Microbial Lipases of Potential in Biocatalysis: Screening, Purification, Molecular Cloning and Heterologous Expression of Actinomycete Lipases in Escherichia Coli

Dharmendra Dheeman (Thesis), Dublin Institute of Technology

Document Type Theses, Ph.D

Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Dublin Institute of Technology, June, 2011.


In recent times, the prospect of lipase catalysis in organic solvents with its associated advantages has received widespread attention. Among the large number of lipases described in the literature, only the enzymes belonging to a narrow range of species have been shown to have adequate stability and biosynthetic capabilities to allow routine use in organic reactions. Moreover, currently known microbial lipases do not always have the desired combination of thermostability and stability in both hydrophobic and hydrophilic organic solvents. Despite the numerous examples of the effective use of lipases, the biocatalysts often need to be optimised to express the desired specificities, stability, and operational properties. Although actinomycetes were recognised through their exogenous lipolytic activities more than a decade ago, lipases from actinomycetes have not been studied as intensively as those from other bacteria. The available genome sequence databases for actinomycetes predict a large number of genes encoding enzymes of different lipolytic activities. Although these data indicate the potential for the synthesis of a broad range of lipolytic enzymes, only a few have been studied and reported to date. The present work was undertaken in this context to discover new lipolytic enzymes from actinomycetes as well as novel bacterial and fungal strains for applications in industrially relevant organic syntheses. A large number of novel bacterial and fungal isolates and actinomycete strains were screened with the objective of finding new lipases with both high thermostability and stability in a broad range of organic solvents. Among several lipase-producing strains screened, Amycolatopsis mediterranei DSM 43304 and Penicillium sp. DS-39 (DSM 23773) were found to produce interesting thermostable, extracellular lipases. Production of lipase from A. mediterranei DSM 43304 was enhanced 12-fold through culture conditions and nutrient source modification. The lipases from A. mediterranei DSM 43304 and Penicillium sp. DSM 23773 were purified to gel-electrophoretic homogeneity and biochemically characterised. The apparent molecular masses of lipases determined by SDS-PAGE were 33 kDa and 43 kDa, respectively. The purified A. mediterranei DSM 43304 was found to be a monomeric protein active on triolein and a broad range of p-NP esters with preference for fatty-acyl chain length of C(8:0). It was most active at 60°C and in alkaline conditions around pH 8.0, with p-NPP as substrate. The lipase showed high stability over a broad pH range of 5.0-12.0 and showed significant thermostability with a half-life of >30 min at 70°C. Hydrolysis of glycerol ester bonds occurred non-specifically at positions sn-1, sn-2 and sn-3. The lipase was strongly resistant, with remarkable activation in most cases, to organic solvents with log P ≥ −1.3–6.6. The purified Penicillium sp. DSM 23773 was optimally active at 45°C in acidic conditions at pH 5.5. The lipase was most active on triolein and exhibited a broad substrate range with a preference for triacylglycerols containing long chain unsaturated fatty acids. It showed no regio-specificity for the ester bond in triolein. The lipase showed significant stability and activation in the presence of organic solvents with log P ≥ 2.0. These features render Penicillium sp. DSM 23773 lipase a potential biocatalyst for applications such as biodiesel production, enzymatic restructuring, by interesterification of different oils and fats, and biodegradation of oil spills in the environment. A. mediterranei DSM 43304 lipase showed significant potential in the synthesis of an industrially important flavour ester isoamyl acetate in free and immobilised states. Celiteimmobilised lipase preparation from Amycolatopsis mediterranei DSM 43304 was used to catalyse the direct esterification reaction between acetic acid and isoamyl alcohol to synthesise isoamyl acetate (banana flavour) in n-hexane. The effects of different operating parameters on molar conversion and initial reaction rates were studied in the absence of mass-transfer limitations. Optimum molar conversion (59%) was obtained at an acetic acid/isoamyl alcohol molar ratio of 2, 1% (v/v) initially added water, 7.5% (w/v) Celiteimmobilised A. mediterranei DSM 43304 at 50°C and 200 rpm. A simplified model, based on a postulated Ping Pong Bi-Bi mechanism, adequately described the kinetics of Celiteimmobilised A. mediterranei DSM 43304 lipase catalysed direct esterification of isoamyl alcohol with acetic acid. The reusability study showed that the Celite-immobilised A. mediterranei DSM 43304 lipase was reusable for up to five cycles without significant decrease in its synthetic potential. Response surface methodology (RSM) based on a 4- variable, 5-level central composite rotatable design (CCRD) was used to study the effect of selected variables viz. enzyme amount (4–12%), substrate molar-ratio (0.2–0.8), temperature (30–50°C) and reaction time (6–18 h) on percentage molar conversion. The results showed that enzyme amount and substrate molar-ratio were the most important variables and temperature and reaction time had less effect on molar conversion (87.5%). Two ORFs encoding putative lipases, showing similar molecular weight and N-terminal sequence homology to purified A. mediterranei DSM 43304 lipase, were cloned and expressed in E. coli BL21(DE3). The inclusion bodies formed were efficiently in vitro refolded and functional native lipases were purified using IMPACT. Although both enzymes share some properties with other lipolytic enzymes, both of them are very stable at high temperatures up to 50°C and also at high pH values. Due to this and other unusual properties of the enzymes such as their high resistance to a variety of substances and polar organic solvents the enzymes are valuable candidates for industrial applications in biocatalysis. The present investigation is the first study on heterologous over-expreesion in E. coli and functional in vitro refolding of lipolytic enzymes from actinomycetes. Thus, the two refolded recombinant enzymes described in this study represent an example of the high potential of putative ORFs, reported in the recently completed actinomycete genomes, as sources of novel lipolytic enzymes for biocatalytic applications.