Effect of Chain Length of Alcohol on the Lipase-Catalyzed Esterification of Propionic Acid in Supercritical Carbon Dioxide

The esterification of propionic acid was investigated using three different alcohols, namely, isopropyl alcohol, isobutyl alcohol, and isoamyl alcohol. The variation of conversion with time for the synthesis of isoamyl propionate was investigated in the presence of five enzymes. Novozym 435 showed the highest activity, and this was used as the enzyme for investigating the various parameters that influence the esterification reaction. The Ping-Pong Bi-Bi model with inhibition by both acid and alcohol was used to model the experimental data and determine the kinetics of the esterification reaction.

Lipase mediated enzymatic degradation of polydioxanone in solution

The enzymatic biodegradation of polydioxanone (PDO) in trifluoroethanol (TFE) at various temperatures (25-55 degrees C) was studied with two different types of lipases, namely immobilized enzyme Novozym 435 and free enzyme porcine pancreas lipase. The biodegradation process was monitored by gel permeation chromatography (GPC). Both enzymes showed the optimum activity at 37 degrees C and Novozym 435 exhibited better thermal stability over the experimental temperature range. A continuous distribution kinetic model was employed to describe the biodegradation process and the model was used to fit the experimental data satisfactorily and obtain kinetic parameters. (C) 2014 Elsevier Ltd. All rights reserved.

Imidazolium based ionic liquid type surfactant improves activity and thermal stability of lipase of Rhizopus oryzae

Lipase and surfactant together form a potent pair in various biotransformation, industrial application and biotechnological studies. The present investigation deals with changes in the activity, stability and structure of lipase from Rhizopus oryzae NRRL 3562 in presence of long chain ionic liquid-type imidazolium surfactant. Both the activity and stability were found to be enhanced in presence of the surfactant at low concentration (1-125 mu M) followed by inhibition at high concentration. The activity increased by 80% and thermal deactivation temperature raised by 2.5 degrees C. Investigations by ultraviolet-visible spectroscopy and circular dichroism revealed structural changes leading to rise in beta-sheet content and lowering of a-helix at low surfactant concentrations. Deactivation at high concentration correlated with greater structural changes depicted by spectroscopic studies. Isothermal titration calorimetric studies showed the binding to be spontaneous in nature involving non-covalent interactions. High negative value of entropy signifies exposure of hydrophobic domains and increase in structural rigidity, which correlates with active site being more accessible and rigid in presence of the surfactant. Application of these surfactants hold greater potential in the field of lipase based biotransformations, enzyme structural modifications and studies. (C) 2015 Elsevier B.V. All rights reserved.

Role of spacer length in interaction between novel gemini imidazolium surfactants and Rhizopus oryzae lipase

An insight into the effects of new ionic liquid-type gemini imidazolium cationic surfactants on the structure and function of the lipases is of prime importance for their potential application. Changes in the activity, stability and structure of Rhizopus ouzae lipase in the presence of novel gemini surfactants, C-16-3-C(16)im]Br-2 and C-16-12-C(16)im]Br-2 were probed in the present study. Surfactant with shorter spacer length, C-16-3-C(i6)im]Br-2 was found to be better in improving the hydrolytic activity and thermal stability of the lipase. For both the surfactants, activation was concentration dependent. CD spectroscopy results showed a decrease in a-helix and an increase in beta-sheet content in the presence of these surfactants. A higher structural change observed in presence of C-16-12-C(16)im]Br-2 correlated with lower enzyme activity. Isothermal titration calorimetric studies showed the binding to be spontaneous in nature based on sequential two site binding model. The forces involved in binding were found to differ for the two surfactants proving that the spacer length is an important factor which governs the interaction. These surfactants could be used as promising components both in enzyme modification and media engineering for attaining the desired goals in biocatalytic reactions. (C) 2015 Elsevier B.V. All rights reserved.

Triacylglycerol lipases of yeast and plants: more than just hydrolases

In the yeast, mobilization of triacylglycerols (TAG) is facilitated by TGL3, TGL4 and TGL5 gene products. Interestingly, experiments using [32P] orthophosphate as a precursor for complex glycerophospholipids revealed that tgl mutants had a lower steady-state level of these membrane lipids. To understand a possible link between TAG lipolysis and phospholipid metabolism, we performed overexpression studies with Tgl3p and Tgl5p which clearly demonstrated that these two enzymes enhanced the level of phospholipids. Domains and motifs search analyses indicated that yeast TAG hydrolases posses a GXSXG lipase motif but also a HX4D acyltransferase motif. Purified Tgl3p and Tgl5p did not only exhibit TAG lipase activity but also catalyzed acyl-CoA dependent acylation of lyso-phosphatidylethanolamine and lyso-phosphatidic acid (LPA), respectively. Search for lipase/hydrolase homologues in the Arabidopsis thaliana genome led to the identification of At4g24160 which possess three motifs that are conserved across the plant species such as GXSXG motif, a HX4D motif and a probable lipid binding motif V(X)3HGF. Characterization of At4g24160 expressed in bacteria revealed that the presence of an acyl-CoA dependent LPA acyltransferase activity. In addition, the purified recombinant At4g24160 protein hydrolyzed both TAG and phosphatidylcholine. We hypothesize that the plant enzyme may be involved in membrane repair. In summary, our results indicate that these TAG lipases play a dual role and thereby contribute to both anabolic and catabolic processes in yeast and plants.

At4g24160, a Soluble Acyl-Coenzyme A-Dependent Lysophosphatidic Acid Acyltransferase

Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the alpha/beta-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX4D acyltransferase motif, and V(X)(3)HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.

Expression of drug resistance markers by spheroplasts ofMycobacterium smegmatis after fusion

Mycobacterial spheroplasts were prepared by treatment of the glycinesensitized cells with a combination of lipase and lysozyme. They were stable for several hours at room temperature but were lysed on treatment with 0.1% sodium dodecyl sulfate. The spheroplasts could be regenerated on a suitable medium. Fusion and regeneration of the spheroplasts were attempted using drug resistant mutant strains ofM. smegmalis. Recombinants were obtained from spheroplast fusion mediated by polyethylene glycol and dimethyl sulfoxide. Simultaneous expression of rccombinant properties was observed only after an initial lag in the isolated clones. This has been explained as due to “chromosome inactivation” in the fused product.

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using H-3]inositol. P-32]orthophosphate, 3H]palmitate and C-14]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids. (C) 2010 Elsevier B.V. All rights reserved.

Enzymatic synthesis of flavors in supercritical carbon dioxide

Commercially important flavor esters of isoamyl alcohol, catalyzed by crude hog pancreas lipase (HPL), were synthesized under solvent-free conditions and in supercritical carbon dioxide. The esters synthesized were isoamyl acetate, isoamyl propionate, isoamyl butyrate, and isoamyl octanoate. Very low yields (3-4%) of isoamyl acetate were obtained, but high yields for the other three esters were obtained under both supercritical and solvent-free conditions. The yields of esters of the even-carbon acids, isoamyl acetate, butyrate, and octanoate, increased with increasing chain length, whereas the yield of isoamyl propionate was higher than that of isoamyl butyrate. The optimum temperature of the reaction was higher under supercritical conditions (45 degreesC) than under solvent-free conditions (35-40 degreesC). The effects of other parameters such as alcohol concentration, water concentration, and enzyme loading were investigated. An increase in the water concentration decreased the conversion significantly in supercritical carbon dioxide but not under solvent-free conditions. The optimum ratio of alcohol to acid was dependent on the extent of inhibition by the acid. Although providing a higher apparent yield by being run in a highly concentrated medium, the overall conversion under solvent-free conditions was lower than that under supercritical conditions for similar enzyme concentrations, indicating that the synthesis of esters in supercritical carbon dioxide might be a viable option.

A novel ``pro-sensitizer'' based sensing of enzymes using Tb(III) luminescence in a hydrogel matrix

Chemically synthesized ``pro-sensitizers'' release the sensitizer in the presence of lipase or beta-glucosidase, triggering a significant luminescence response from a lanthanide based hydrogel.

Enzymatically degradable EMI shielding materials derived from PCL based nanocomposites

In this study, two different types of multiwall carbon nanotubes (MWNTs) namely pristine (p-MWNTs) and amine functionalized (a-MWNTs) were melt-mixed with polycaprolactone (PCL) to develop biodegradable electromagnetic interference (EMI) shielding materials. The bulk electrical conductivity of the nanocomposites was assessed using broadband dielectric spectroscopy and the structural properties were evaluated using dynamic mechanical thermal analysis (DMTA). Both the electrical conductivity and the structural properties improved after the addition of MWNTs and were observed to be proportional to the increasing fractions in the nanocomposites. The shielding effectiveness of the nanocomposites was studied using a vector network analyzer (VNA) in a broad range of frequencies, X-band (8 to 12 GHz) and K-u-band (12 to 18 GHz) on toroidal samples. The shielding effectiveness significantly improved on addition of MWNTs, more in the case of p-MWNTs than in a-MWNTs. For instance, at a given fraction of MWNTs (3 wt%), PCL with p-MWNTs and a-MWNTs showed a shielding effectiveness of -32 dB and -29 dB, respectively. Moreover, it was observed that reflection was the primary mechanism of shielding at lower fractions of MWNTs, while absorption dominated at higher fractions in the composites. As one of the rationales of this work was to develop biodegradable EMI shielding materials to address the challenges concerning electronic waste, the effect of different MWNTs on the biodegradability of PCL composites was assessed through enzymatic degradation. The enzymatic degradation of the samples cut from the hot pressed films by bacterial lipase was investigated. It was noted that a-MWNTs exhibited almost similar degradation rate as the control PCL sample; however, p-MWNTs showed a slower degradation rate. This study demonstrates the potential use of PCL-MWNT composites as flexible, light weight and eco-friendly EMI shielding materials.