Identification and characterization of a hydrophobin Vmh3 from Pleurotus ostreatus.

Hydrophobins (HPs) are relatively small surface-active proteins of fungal origin. Being an industrially important protein, isolation of new molecules from GRAS (Generally Regarded as Safe) strains like mushrooms is the need of the time. In the present work, hydrophobin Vmh3-1 is isolated, purified, and identified from a culture broth and vegetative mycelia of Pleurotus ostreatus grown in a Potato dextrose broth (PDB) in static culture conditions. Purified proteins from the broth and the cell wall showed bands of 11 kDa and 17 kDa when analyzed on SDS-PAGE. Hydrophobin Vmh3-1 was identified in purified protein samples by the Orbitrap-HR-LC-MS/MS analysis with a maximum of 66% sequence coverage. The amphipathic nature of the protein was revealed by an increase in the water contact angle (WCA) of the hydrophilic surface of glass by 87% as well as a decrease in the WCA of the hydrophobic surface of Teflon by 19%. The emulsification property was tested with food-grade oils and Hexane. A maximum activity (EI 24) of 87.64% was recorded for Sunflower oil. In CD (Circular dichroism) spectra, Vmh3-1 showed the typical spectra of hydrophobin with a dominance of ß-sheets (51%) in the secondary structure and a minimum percentage of the α-helix (2%). The protein did not show a self-aggregating property on vigorous shaking making it suitable for numerous industrial applications. The identification of Vmh3-1 with detailed amino acid sequencing and the characterization of the protein to evaluate its potential in surface modifications for various industrial applications is demonstrated herein for the first time.

Exploiting Phosphate-Starved cells of Scenedesmus sp. for the Treatment of Raw Sewage.

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 106 cells mL-1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 106 cells mL-1.

Production of polyunsaturated fatty acids in recombinant Lipomyces starkeyi through submerged fermentation.

Long chain polyunsaturated fatty acids (LCPUFA) are known to play an important role in human health and nutrition. Considering the limitation of LCPUFA sources, it is necessary to search new avenues for their production. Oleaginous yeasts are an attractive target for harvesting single cell oil, mainly because of the ease of cultivation with cheaper raw material. Lipomyces starkeyi is one such oleaginous yeast, which can accumulate oil to the extent of 60% of its biomass and where genetic transformation can be achieved. In our earlier work, Δ15 desaturase gene (AEP37840) from flax was transformed into L. starkeyi. In the present work, we report optimization of medium for the production of ω-3 enriched oil from this transformed yeast. A basic medium containing 20 g/l glucose as a carbon source and 10 g/l yeast extract as a nitrogen source was used during fermentation. At regular time intervals, glucose was fed to maintain high C:N ratio (65:10) during fermentation. Under the most favorable conditions, dry biomass and total lipid content were 18 and 7.29 g/l, respectively. Prior to genetic transformation, L. starkeyi contained 56.03 mg/l DHA along with 71.4 mg/l EPA and 42.2 mg/l ALA. Genetic engineering of this yeast resulted in a strain that produced 1080 mg/l DHA (17.4%) along with 74.28 mg/l EPA and 126.72 mg/l ALA possibly through modification of PUFA biosynthetic pathway. To the best of our knowledge, this is a first report of DHA enrichment and opens up avenues for LCPUFA production through L. starkeyi.

Media optimization for biosurfactant production by Rhodococcus erythropolis MTCC 2794: artificial intelligence versus a statistical approach.

This paper entails a comprehensive study on production of a biosurfactant from Rhodococcus erythropolis MTCC 2794. Two optimization techniques--(1) artificial neural network (ANN) coupled with genetic algorithm (GA) and (2) response surface methodology (RSM)--were used for media optimization in order to enhance the biosurfactant yield by Rhodococcus erythropolis MTCC 2794. ANN and RSM models were developed, incorporating the quantity of four medium components (sucrose, yeast extract, meat peptone, and toluene) as independent input variables and biosurfactant yield [calculated in terms of percent emulsification index (% EI(24))] as output variable. ANN-GA and RSM were compared for their predictive and generalization ability using a separate data set of 16 experiments, for which the average quadratic errors were approximately 3 and approximately 6%, respectively. ANN-GA was found to be more accurate and consistent in predicting optimized conditions and maximum yield than RSM. For the ANN-GA model, the values of correlation coefficient and average quadratic error were approximately 0.99 and approximately 3%, respectively. It was also shown that ANN-based models could be used accurately for sensitivity analysis. ANN-GA-optimized media gave about a 3.5-fold enhancement in biosurfactant yield.

Enhanced production of amidase from Rhodococcus erythropolis MTCC 1526 by medium optimisation using a statistical experimental design.

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket-Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.

Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574.

The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L(-1)) and achieved a critical micelle concentration (CMC) value of 120 mg L(-1).

Immobilization of Candida rugosa lipase on poly(allyl glycidyl ether-co-ethylene glycol dimethacrylate) macroporous polymer particles.

Macroporous polymer particles containing surface epoxy groups were synthesized for immobilization of Candida rugosa lipase (CRL). The effect of incorporation of two different sets of monomers [allyl glycidyl ether (AGE) and glycidyl methacrylate (GMA)] and the effect of crosslinking density on immobilization of lipase were studied. AGE-co-EGDM polymers gave higher binding and expression of lipase than GMA-co-EGDM polymers. Optimization of immobilization parameters was done with respect to immobilization time and enzyme loading. Amongst AGE-co-EGDM polymer series, AGE-150 polymer found to give maximum lipase activity yield and therefore evaluated for temperature, pH and storage stability. Under optimum conditions, AGE-150 polymer gave 78.40% of activity yield. Immobilized lipase on AGE-150 showed a broader pH, higher temperature and excellent storage stability.