Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.
Lipases are versatile biocatalysts that can perform innumerable different reactions. Their enantio-, chemo- and stereo-selective nature makes them an important tool in the area of organic synthesis. Unlike other hydrolases that work in aqueous phase, lipases are unique as they act at the oil/water interface. Besides being lipolytic, lipases also possess esterolytic activity and thus have a wide substrate range. Hence, the lipase assay protocols hold a significant position in the field of lipase research. Lipase activity can be estimated using a wide range of assay protocols that differ in terms of their basic principle, substrate selectivity, sensitivity and applicability. As the value of these enzymes continues to grow and new markets are exploited, development of new or improved enzymes will be a key element in the emerging realm of biotechnology. Hence, development of faster and simpler protocols incorporating newer and more specific substrates is the need of the hour. In this endeavour, methods that could be adopted for molecular screening occupy an important position. Here, an overview of the lipase assay protocols is presented with emphasis on the assays that can be adopted for the molecular screening of these biocatalysts.
Aim: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor.
Methods and Results:
Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0·1% KH2PO4, 0·5% NH4Cl, 0·24% (NH4)2HPO4, 0·01% MgSO4.7H2O and 1% emulsified palm oil, at 45 °C and pH 7·0, agitated at 250 rev min−1 with 6‐h‐old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml−1) and an approximately three fold increase in specific activity (160 U mg−1) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables.
Conclusions: The maximum lipase production was 50 U ml−1, with specific activity 160 U mg−1 protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply.
Significance and Impact of the Study: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.
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