Aims:  Isolation of bacterial antagonist for use in the biological control of phytopathogenic fungi like rice blast fungus, Magnaporthe grisea, and to further purify and characterize the antifungal molecule produced by the antagonist. Methods and Results:  Bacterial antagonist exhibiting highest antifungal activity against the rice blast fungus M. grisea was isolated from soil and identified as Bacillus licheniformis BC98. Besides M. grisea, the isolate also inhibited the growth of other phytopathogens such as Curvularia lunata and Rhizoctonia bataticola. Biologically active fractions were isolated from the culture filtrate and further fractionated by reverse‐phase high‐performance liquid chromatography (HPLC) enabling detailed structural characterization of a component of molecular mass 1035 Da. The active peptide was identified as surfactin after 500 MHz 1H NMR analysis. Microscopic analysis of the effect of the antagonist on M. grisea revealed bulbous hyphae showing patchy and vacuolated cytoplasm when observed under the electron microscope. Conclusions:  The antagonistic lipopeptide secreted by B. licheniformis BC98 and identified as surfactin, induced morphological changes in M. grisea, inhibiting its further growth, and thus exhibiting fungicidal activity. Significance and Impact of the Study:  The antagonist inhibits germination of M. grisea, a potent rice phytopathogen, and therefore appears to be a potential candidate for control of rice blast disease.

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The formation of local structure, in short peptides has been probed by examining cleavage patterns and rates of proteolysis of designed sequences with a high tendency to form beta-hairpin structures. Three model sequences which bear fluorescence donor and acceptor groups have been investigated: [see text]. Fluorescence resonance energy transfer (FRET) provides a convenient probe for peptide cleavage. MALDI mass spectrometry has been used to probe sites of cleavage and CD spectroscopy to access the overall backbone conformation using analog sequences, which lack strongly absorbing donor and acceptor groups. The proteases trypsin, subtilisin, collagenase, elastase, proteinase K and thermolysin were used for proteolysis and the rates of cleavage determined. Peptide 3 is the most susceptible to cleavage by all the enzymes except thermolysin, which cleaves all three peptides at comparable rates. Peptides 1 and 2 are completely resistant to the action of trypsin, suggesting that beta-turn formation acts as a deterrent to proteolytic cleavage.

A synthetic collagenase substrate containing the internal peptide sequence--Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro--has been synthesized, with an N-terminus 4-((4-(dimethylamino)phenyl)azo)-benzoyl (DABCYL) group and C-terminus 5-[2-(acetamido)ethylamino] naphthalene-1-sulfonic acid (AEDANS) moiety resulting in internal quenching of AEDANS fluorescence. Peptide bond hydrolysis results in a large increase in fluorescence at 490 nm upon excitation at 336 nm. The substrate is cleaved exclusively by Clostridium histolyticum collagenase and is completely resistant to attack by proteases like thermolysin, proteinase K, and trypsin. K(m) and V(max) values for substrate hydrolysis by collagenase have been determined, establishing the peptide as one of the best binding substrates for the enzyme. MALDI mass spectrometry using a derivative of the substrate establishes that the sites of cleavage lie within the collagen like domain. The CD spectrum of an analog peptide lacking the donor and acceptor groups reveals spectral features that are reminiscent of weak polyproline structures.

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.