Gurvinder Kaur

Extracellular enzyme production in Metarhizium anisopliae isolates

Extracellular enzymes produced by Metarhizium anisopliae are believed to play a key role in cuticle hydrolysis. The in-vitro production of cuticle-degrading enzymes, such as chitinase, proteinase, caseinase, lipase and amylase in fourteen isolates of M. anisopliae exhibited significant natural isolate variability. The isolates were also evaluated for chitinase and proteinase enzyme assays in order to quantify the enzyme production. The growth characteristics and colony morphology of the isolates showed variation and few isolates formed sectors and the colonies were either fluffy or powdery. Among the isolates studied, isolate UM2 was found to show good consistence with the results on enzyme measurements as well as the growth characteristics and colony morphology. Such characterization of isolate variability could rationally be used in the selection of isolates for the production of improved myco-pesticides in the integrated pest management programs.

Optimization of different process variables for the production of an indolizidine alkaloid, swainsonine from Metarhizium anisopliae

Swainsonine is a polyhydroxylated indolizidine alkaloid having anticancer, antimetastatic, antiproliferative and immunomodulatory activities and also potential therapeutic applications against AIDS. In the present study, ten isolates of M. anisopliae were screened and enzyme assayed for the production of swainsonine in different media (Complex oatmeal, Czapekdox media with and without lysine (8% w/v) and Sabouraud dextrose broth (SDB)). Among these strains, ARSEF 1724 (UM8) was found to produce highest amount of swainsonine (1.34 μg/l) after 72 h of incubation under shake flask conditions at 180 rpm and 28 °C in complex oatmeal media. In order to maximize the yield of swainsonine the media composition including macro and micronutrients were optimized. The process variables including the chemical factors like carbon sources, nitrogen sources of both organic and inorganic nature and pH with constant inoculum size (1 × 108 spores/ml) were screened using classical one‐factor‐at‐a‐time (OFAT) approach to find their optimum levels. The present study shows that the nutrient requirement is specific for each strain of Metarhizium. Oatmeal extract (6%) was found to be the best supporting media along with nitrogen source, glucose (2%) as best carbon source and pH (∼5) as the best for swainsonine production. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Response surface methodology for optimizing process parameters for the mass production of Beauveria bassiana conidiospores

Beauveria bassiana is an insect pathogenic fungus and is currently being exploited as an effective commercial mycoinsecticide. Solid state fermentation is the most efficient way to mass produce B. bassiana conidiospores on solid substrates. Statistical optimization strategy was adopted to maximize the conidia production through solid state fermentation. Four substrates viz., rice (polished), crushed sorghum, wheat bran and rice bran at variable moisture content and yeast extract concentration were used. A full factorial central composite design was used to design the experiment and response surface method was used to study the optimal parameters required for large scale production. Optimization of the two most important factors like moisture content and yeast extract concentration at varied levels conferred best conidial yield of 28.8 × 10 9 /gm for the mixed substrate rice + wheat bran at 35% moisture content and 1.5% yeast extract concentration.

Production, HPLC analysis, and in situ apoptotic activities of swainsonine toward lepidopteran, Sf-21 cell line

Swainsonine, a secondary metabolite from Metarhizium anisopliae has been extensively studied in the complementary areas of therapeutics and toxicology. This work aims to develop a simple UV‐HPLC method of analyses for swainsonine in Metarhizium fermentation broth and to explore its in situ entomotoxic activities. The partially purified broth was quantitatively analyzed using middle UV (205 nm)‐reverse phase HPLC method with different mobile phases and gradient programmes. Swainsonine was eluted as single peak at (te) 6.0–6.9 min with average concentration of 4.04 ± 0.52 μg/mL using optimal mobile phase (0.1% trifluoroacetic acid in water and acetonitrile). The mass spectrometry analysis further indicated the characteristic MS1 species for swainsonine, [M+H]+ 174.30 in corresponding HPLC peaks. The antiproliferative effects of swainsonine on lepidopteran, Sf‐21 cells were determined through 3‐(4, 5‐dimethylthia‐zol‐2‐yl)−2, 5‐diphenyl tetrazolium bromide (IC50 standard = 3.90 μM and IC50 purified = 5.27 μM) and trypan blue dye exclusion (IC50 standard = 6.91 μM and IC50 purified = 8.67 μM) assays. The fluorescence activated cell sorting evaluation of Sf‐21 cells showed nearly 35% and 42% of population in various apoptotic stages at 36 h, when treated with standard and purified swainsonine, respectively. The morphodimensional field emission scanning electron and atomic force microscopic analyses further confirmed the characteristic apoptotic features like membrane blebbings, ruptures and volume shrinkage in the lepidopteran cells after 24–36 h of post‐treatment incubation. The study describes the potential entomotoxic activities of swainsonine and its role in the virulence of Metarhizium spp.

Optimization of different factors for efficient protoplast release from entomopathogenic fungusMetarhizium anisopliae

Optimization of different factors for efficient protoplast release fromMetarhizium anisopliae was investigated. Factors like culture media, age of the mycelium, incubation time, different enzymatic combinations and osmotic stabilizer were studied. Mycelium harvested at 40th h in Sabouraud Dextrose broth showed the best protoplast yield over the other media and age of mycelium tested. An incubation time of 3 h and Lysing enzyme at a concentration of 10 mg/ml was the best among the different enzymatic concentrations and combinations tested and yielded a protoplast release of 7.3×108 protoplasts/ml. The most suitable osmotic stabilizer for efficient protoplast release was 0.7M KCl.

Effects of carbon and nitrogen sources on the induction and repression of chitinase enzyme fromMetarhizium anisopliae isolates

Metarhizium anisopliae, an entomopathogenic hyphomycete, is being used effectively in Integrated Pest Management (IPM) system. Foliar application of these fungi is quite satisfactory as it invades its host by adhering to insect cuticles and formation of penetration structures called appresoria, which produces various extracellular enzymes, including chitinase that causes the insect cuticle breaching. The induction and repression mechanism of chitinase activity is not entirely understood and activity of this enzyme is different in response to different carbon and nitrogen sources. This report illustrates the effect of two carbon sources viz. colloidal chitin and dextrose and a nitrogen source, yeast extract on the chitinase production of fourteenM. Anisopliae isolates. The chitinase activity varied among the isolates and the different media used. A high enzymatic activity was observed in the medium containing an extra nitrogen source (yeast extract) followed by the medium containing colloidal chitin as a sole source of carbon and nitrogen. The exochitinase activity and the chitinase activity gel were also studied for the isolates showing high chitinase enzyme production. An array of chitinase isozymes were observed on chitinase activity gel with a common 14.3 kDa enzyme for all the isolates.

The antileukaemic cell cycle regulatory activities of swainsonine purified from Metarhizium anisopliae fermentation broth

Swainsonine is a Metarhizium secondary metabolite known differentially for its specific mannosidase inhibitory, toxic and therapeutic activities. Here, the standard and purified swainsonine from Metarhizium anisopliae fermentation broth were comparatively evaluated for their in situ antileukaemic activities in human promyelocytic cell line, HL-60. Both the standard (IC50 = 6.96 μM) and purified (IC50 = 9.50 μM) compounds inhibited the leukaemic cell proliferation without inflicting cell membrane disruption at 48 h of post-treatment incubation. The DNA cell cycle analysis showed approximately 48.81% and 60.72% of the treated cells arrested in the synthetic phase (S-phase) at 36 and 48 h, respectively, upon treatment with IC50 concentration of the purified swainsonine. However, only 29.62% of cells were arrested in S-phase with standard swainsonine at 48 h, suggesting the comprehensive action of certain other metabolites sharing the similar paradigm of antiproliferative properties in Metarhizium broth extract.

Preparative-cum-quantitative mass-directed analysis of swainsonine and its in situ activity against Sf-21 cell line

Swainsonine is a polyhydroxy indolizidine alkaloid with various research and potential therapeutic applications. In this work, swainsonine was partially purified (2.5-folds) with acetone-methanol solvent system from Metarhizium anisopliae fermentation broth. The partially purified broth was further subjected to mass-directed preparative-cum-quantitative analysis. Swainsonine was eluted as MS1 fraction [M + H](+) 174.36 ± 0.21 at 4.91 ± 0.04 min with calculated yield of 7.85 ± 1.59 μg mL(-1) corresponding to 3.74 × 10(5) counts. In situ antiproliferative activity of standard and purified swainsonine fractions was tested against Spodoptera frugiperda, Sf-21 cell line with IC50 values of 2.96 μM and 3.28 μM, respectively, at 36 h. This analytical procedure for purification and quantitative analysis of swainsonine may ensure its suitability for routine laboratory studies and research.