Evaluation of acrylamide production by Rhodococcus rhodochrous (RS-6) cells immobilized in agar matrix.

AIMS: The efficiency of acrylamide production was examined with immobilized cells of Rhodococcus rhodochrous (RS-6) containing NHase. METHODS AND RESULTS: Different entrapment matrices such as agar, alginate and polyacrylamide were used. Various immobilization parameters like agar concentration, cell concentration and reaction conditions affecting the bioconversion process using suitable matrices were determined. The cells immobilized with agar matrix were found to be most effective for acrylonitrile conversion. The bioconversion was more efficient in beads prepared with 2% agar and 5% (v/v) cell concentration. The entire conversion of acrylonitrile to acrylamide with agar entrapped cells was achieved in 120 min at 15°C. The agar entrapped R. rhodochrous (RS-6) cells exhibited 8% (w/v) tolerance to acrylonitrile and 35% tolerance to acrylamide. The immobilized cells also retained 50% of its conversion ability up to seven cycles. The laboratory-scale (1 L) production resulted in 466 g L-1 accumulation of acrylamide in 16 h. CONCLUSIONS: The cells immobilized in agar showed better stability and biocatalytic properties and increased reusability potential. SIGNIFICANCE AND IMPACT OF THE STUDY: The agar-immobilized Rhodococcus rhodochrous (RS-6) cells showed enhanced tolerance for both the substrate and product and is economical for the large-scale production of acrylamide.

Response surface methodology: An effective optimization strategy for enhanced production of nitrile hydratase (NHase) by Rhodococcus rhodochrous (RS-6).

Nitrile hydratase is an enzyme which catalyze the hydration of nitriles into amide and their role as catalysts for acrylamide production in industries are well known. The present study aims at statistically optimizing physiological and nutritional parameters for NHase production from Rhodococcus rhodochrous (RS-6). The effect of incubation period, temperature, pH, carbon and nitrogen sources on the production of NHase was investigated by one factor at a time strategy. Further optimization process was carried out by response surface methodology for studying the interactive effect of these variables using central composite design. The optimized levels of variables obtained by statistical analysis were: incubation period 48 h, temperature 33 °C, pH 7.0, glycerol 1% and urea 0.75%, which resulted in maximum NHase production. The results of ANOVA were significant with the F-value of the model being 296.78, value of R 2 is 0.9983 and the lack of fit test was not significant. The contour and response surface plots showed significant interaction between the variables. The NHase yield was enhanced up to 6.22 fold by statistical optimization using RSM. Thus, the developed experimental design is effective towards process optimization for NHase production from R. rhodochrous (RS-6).

Absorption and Reduction of Chromium by Fungi.

Biosorption has gained increased attention as a reliable and proven technology for the remediation of industrial effluents rich in chromium. The present study was planned to isolate potential fungi from effluents contaminated sites and assess their efficiency for the absorption and reduction of chromium. Two species of Aspergillus and a species of Trichoderma which were isolated from contaminated sites and exhibited resistance to 10 mM of chromium on agar were chosen for the study. A biosorbent was designed by growing these fungal isolates on luffa sponge under shaken condition. The absorption and reduction of chromium, by the designed biosorbent was determined by Atomic Absorption Spectrophotometry and UV Visible Spectrophotometer. Actively growing fungi on luffa sponge showed better absorption (21%-25%) and reduction (28%-35%) capacity when compared to heat killed biosorbent in all fungi tested within 24 h of incubation. Interestingly, there was a liner increase in the absorption and reduction (85%-100%) of chromium by the biosorbent designed by using A. niger.

A simple, efficient and rapid screening technique for differentiating nitrile hydratase and nitrilase producing bacteria.

Nitrile hydrolyzing enzymes catalyze the hydration of nitrile compounds to corresponding amides and acids. Bacteria, isolated from soil samples were screened for nitrile hydrolyzing enzymes by simple dye based 96 well plate and nesslerization method. Bromothymol blue was used as an indicator for the detection of amides and acids based on colour change of the indicator dye from blue to dark green or yellow. The screening assay also differentiates between nitrile hydratase (NHase) and nitrilase producing bacteria. Among the 108 bacterial strains screened for enzyme activity, six strains were positive for NHase activity and eleven strains were positive for nitrilase activity based on their ability to degrade acrylonitrile into products. The strain showing maximum NHase activity in quantitative assay was identified as Rhodococcus rhodochrous. The modified method developed by us would be useful for rapid screening of nitrile degrading bacteria potent for acrylamide/acrylic acid production when acrylonitrile is supplied as substrate.

Growth inhibitory and proapoptotic effects of l-asparaginase from Fusarium culmorum ASP-87 on human leukemia cells (Jurkat).

The objective of this study was to evaluate the anticancer properties of l-asparaginase purified from fungal isolate Fusarium culmorum ASP-87 against human T-cell leukemia cell line (Jurkat). The growth inhibitory and proapoptotic effects of purified l-asparaginase on Jurkat cell lines were investigated by determining its influence on cell viability, colony formation, DNA fragmentation, and cell cycle progression. The results revealed that purified l-asparaginase showed significant decrease in cell survival with IC50 value of 90 µg/mL (9 IU/mL). The enzyme inhibited colony formation and showed characteristic laddering pattern on agarose gel thereby confirming the induction of apoptosis. Further, cell cycle analysis revealed that the enzyme induced apoptotic cell death by arresting the growth of cells at G2 -M phase. However, the enzyme did not elicit any toxic effects on human erythrocytes. l-asparaginase purified from F. culmorum ASP-87 showed significant and selective cytotoxic and apoptotic effects on human T-cell leukemic cells in dose-dependent manner. Results of the study give leads for the anticancer effects of fungal l-asparaginase and its potential usefulness in the chemotherapy of leukemia.

Harnessing indigenous plant seed oil for the production of bio-fuel by an oleaginous fungus, Cunninghamella blakesleeana- JSK2, isolated from tropical soil.

Cunninghamella blakesleeana- JSK2, a gamma-linolenic acid (GLA) producing tropical fungal isolate, was utilized as a tool to evaluate the influence of various plant seed oils on biomass, oleagenicity and bio-fuel production. The fungus accumulated 26 % total lipid of their dry biomass (2 g/l) and 13 % of GLA in its total fatty acid. Among the various plant seed oils tested as carbon sources for biotransformation studies, watermelon oil had an effect on biomass and total lipid increasing up to 9.24 g/l and 34 % respectively. Sunflower, pumpkin, and onion oil increased GLA content between 15-18 %. Interestingly, an indigenous biodiesel commodity, Pongamia pinnata oil showed tremendous effect on fatty acid profile in C. blakesleeana- JSK2, when used as a sole source of carbon. There was complete inhibition of GLA from 13 to 0 % and increase in oleic acid content, one of the key components of biodiesel to 70 % (from 20 % in control). Our results suggest the potential application of indigenous plant seed oils, particularly P. pinnata oil, for the production of economically valuable bio-fuel in oleaginous fungi in general, and C. blakesleeana- JSK2, in particular.