Vikram Sahai

Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor

Alkaline protease production in Bacillus mojavensis was improved up to 4.2-fold in a 14 l bioreactor during validation of a predicted statistical model. The final enzyme yield in the bioreactor was 2389 U ml−1 obtained within 10–12 h compared to 558 U ml−1 after 24 h in shake flask cultures. Analysis of variance (ANOVA) of face-centered central composite design showed a high coefficient of determination (R2) value of 0.9473, thus ensuring a satisfactory adjustment of the quadratic model with the experimental data. The coordinates of three factors (casamino acids, inoculum age and agitation) were positive, whereas negative coordinates were obtained for other two factors (glucose and incubation time). Protease production was subjected to catabolite repression by glucose (>2 mg ml−1). The response surface curves predicted increased levels of casamino acids (12 mg ml−1) and midlevel of glucose (2 mg ml−1) as best C/N combination for optimal enzyme production (1133 U ml−1) within 18 h at high agitation rates (>250 rpm) and low inoculum density (A550 nm≅0.250). Protease production was drastically reduced at low agitation rates. The present study provides useful information about the regulation of protease synthesis through manipulation of various physicochemical factors.

Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

Aim: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor.

Optimization of xylanase production by Melanocarpus albomyces IIS68 in solid state fermentation using response surface methodology

Xylanase production by the thermophilic fungus, Melanocarpus albomyces IIS68, during solid state fermentation of wheat straw was studied and the effects of various variables were observed. Using the response surface methodology and the multivariant statistical approach, the optimum levels of the variables affecting xylanase production were determined. The optimum levels of the variables were 600-850 microm particle size, 43 h inoculum age, 1.37% Tween 80, 86% initial moisture content, 5.1% urea, 0.74% yeast extract and a harvest time of 96 h. Under these optimized conditions, xylanase activity of 7760 U/g initial dry substrate was obtained which was in very good agreement with the value predicted by the quadratic model (7890 U/g initial dry substrate).

Optimization of compactin production in chemically defined production medium by Penicillium citrinum using statistical methods

Abstract Production of compactin by Penicillium citrinum was optimized using Plackett–Burman and central composite rotatable design. Out of 11 factors studied by Plackett–Burman design, six influenced the compactin production. Central composite rotatable design was used to optimize the significant factors and response surface plots were generated. Using these response surface plots and point prediction, optimized values of the factors were determined and a maximum compactin production of 490 mg/l was predicted. An experimental study using the optimized parameters resulted in production of 456 mg compactin per l, 93% of the predicted value.

Compactin—a review

Compactin, a hypocholesterolemic molecule, is a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase, which is a regulatory enzyme for cholesterol biosynthesis. The structural similarity and high affinity of the acid form of compactin and HMG, the natural substrate of enzyme, results in specific and effective inhibition of this enzyme. Inhibition results in reduced levels of mevalonic acid in the body, leading to pleiotropic effects. Various fungi have been used for the commercial production of compactin. Using different strategies for improving production levels, yields have been increased to around 900 times of the amount originally produced. Recently, the gene sequence responsible for compactin production has been cloned and sequenced. This review deals with the chemistry, mode of action, pharmacology, biosynthesis, and production of compactin. A comparative study of various reports dealing with the production of compactin is also included.

Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production.

A chemically defined medium was optimized for the maximum biomass production of recombinant Pichia pastoris in the fermentor cultures using glycerol as the sole carbon source. Optimization was done using the statistical methods for getting the optimal level of salts, trace metals and vitamins for the growth of recombinant P. pastoris. The response surface methodology was effective in optimizing nutritional requirements using the limited number of experiments. The optimum medium composition was found to be 20 g/L glycerol, 7.5 g/L (NH4)2SO4, 1 g/L MgSO4.7H2O, 8.5 g/L KH2PO4, 1.5 mL/L vitamin solution and 20 mL/L trace metal solution. Using the optimized medium 11.25 g DCW/L biomass was produced giving a yield coefficient of 0.55 g biomass/g of glycerol in a batch culture. Chemostat cultivation of recombinant P. pastoris was done in the optimized medium at different dilution rates to determine the kinetic parameters for growth on glycerol. Maximum specific growth rate of 0.23 h(-1) and Monod saturation constant of 0.178 g/L were determined by applying Monod model on the steady state data. Products of fermentation pathway, ethanol and acetate, were not detected by HPLC even at higher dilution rates. This supports the notion that P. pastoris cells grow on glycerol by a respiratory route and are therefore an efficient biomass and protein producers.

Statistical media optimization and production of ITS α-amylase from Aspergillus oryzae in a bioreactor

The production of an intermediate temperature-stable (ITS) α-amylase from Aspergillus oryzae was studied by using a central composite design with three independent variables, viz., starch, yeast extract, and K2HPO4. The model equation provided a suitable model for the response surface for α-amylase production, and, from the optimal concentrations of the medium components, a model was predicted, which was then used for enzyme production in a 150-L bioreactor. In the bioreactor studies, the enzyme yields (161 U/ml) were similar to that of the shake flask (133 U/ml); however, the time required for maximum α-amylase production in the bioreactor was reduced to 48 h compared with 120 h in shake flask cultures. An increased level of phosphate in the medium and low inoculum size were necessary to control the excessive foaming in the bioreactor; however, control of the pO2 level and agitation was not mandatory for enzyme production. The peak enzyme production coincided with the increase in pH of the fermentation broth and was maximal when the pH of the system was above 7.5. Thus, in the present study, pH acted as an indicator of the initiation or end of the enzyme synthesis or of the fermentation cycle.

High-density spore production of Piriformospora indica, a plant growth-promoting endophyte, by optimization of nutritional and cultural parameters

Piriformospora indica is an axenically cultivable root endophytic fungus which exerts plant growth promoting effects on its host plants. To enable commercial production of its spores, the medium composition and culture conditions have been optimized in a 14 L bioreactor such that they result in maximum biomass during growth phase and in maximum spore yield during subsequent sporulation phase. Maximum spore yields were obtained with modified Kaefer medium using a glucose deprivation strategy. An enhancement of 100% in overall biomass productivity (0.18 g L(-1) h(-1)) and reduction of about 70% in the time (60 h) required to achieve the maximum spore yield (9.25×10(7) spores/mL) was achieved in comparison to the original Kaefer medium. The high spore yield obtained in the present study seems to be economical for commercial production of P. indica.

Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals

Human interferon-alpha 2b (IFN-alpha2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-alpha2b, Saccharomycescerevisiae MF-alpha factor prepro sequence and a mutated alpha prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-alpha2b into the culture medium of P. pastoris. The native secretion signal of IFN-alpha2b did not secrete protein into the culture medium of P. pastoris. The alpha prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-alpha2b (200 mg/l) into the culture medium, with the same amino acid sequence as that of the native IFN-alpha2b secreted by human lymphocytes. The full alpha prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-alpha2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full alpha prepro sequence was used for the secretion of IFN-alpha2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal.

Application of inorganic carrier-based formulations of fluorescent pseudomonads and Piriformospora indica on tomato plants and evaluation of their efficacy

Aims:  Fluorescent pseudomonads are widely used as bioinoculants for improving plant growth and controlling phytopathogenic fungi. Piriformospora indica (Pi), a symbiotic root endophyte, also has beneficial effects on a number of plants. The present study focuses on the improvement of growth yields of tomato plants and control of Fusarium wilt using inorganic carrier‐based formulations of two fluorescent pseudomonad strains (R62 and R81) and Pi.