Substrate Report for: Trihexanoin

Lipases from Bacillus thermocatenulatus are a member of superfamily of alpha/beta hydrolase, but there are structural differences between them. In this work, we focused on the alpha5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical alpha/beta hydrolase fold. In silico study showed that the alpha5 helix is a region that causes disorder in BTL2 protein. So, the alpha5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Deltaalpha5-BTL2) has been expressed in Pichia pastoris yeast. The alpha5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.

With the aim of determining the fatty acid (FA) selectivity of lipases, a mixture of oleic acid and monoacid triacylglycerols (TAGs) including tricaproin (T6), tricaprylin (T8), tricaprin (T10), trilaurin (T12), trimyristin (T14), tripalmitin (T16) and tristearin (T18) was used as the substrate in acidolysis performed in hexane. Three immobilized lipases, namely, Lipozyme TL IM from Thermomyces lanoginosus, Lipozyme RM IM from Rhizomucor miehei and Novozym 435 from Candida antarctica, were used as biocatalyst. The effects of operating variables including the mole ratio of oleic acid to monoacid TAG, temperature, enzyme dosage and reaction time on incorporation were also investigated. Significantly different incorporation rates were obtained for different TAGs used (P < 0.05). Incorporation of oleic acid into TAGs except tricaproin and tricaprylin was higher for all the TAGs with Lipozyme TL IM catalyzed reactions than those of other two enzymes tested. Incorporation of oleic acid decreased as the acyl chain length of FA in the TAG increased with Novozyme 435 catalyzed acidolysis. Compared to the other substrate mixtures, the highest incorporation was observed for oleic acid and tricaproin mixture with three lipases tested. It was shown that the FA selectivity of the lipases is strongly dependent on the acyl chain length of FA in a TAG.

Beside their inhibitory effect upon lipase adsorption, bile salts at low concentration (around 0.2 mM) have repeatedly been shown to enhance lipolysis slightly. From the data reported in this paper, this activation has been attributed to a stabilization of the adsorbed lipase brought about by low concentrations of bile salts. This hypothesis relies on the following observations. (a) For a given temperature, the activation by bile salts depends on the substrate. It is maximum for trihexanoin (trihexanoylglycerol) and does not exist for tripropionin (tripropionylglycerol). (b) In the absence of bile salts, the optimal activities are obtained for different temperatures depending on the substrate. (c) For a given substrate, the activation by a low concentration of bile salts depends on the temperature. It increases when the temperature is raised (up to 35-40 degrees C) and completely disappears at a sufficiently low temperature (around 10 degrees C). (d) This temperature effect does not seem to be due to a modification of the physical parameters of the interface as measured by the interfacial tension. (r) Colipase, like bile salts, increases the lipase activity on short-chain triglycerides but only at high temperature when lipase denaturation occurs. It has no influence upon the activity when the temperature is sufficiently low (around 10 degrees C).

Lipases from Bacillus thermocatenulatus are a member of superfamily of alpha/beta hydrolase, but there are structural differences between them. In this work, we focused on the alpha5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical alpha/beta hydrolase fold. In silico study showed that the alpha5 helix is a region that causes disorder in BTL2 protein. So, the alpha5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Deltaalpha5-BTL2) has been expressed in Pichia pastoris yeast. The alpha5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.

With the aim of determining the fatty acid (FA) selectivity of lipases, a mixture of oleic acid and monoacid triacylglycerols (TAGs) including tricaproin (T6), tricaprylin (T8), tricaprin (T10), trilaurin (T12), trimyristin (T14), tripalmitin (T16) and tristearin (T18) was used as the substrate in acidolysis performed in hexane. Three immobilized lipases, namely, Lipozyme TL IM from Thermomyces lanoginosus, Lipozyme RM IM from Rhizomucor miehei and Novozym 435 from Candida antarctica, were used as biocatalyst. The effects of operating variables including the mole ratio of oleic acid to monoacid TAG, temperature, enzyme dosage and reaction time on incorporation were also investigated. Significantly different incorporation rates were obtained for different TAGs used (P < 0.05). Incorporation of oleic acid into TAGs except tricaproin and tricaprylin was higher for all the TAGs with Lipozyme TL IM catalyzed reactions than those of other two enzymes tested. Incorporation of oleic acid decreased as the acyl chain length of FA in the TAG increased with Novozyme 435 catalyzed acidolysis. Compared to the other substrate mixtures, the highest incorporation was observed for oleic acid and tricaproin mixture with three lipases tested. It was shown that the FA selectivity of the lipases is strongly dependent on the acyl chain length of FA in a TAG.

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90 degrees C and between pH 6.5 and 10; the half-life of the enzymes at 75 degrees C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53-2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.

Beside their inhibitory effect upon lipase adsorption, bile salts at low concentration (around 0.2 mM) have repeatedly been shown to enhance lipolysis slightly. From the data reported in this paper, this activation has been attributed to a stabilization of the adsorbed lipase brought about by low concentrations of bile salts. This hypothesis relies on the following observations. (a) For a given temperature, the activation by bile salts depends on the substrate. It is maximum for trihexanoin (trihexanoylglycerol) and does not exist for tripropionin (tripropionylglycerol). (b) In the absence of bile salts, the optimal activities are obtained for different temperatures depending on the substrate. (c) For a given substrate, the activation by a low concentration of bile salts depends on the temperature. It increases when the temperature is raised (up to 35-40 degrees C) and completely disappears at a sufficiently low temperature (around 10 degrees C). (d) This temperature effect does not seem to be due to a modification of the physical parameters of the interface as measured by the interfacial tension. (r) Colipase, like bile salts, increases the lipase activity on short-chain triglycerides but only at high temperature when lipase denaturation occurs. It has no influence upon the activity when the temperature is sufficiently low (around 10 degrees C).