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The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 °C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 °C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

This study investigates the potential of microbial polysaccharides (MP) gellan gum and pullulan to reduce oil uptake in a deep-fried snack (Namkeen). The concentration of MP in refined wheat flour varied between 1-10% (w/w) and the frying time and temperature are optimized. The results reveal that MP added namkeens showed a maximum relative oil reduction of 33.87% with gellan gum and 26.93% with pullulan at 10% (w/w). Gellan gum and pullulan result in oil reduction of 23.19% and 15.97% with 5% (w/w). The results reveal that gellan gum added namkeens exhibited 1.45 times lower oil uptake than pullulan added namkeen indicating potential oil reduction capabilities. The hardness of namkeen is observed to increase with an increase in MP concentration. Sensorial and textural characteristics of control and MP incorporated fried samples are comparable up to 5% (w/w) with MP concentration. The results for the control samples and MP incorporated samples are statistically significant. Furthermore, overall sensorial acceptability of pullulan added namkeens (7.74 ± 0.53) is slightly higher than that of gellan gum added namkeens (7.64 ± 0.36). Practical Application: Low-fat diet and functional foods are expected to help in managing the conditions of hyperlipidaemia. This study focuses on the potential of MP in reducing the oil content of deep-fried snacks. The efficacy of MP gellan gum and pullulan as functional ingredients for oil reduction in fried snacks demonstrates its aid in formulating low-fat foods thereby retaining sensorial characteristics.

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Activity staining or zymography is a technique to detect enzymes based on their function/activity toward a specific substrate. Multiple enzyme-producing microbes secrete enzymes along with other proteins at varying time points during fermentation. The technique of zymography can be used to detect functionality of enzymes in complex protein/other enzyme mixtures. The protein bands corresponding to specific enzyme among other enzymes/proteins can be located by polyacrylamide gel electrophoresis (PAGE) followed by zymogram analysis. This can be employed to locate the secretion pattern of protein/enzyme from intracellular region to extracellular medium. Here we describe simple method for detection and cellular localization of esterases and protease secreted by single microbial strain in one PAGE gel.

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