Methods for detection and characterization of lipases: A comprehensive review

Abstract

Microbial lipases are very prominent biocatalysts because of their ability to catalyze a wide variety of reactions in aqueous and non-aqueous media. The chemo-, regio- and enantio-specific behaviour of these enzymes has caused tremendous interest among scientists and industrialists. Lipases from a large number of bacterial, fungal and a few plant and animal sources have been purified to homogeneity. This article presents a critical review of different strategies which have been employed for the detection, purification and characterization of microbial lipases.

Introduction

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 (Gupta et al., 2004). Lipases are also defined as glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides (Joseph et al., 2008). Novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agro-chemicals, and flavour compounds (Jaeger and Eggert, 2002).

Taxonomically close strains may produce lipases of different types. There are many microorganisms known to produce different lipases (Iizumi et al., 1990). Microbial lipases possessing either high alkalophilic or thermophilic properties have been reported from Alcaligenes (Kakusho et al., 1982). Pseudomonas fragi (Nishio et al., 1987) P. nitroreducens (Watanabe et al., 1977) and other Pseudomonas strains were isolated from soil that produced lipase having different characteristics. Choo et al. (1998) isolated a psychrotrophic Pseudomonas strain from Alaskan soil, which produced a cold-adapted lipase at low temperatures. The oily environment (sewage, rubbish dump sites and oil mill effluent) may provide a good environment for lipolytic microorganisms to flourish and for isolation of lipase producing microorganisms (Rahman et al., 2007).

When screening bacteria for lipase production both culture pH and assay pH are important parameters. The stability depends upon the presence of substrate (Andersson et al., 1979, Watanabe et al., 1977). Almost all microbial lipases can be regarded as acid lipases or neutral lipases if they are classified by their optimum pH value for the lipolytic activity (Iizumi et al., 1990). Quantitative analysis is important to compare the lipolytic activities of various isolates. There is no single universal method of lipase assay. The choice of a particular method will depend on the user’s own specific requirements. For the assay of any enzyme, the sensitivity, availability of substrates, and ease of the procedure have to be considered (Hendrickson, 1994).

Growth conditions such as availability of carbon and nitrogen sources, the presence of activators, stimulators, inhibitors, surfactants, incubation temperature, pH, and the level and source of inoculum (Hadeball, 1991) and oxygen tension (Chartrain et al., 1993) can influence the synthesis of lipase. According to Shelley et al. (1987b) three factors must work together if a lipase- positive bacterium is to be detected: (1) the organism must grow; (2) the organism must produce or release lipase under the prevailing growth conditions; and (3) the detection method used must be of sufficient sensitivity.

One important aspect of lipolytic enzymes is the unique physicochemical character of the reactions they catalyse at lipid–water interfaces, involving interfacial adsorption and subsequent catalysis sensu stricto. Most of the lipases are water-soluble enzymes acting on water-insoluble substrates (supersubstrates). Due to this heterogeneous character it is difficult to accurately quantitate both the amount of interface (specific surface) as well as the interfacial parameters (such as the interfacial tension, surface viscosity, surface potential, etc.). Lipolytic process depends on the “interfacial quality” of the substrate (Small, 1997, Dahim and Brockman, 1998, Panaitov and Verger, 2000). The emulsification of the water-insoluble substrates, which requires the presence at the interface of surface active amphiphiles such as detergents, other lipids, proteins, etc., can therefore drastically influence lipase activity measurements (Gargouri et al., 1986).

Lipases with new specificities are needed and the engineering of cloned enzymes as well as the isolation of new lipases from natural sources is therefore of increasing potential value. Reliable, convenient, and sensitive assays to detect a true lipase activity in cellular homogenates are required. As with all reactions catalysed by enzymes, activity measurements can be carried out using various physicochemical methods (by monitoring the disappearance of the substrate or the release of the product) (Beisson et al., 2000).