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In this study, extracellular inulinase from Bacillus sp. 11/3 was partially purified and concentrated using aqueous two‐phase system (ATPS). Two different phase forming salts and four types of polyethylene glycol (PEG) were used. Binodal curves and tie‐length lines (TLLs) for eight ATPS were developed. For inulinase purification, concentrations of PEG and salt according to binodal curves (between 17 and 26%) were chosen. All ATPSs for inulinase purification were characterized. An ATPS consisted of 26% PEG1000 and 26% MgSO4 was found to be the most suitable for inulinase purification. This ATPS has 28.47% TLL, 1.03 of volume ratio, purification factor of 4.65 fold and recovery yield of 66.17%. On the SDS‐PAGE electrophoresis two protein bands with molecular weight of around 24 and 56 kDa were observed. The partially purified enzymes had optimal activity at pH 8.0 and 6.5, optimal temperature at 30 and 70°C and kinetic parameters Km = 26.32 mmol and Vmax = 526 mmol/min.

Background: Rhizopus arrhizus is a potential microorganism for lipase production. Solid-state fermentation is used for microbial biosynthesis of enzymes, due to advantages, such as high productivity, utilization of abundant and low-cost raw materials, and production of enzymes with different catalytic properties. Objective: The objective of the research is optimization of the conditions for lipase production in solid-state fermentation by Rhizopus arrhizus in a nutrient medium, containing agroindustrial wastes. Method: Biosynthesis of lipase in solid-state fermentation by Rhizopus arrhizus was investigated. The effect of different solid substrates, additional carbon and nitrogen source, particles size and moisture content of the medium on enzyme production was studied. Response surface methodology was applied for determination of the optimal values of moisture content and tryptone concentration. A procedure for efficient lipase extraction from the fermented solids was developed. Results: Highest lipase activity was achieved when wheat bran was used as a solid substrate. The addition of 1% (w/w) glucose and 5% (w/w) tryptone to the solid medium significantly increased lipase activity. The structure of the solid medium including particles size and moisture content significantly influenced lipase production. A mathematical model for the effect of moisture content and tryptone concentration on lipase activity was developed. Highest enzyme activity was achieved at 66% moisture and 5% (w/w) tryptone. The addition of the non-ionic surfactant Disponyl NP 3070 in the eluent for enzyme extraction from the fermented solids increased lipase activity about three folds. Conclusion: After optimization of the solid-state fermentation the achieved 1021.80 U/g lipase activity from Rhizopus arrhizus was higher and comparable with the activity of lipases, produced by other fungal strains. The optimization of the conditions and the use of low cost components in solid-state fermentation makes the process economicaly effective for production of lipase from the investigated strain Rhizopus arrhizus.

Aims:The aim of this study is optimization of the concentrations of carbon and nitrogen sources for lipase production by Rhizopus arrhizus using response surface methodology. Study Design: For this work optimal 2 2 composite design was used for studying the optimal concentrations of corn starch and tryptone for lipase production by submerged fermentation. A series of planned experiments in three replications was carried out and a mathematical model was developed which was used to describe the process. Optimal levels of studied independent variables were calculated by using the model and the conversion rate. Place and Duration of Study: This study is a part of PhD dissertation developed in University of Original Research ArticleFood Technologies, Bulgaria, Department of Biochemistry and Molecular Biology. Methodology: Maximum lipase activity was achieved by an optimization of some components of the fermentation medium. Corn starch (in concentrations 5.0, 10.0, 15.0 g.dm -3) and tryptone (2.0, 5.0, 8.0 g.dm -3 ) as independent variables were chosen. Lipase activity was determined by a spectrophotometric assay using synthetic substrate p-nitrophenyl palmitate. Results: A planned mathematical experiment was carried out and a regression model was developed. The value of R 2 was 95.65% which showed that the model had high correlation with the experimental results. The effects of every independent variable had an optimal value while the interaction effects led toenhancement of lipolytical activity. In this case the enzyme activity increased rapidly to 1100 U.dm -3. For lipase activity above that value, large enhancements of the corn starch and tryptone concentrations were needed. In order to use the medium substrates properly there the conversion rate was calculated and it was also considered for the optimization. Conclusion: By carrying out an optimal composite design a mathematical model was derived, with the aid of which, optimum values of tryptone (6.6 g.dm -3) and corn starch (10.5 g.dm -3) were determined, when the conversion rate and the first derivative of enzyme activity were considered. Those results were confirmed by triplicate experiments at the optimal concentrations. Lipase activity of Ȳ=1340.74 U.dm -3 was achieved, which was very close to the predicted one Ŷ=1235.26 U.dm -3.