate but 6 M NaCl did not inactivate them. Using 2 M of sodium phosphate, the inactivation of the 3 immobilized enzymes still was very significant even at 25 °C but at lower rate than with higher phosphate concentration. Thermal stress inactivations of the immobilized enzymes revealed that even 100 mM sodium phosphate produced a significant decrease in enzyme stability; this effect was less pronounced for Lecitase but dramatic for CALB. While 6 M NaCl presented slightly positive (LU) or negative (TLL) effects on their thermal stabilities of, CALB was thermally stabilized under the same conditions. Results were very different using free enymes. Fluorescence spectroscopy revealed dramatic structural rearrangements of the immobilized enzymes in the presence of high phosphate concentration. From these results, the use of sodium phosphate does not seem to be recommended for studies on thermal stability of lipases, although this should be verified for each enzyme and immobilized preparation.     

Latent TGF-β-binding proteins

The LTBPs (or latent transforming growth factor β binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, − 2, − 3, and − 4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here.The LTBPs were first identified as forming latent complexes with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans-golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.     

Age-specific digestion of Tenebrio molitor (Coleoptera: Tenebrionidae) and inhibition of proteolytic and amylolytic activity by plant proteinaceous seed extracts

Tenebrio molitor L. (Coleoptera: Tenebrionidae), is an international and serious pest of stored products. So far nothing is known about the activity for each growth stage digestive enzyme regarding this insect species. Thus, the aim of the current study was to get in depth analysis of the stage specific digestion and to investigate the effect of cereal (wheat cultivars including MV17, Aflak, Sivand, Saymon, and Zare) and legume (bean) seed extracts on the two main digestive enzymes i.e. α-amylases and proteases. Therefore, gut enzymes were extracted using distilled water and wheat cultivars and bean seed proteinaceous compounds were extracted using 0.1 M NaCl. Results showed that a steady state increase in the number and amount of digestive enzyme activities from first to fourth instar larvae was seen in both enzyme and in gel assays. In the first instar larvae (L1) only one band of α-amylase activity was seen (A1), whereas in the second (L2), third (L3), fourth (L4) and fifth (L5) instar larvae as well as in the adult (A) more than one amylase band (up to 4 isoenzymes) was seen. The same pattern was observed for α and β glucosidases and proteases. Probit analysis showed that bean and MV17 inhibited the amylase activity with an I₅₀ of 9.73 and 7.4 μg, respectively. The same cultivar seed extract inhibited protease activity with I₅₀s of 11.54 and 6.5 μg proteins. It is concluded that proteinaceous extract of cereals and bean seeds have a strong potential to be used in this pest management.     

Site-directing an intense multipoint covalent attachment (MCA) of mutants of the Geobacillus thermocatenulatus lipase 2 (BTL2): Genetic and chemical amination plus immobilization on a tailor-made support

Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst⁻¹ min⁻¹) as the reference in the production of PUFAs.     

Immunolocalization of RhoA and RhoB GTPases in pleomorphic adenoma of the parotid

The pleomorphic adenoma of the parotid (PA) is characterized by the high tissues diversity. Rho GTPases participate in signal transduction pathways that regulate several biological processes, including cell differentiation. A quantitative analysis of RhoA and RhoB GTPases immunoexpression was performed in healthy parotids and in 23 PA cases, predominantly epithelial (PE) or mesenchymal (PM), followed by Student's t test. In PE cases, RhoA immunoexpression was higher in sheets and trabeculae (p < 0.05), whereas RhoB only in sheets (p < 0.05). In normal parotids, RhoA and RhoB were not detected in acinar cells. Ducts have expressed RhoA and RhoB in normal parotids and PA. RhoB was detected in myxoid and chondromyxoid cells. Normal parotids do not express RhoA and RhoB proteins in acinar cells, indicating a lack of function in secretory cells. Despite RhoA and RhoB GTPases are different in their biological roles, no significant difference in immunoexpression of the RhoA and RhoB GTPases in epithelial and mesenchymal structures of PA.