Ashis K. Mukherjee

Neutralisation of lethality, myotoxicity and toxic enzymes of Naja kaouthia venom by Mimosa pudica root extracts

Aqueous and alcoholic extracts of dried roots of Mimosa pudica were tested for their inhibitory activity on lethality, myotoxicity and toxic enzymes of Naja kaouthia venom. The aqueous extract, particularly the normal water extract, displayed a significant inhibitory effect on the lethality, myotoxicity and tested enzyme activities of venom compared with alcoholic extracts. The present finding suggests that aqueous extracts of M. pudica root possess compound(s), which inhibit the activity of cobra venom.

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase

Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe3O4 superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25 degrees C and pH 7.2 of the reaction mixture before addition of H2O2 (3% w/w), 2% (w/v) PEG(6000) and 0.062:1 molar ratio of PEG to FeCl2 x 4H2O. Further statistical optimization using response surface methodology yielded an R2 value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

Some biochemical properties of Russell's viper (Daboia russelli) venom from Eastern India: correlation with clinico-pathological manifestation in Russell's viper bite.

In the present study, some biochemical properties and pathological effects of Daboia russelli venom from Burdwan district of West Bengal, eastern India are presented. The clinical features of Russells viper envenomation observed in patients admitted to Burdwan Medical College & Hospital are also reported. In vitro, whole venom exerts strong trypsin inhibitory, phospholipase A2 and procoagulant activities in addition to moderate adenosine monophosphatase and adenosine triphosphatase activities. Lethality (LD50) of this venom sample is 0.7 mg kg (i.v.) of mice. Significant local tissue damaging effects including edema, hemorrhage and necrosis are observed in experimental animal models. An increase in the level of serum enzymes, such as aspartate transaminase, alkaline phosphatase, creatine phosphokinase, lactate dehydrogenase after D. russelli venom injection in albino rats is indicative of cell or tissue damage. High incidence of intravascular hemolysis in addition to hemostasis, haemoptysis and haematuria are observed as the most prominent features of RVV envenomation from this part of India. The present study reinforces the hypothesis that variation in the venom composition of RVV from eastern India with respect to venom samples of Russells vipers from other parts of India is responsible for the differences in the clinical manifestation in patients from eastern India.

Purification and characterization of an anticoagulant phospholipase A2 from Indian monocled cobra (Naja kaouthia) venom

An anticoagulant, non-toxic phospholipase A(2) was isolated from the venom of Indian monocled cobra (Naja kaouthia) by a combination of ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. This purified protein named NK-PLA(2)-I, had a subunit molecular mass of 13.6 kDa and migrated as a dimer under non-reduced condition in SDS-PAGE. NK-PLA(2)-I was a highly thermostable protein requiring basic pH optima for its catalytic activity and showed preferential hydrolysis of phosphotidylcholine. This protein exhibited higher anticoagulant, indirect hemolysis, liver and heart tissue damaging activity but exerted less toxicity, direct hemolysis, edema and lung tissue damaging activity as compared to whole venom. Treatment of NK-PLA(2)-I with rho-BPB, TPCK, PMSF, antivenom and heating had almost equal effect on PLA(2), and other pharmacological properties except in vitro tissue damaging activity. Current investigation provides a fairly good indication that NK-PLA(2)-I induces various pharmacological effects by mechanisms, which are either dependent or independent of its catalytic activity.

Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell's Viper (Daboia russelii russelii) and assessment of its therapeutic potential

BACKGROUND Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates. METHODS A 51.3kDa thrombin-like serine protease (Russelobin) purified from the venom of Russells Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice. RESULTS Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α2-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α2MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity. CONCLUSIONS Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy. GENERAL SIGNIFICANCE Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.

Ecological significance and some biotechnological application of an organic solvent stable alkaline serine protease from Bacillus subtilis strain DM-04

An organic solvent stable, alkaline serine protease (Bsubap-I) with molecular mass of 33.1 kDa, purified from Bacillus subtilis DM-04 showed optimum activity at temperature and pH range of 37-45 degrees C and 10.0-10.5, respectively. The enzyme activity of Bsubap-I was significantly enhanced in presence of Fe(2+). The thermal resistance and stability and of Bsubap-I in presence of surfactants, detergents, and organic solvents, and its dehairing activity supported its candidature for application in laundry detergent formulations, ultrafiltration membrane cleaning, peptide synthesis and in leather industry. The broad substrate specificity and differential antibacterial property of Bsubap-I suggested the natural ecological role of this enzyme for the producing bacterium.

Purification and biochemical characterization of a thermostable, alkaliphilic, extracellular α-amylase from Bacillus subtilis DM-03, a strain isolated from the traditional fermented food of India

Bacillus subtilis strain DM‐03, which is isolated from starter culture used for the production of alcohol by local Assam tribes, grows optimally at 52–55 °C and secretes a significant amount of α‐amylase at pH 8.0 into the culture media. This α‐amylase, purified by ion‐exchange, gel‐filtration and reverse‐phase HPLC, constitutes 2.9% of the total extracellular protein. This purified enzyme, named Bsamy‐I, has a subunit with molecular mass of 42.8 kDa as determined by SDS/PAGE, and optimum temperature and pH values at 52–55 °C and 9.0 respectively, which makes it ideal for use in the detergent industries. Maximum α‐amylase production is obtained by using soluble starch and NH4Cl as carbon and nitrogen sources respectively. Thermostability of the enzyme is evident from heating the enzyme at 95 °C for 10 min, which results in a loss of 60% of the original enzyme activity. 4‐Bromophenacyl bromide and PMSF at 4 and 1.5 mM concentration respectively completely abolish the enzymic activity, documenting the essential role of histidine and carboxylic residues in the catalytic process.

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K(m) and V(max) values of Bafibrinase towards fibrin were determined as 0.24 μM and 2.8 μmol/min, respectively. It showed a K(m) value of 0.139 mM towards a chromogenic substrate for plasmin (D-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,β-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.

A statistical approach for the enhanced production of alkaline protease showing fibrinolytic activity from a newly isolated Gram-negative Bacillus sp. strain AS-S20-I.

An alkaline-fibrinolytic protease-producing bacterial strain (AS-S20-I) isolated from a soil sample in Assam was a Gram-negative rod and grown at temperatures ranging from 25 to 55 °C, and pH 6.5 to 11.0. Taxonomic identification of isolated strain by polyphasic approach (phenotypic characterization, chemotaxonomic properties, and ribotyping data of the strain) suggested that it belongs to the genus Bacillus, for which the name Bacillus sp. strain AS-S20-I (MTCC 8961) was proposed. The initial screening by using Plackett-Burmans design demonstrated that among the tested factors, casein, ammonium sulphate and pH of the medium significantly (p < 0.05) enhanced the protease (fibrinolytic enzyme) yield in submerged fermentation. Further optimization of fibrinolytic protease production by Bacillus. sp. strain AS-S20-I in SmF by applying RSM was achieved as 749.0 × 10(3)UL(-1) in the presence of 3.0% (w/v) casein and 0.12% (w/v) ammonium sulphate at pH 10.9 and 45 °C. This was a 4.0-fold increase in yield compared with that obtained before applying the Plackett-Burman and RSM experimental design. The protease preparation preferentially degraded the fibrin (specific activity 2408.0 ± 70.0 U mg(-1); mean ± S.D.) suggesting that its future application in pharmaceutical industry as thrombolytic and anticancer drugs is highly promising.

Characterization of a novel pro-coagulant metalloprotease (RVBCMP) possessing α-fibrinogenase and tissue haemorrhagic activity from venom of Daboia russelli russelli (Russell's viper): Evidence of distinct coagulant and haemorrhagic sites in RVBCMP

A novel, basic pro-coagulation metalloprotease (Russells viper basic coagulant metalloprotease, RVBCMP) with an approximate molecular weight of 15kDa was purified from the venom of Daboia russelli russelli (Russells viper) from eastern India. RVBCMP exerted dose-dependent coagulation of platelet-poor human plasma; however, RVBCMP possessed less coagulant activity as compared with the coagulant activity of crude Russells viper venom (RVV). RVBCMP did not show oedema induction, direct haemolysis of washed erythrocytes, hydrolysis of human plasma albumin or globulin, and thrombin-like activity, but exhibited caseinolytic, alpha-fibrinogenolytic, and liver tissue haemorrhagic activities. Inhibition of coagulant and protease activities of RVBCMP by EDTA suggested a metalloprotease nature of this protein. RVBCMP showed antigenicity as was evident from the immunoblotting experiment. None of the tested plant extracts, except Leucus lavandulaefolia, inhibited the coagulant or haemorrhagic activity of RVBCMP. Interestingly, aqueous extracts of the tested plants as well as the commercial polyvalent antivenom raised against crude RVV differentially inhibited the coagulant and tissue haemorrhagic activity of RVBCMP. The current investigation provides a fairly good indication that RVBCMP possesses a distinct, perhaps overlapping, site for coagulant and tissue haemorrhagic activity.