BARD1 is an ATPase activating protein for OLA1

OLA1 is a P-loop ATPase, implicated in centrosome duplication through the interactions with tumor suppressors BRCA1 and BARD1. Disruption of the interaction of OLA1 with BARD1 results in centrosome amplification. However, the molecular interplay and mechanism of the OLA1-BARD1 complex remain elusive. Here, we use a battery of biophysical, biochemical, and structural analyses to elucidate the molecular basis of the OLA1-BARD1 interaction. Our structural and enzyme kinetics analyses show this nucleotide-dependent interaction enhances the ATPase activity of OLA1 by increasing the turnover number (k_cat). Unlike canonical GTPase activating proteins that act directly on the catalytic G domain, the BARD1 BRCT domain binds to the OLA1 TGS domain via a highly conserved BUDR motif. A cancer related mutation V695L on BARD1 is known to associate with centrosome abnormality. The V695L mutation reduces the BARD1 BRCT-mediated activation of OLA1. Crystallographic snapshot of the BRCT V695L mutant at 1.88 Å reveals this mutation perturbs the OLA1 binding site, resulting in reduced interaction. Altogether, our findings suggest the BARD1 BRCT domain serves as an ATPase activating protein to control OLA1 allosterically.     

Microdomains in mixed monolayers of oleanolic and stearic acids: thermodynamic study and BAM observation at the air-water interface and AFM and FTIR analysis of LB monolayers

Monolayers of oleanolic acid (OLA) mixed with stearic acid (SA) were studied at the air-water interface. The surface pressure-area (pi-A) isotherms, measured over the whole composition range, and BAM observations were used to investigate the phase behaviour and self-organization of these components in a two-dimensional structure. Pure OLA forms a very compressible monolayer, and BAM observation revealed the coexistence of large and irregular solid domains of different thickness dispersed in a gas matrix, compatible with the two most probable orientations of the OLA molecule at the interface. Mixtures of OLA/SA form condensed monolayers from low surface pressures and the thermodynamic analysis indicates that OLA molecules, in the presence of the long-chain SA, orient with the major axis almost perpendicular to the interface. Langmuir-Blodgett (LB) monolayers of pure SA and mixtures were further characterized by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR). AFM images of LB mixed monolayers evidenced microphase separation, not observable by BAM. The SA rich domains are 4-6A thicker than those rich in OLA. The FTIR spectra of mixed LB films on CaF2 substrates showed that OLA does not perturb the all-trans conformation of the SA long alkyl chains, up to a mole fraction of 0.4. The carbonyl-stretching band of OLA suggests that the carboxylic groups of neighbour OLA molecules are involved in hydrogen bonds, forming dimers, as in pure solid phase OLA. These interactions seem to prevail over the OLA-water hydrogen bonds.     

Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry

The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.     

Role of arachidonic acid metabolism in Stat5 activation induced by oleic acid in MDA-MB-231 breast cancer cells

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. In breast cancer cells, the free fatty acid oleic acid (OLA) induces proliferation, migration, invasion and an increase of MMP-9 secretion. However, the role of OLA on Stat5 activation and the participation of COX-2 and LOXs activity in Stat5 activation induced by OLA remain to be investigated. We demonstrate here that stimulation of MDA-MB-231 breast cancer cells with 100 μM OLA induces Stat5 phosphorylation at Tyr-694 and an increase of Stat5–DNA complex formation. The Stat5 DNA-binding activity requires COX-2, LOXs, metalloproteinases and Src activities. In addition, OLA induces cell migration through a Stat5-dependent pathway. In summary, our findings establish that OLA induces cell migration through a Stat5-dependent pathway and that Stat5 activation requires AA metabolites in MDA-MB-231 breast cancer cells.     

Water-stable all-biodegradable microparticles in nanofibers by electrospinning of aqueous dispersions for biotechnical plant protection

Pheromone eluting oligolactide (OLA) microcapsules immobilized in electrospun biodegradable polyester nanofibers were obtained by electrospinning of aqueous dispersions of the microcapsules. OLA was prepared by conventional melt polycondensation of lactic acid. Following the protocol of the solvent displacement method, OLA was dissolved in acetone and mixed with Brij S20 and the pheromone of the European grape vine moth, Lobesia Botrana, (E,Z)-7,9-dodecadien-l-yl acetate (DA). Up to 32 wt % of this mixture could be dispersed in water with colloidal stability of several weeks without any sedimentation. Without DA as well as OLA, no stable dispersions of OLA in water were obtained. Replacement of DA by classical hydrophobes typically used for miniemulsions did not yield stable dispersions, but the addition of octyl acetate, which shows structural similarity to DA, yielded stable dispersions in water up to 10 wt %. Dispersions of OLA/DA were successfully electrospun in combination with an aqueous dispersion of a biodegradable block copolyester resulting in water-stable nanofibers containing OLA/DA microcapsules. Release of DA from microcapsules and fibers was retarded in comparison with non-encapsulated DA, as shown by model studies.     

New amphiphilic lactic acid oligomer-hyaluronan conjugates: synthesis and physicochemical characterization

The "grafting onto" strategy was used to conjugate DL-lactic acid oligomers (OLA) to hyaluronan (HA) for the sake of developing novel degradable HA-based self-assembling polymeric systems. Grafting was achieved by reacting COCl-terminated OLA with cetyltrimethylammonium hyaluronate (CTA-HA) in dimethyl sulfoxide (DMSO). The resulting CTA-HAOLA conjugates were purified and turned to sodium form (Na-HAOLA) by dissolution in a phosphate buffer-DMSO mixture and successive dialyses against DMSO, ethanol, and water. In contrast, when the same protocol was applied to CTA-HAOLA, phase separation with gel formation was observed. The solution phase was composed of Na-HAOLA whereas the gel phase was made of mixed CTA-Na-HAOLA salt with ca. 25% of the carboxyl groups neutralized by CTA. Gelation was assigned to intramolecular hydrophobic associations between OLA and cetyl alkyl chains that complemented electrostatic interactions between CTA and HA COO- groups synergistically. Therefore, the corresponding stabilized CTA ions required more drastic conditions to be released. Under the selected dialysis conditions, the CTA-Na-HAOLA gels formed tiny tubes. Na-HAOLA and CTA-Na-HAOLA were characterized by FTIR, one-dimensional 1H and two-dimensional 1H NMR. The extent of grafting was ca. 5% per disaccharidic repeating unit, regardless of the molecular weight, as determined by NMR and capillary zone electrophoresis. Amphiphilic Na-HAOLA molecules were aggregated and formed spherical species in water according to size exclusion chromatography combined with multiangle laser light scattering detection. The critical aggregation concentration ranged between 0.2 and 0.35% (w/v), depending of the molecular weight of the parent hyaluronan.     

A LB film morphological study with reference to biopolymer–surfactant interaction taking gelatin–CTAB system as a model

The interaction of gelatin with cetyltrimethylammonium bromide (CTAB) studied at pH 9 and an ionic strength of 0.005, produces an interfacial surface active gelatin–CTAB (monomer) complex (GS_n^I), a surface inactive gelatin–CTAB (micelle) complex in bulk (GS_m^B), followed by coacervation, and its solubilization in micellar solution of CTAB. We have herein attempted to probe the interfacial morphological changes of gelatin and its CTAB complexes, and not the bulk properties like coacervation and/or micellar solubilization. The morphologies of pure gelatin and CTAB films and that of gelatin–CTAB interacted complex at the interface have been investigated using LB, SEM, AFM and ellipsometric techniques. The stability of the gelatin monolayer at varied concentrations with and without CTAB has been examined. The SEM images of stabilized films of gelatin and gelatin–CTAB complex have witnessed compact smooth as well as rough surfaces with formation of distinct domains. Drastic morphological change in the film before the critical aggregational concentration of CTAB (T₂) has been in line with an initial abrupt decrease in surface tension. This has been corroborated by AFM measurements, which along with morphology demonstration has provided information on the diameter of the ensembles formed and roughness of the LB films constituted of pure components and their complexes. Thickness of the film was at its maximum in the domain region, as corroborated by ellipsometric technique. Such an elaborate interfacial monolayer and film morphology study of biopolymer-amphiphile system has been rarely documented in literature.     

Gelatin blending improves the performance of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films for biomedical application

To improve the performance of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), gelatin was blended with PHBHHx at different ratios. With increasing gelatin content, the weight loss of gelatin/PHBHHx blend in simulated body fluid at 37 degrees C was accelerated. After 2 months, there was about 15% weight loss in PHBHHx blending with 30% gelatin. Scanning electron microscopy and X-ray diffraction results showed that gelatin blending increased the surface porosity and decreased the crystallinity, which may be responsible for the acceleration of the weight loss. Second harmonic generation results indicated that 10% gelatin blending had less disruption to PHBHHx spatial structure, resulting in better tensile mechanical properties. At the same time, increased surface porosity and decreased crystallinity caused by gelatin incorporation may be beneficial for cell growth compared with pure PHBHHx. All these indicated that gelatin incorporation may improve the performances of PHBHHx to meet the need of different situations during medical implantation.     

Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10–100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3±1.11% and 68.8±1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5–10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.     

Physical properties of nisin-incorporated gelatin and corn zein films and antimicrobial activity against Listeria monocytogenes

Edible films of gelatin and corn zein were prepared by incorporating nisin to the film-forming solutions. Corn zein film with nisin of 12,000 IU/ml had an increase of 11.6 MPa in tensile strength compared with the control, whereas gelatin film had a slight increase with the increase of nisin concentration added. Water vapor permeability for both corn zein and gelatin films decreased with the increase of nisin concentration, thus providing a better barrier against water. Antimicrobial activity against Listeria monocytogenes increased with the increase of nisin concentration, resulting in 1.4 log cycle reduction for corn zein film and 0.6 log cycle reduction for gelatin film at 12,000 IU/ml. These results suggest that incorporation of nisin into corn zein and gelatin films improve the physical properties of the films as well as antimicrobial activity against pathogenic bacteria during storage, resulting in extension of the shelf life of food products by providing with antimicrobial edible packaging films.     

Elastic Networks of Protein Particles

This paper describes the formation and properties of protein particle suspensions. The protein particles were prepared by a versatile method based on quenching a phase-separating protein–polysaccharide mixture. Two proteins were selected, gelatin and whey protein. Gelatin forms aggregates by means of reversible physical bonds, and whey protein forms aggregates that can be stabilized by chemical bonds. Rheology and microscopy show that protein particles aggregate into an elastic particle gel for both proteins. Properties similar to model systems of synthetic colloidal particles were obtained using protein particle suspensions. This suggests that the behaviour of the particle suspensions is mainly governed by the mesoscopic properties of the particle networks and to a lesser extent on the molecular properties of the particles.     

Preparation of gelatin microbeads with a narrow size distribution using microchannel emulsification

The purpose of this study was to prepare monodisperse gelatin microcapsules containing an active agent using microchannel (MC) emulsification, a novel technique for preparing water-in-oil (W/O) and oil-in-water (O/W) emulsions. As the first step in applying MC emulsification to the preparation of monodisperse gelatin microcapsules, simple gelatin microbeads were prepared using this technique. A W/O emulsion with a narrow size distribution containing gelatin in the aqueous phase was created as follows. First, the aqueous disperse phase was fed into the continuous phase through the MCs at 40°C (operating pressure: 3.9 kPa). The emulsion droplets had an average particle diameter of 40.7 μm and a relative standard deviation of 5.1%. The temperature of the collected emulsion was reduced and maintained at 25°C overnight. The gelatin microbeads had a smooth surface after overnight gelation; the average particle diameter was calculated to be 31.6 μm, and the relative standard deviation, 7.3%. The temperature was then lowered to 5°C by rapid air cooling and finally dried. The gelatin beads were dried and could be resuspended well in iso-octane. The had an average particle diameter of 15.6 μm, and a relative standard deviation of 5.9%. Using MC emulsification, we were able to prepare gelatin microbeads with a narrow size distribution. Since this emulsification technique requires only a low-energy input, it may create desirable experimental conditions for microencapsulation of unstable substances such as peptides and proteins. This method is promising for making monodisperse microbeads.     

Preparation and characterizations of self-assembled PEGylated gelatin nanoparticles

The PEGylated gelatin nanoparticles were prepared by self-assembling method and characterized. The gelatin drug carrier was proposed as a targeting drug delivery system with the hypothesis that the gelatin carrier could be degraded by the matrix metalloprotease (MMP) and release the anticancer drug loaded inside carriers around the cancer site. The gelatin nanoparticles proposed in this study were composed of deoxycholic acid (DOCA), monomethoxy polyethylene glycol (MPEG), and gelatin. The carboxyl groups of DOCA and carboxylated MPEG were coupled with amine group of gelatin by dichlorohexylcarbodiimide (DCC) method. One molecule of gelatin coupled with 205 molecules of MPEG and 275 molecules of DOCA. The synthesized gelatin/DOCA/MPEG conjugates (GDM) were ultrasonicated to produce self-assembled nanoparticles. DOCA acted as the hydrophobic core, thereby aggregating gelatin molecules and hydrophilic MPEG chains located at the surface of the nanoparticles. The concentration of GDM, intensity of sonication, sonication time and temperature, all affected to control the particle size in the ultrasonication. The optimum condition was obtained as 1.0 mg/mL of GDM, 28 W for sonication intensity, 3 min of sonication time, and room temperature. The size distribution of particle was found to be 100–1000 nm in this condition. The particles which had a broad size distribution were filtered by 0.2 μm membrane. The product yield of particles having below 200 nm of size was about 30%. After filtration, an average diameter of GDM nanoparticle was 176 nm (155–200 nm).     

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.     

Biomacromolecules electrostatic self-assembly on 3-dimensional tissue engineering scaffold

A poly(ethylenimine) (PEI) was employed to obtain a stable positively charged surface on a poly(D,L-lactide) (PDL-LA) tissue engineering scaffold. An extracellular matrix (ECM)-like biomacromolecule, gelatin, was selected as polyelectrolyte and deposit alternately with PEI on the activated PDL-LA scaffold via ESA technique. The zeta-potential result showed alternating charge of polyelectrolytes (PEI/gelatin) layering on PDL-LA microspheres. Quartz crystal microbalance (QCM) measurement further verified the gradual deposition of PEI/gelatin on the PDL-LA thin film. The combination of PEI aminolysis and the layer-by-layer technique was then explored to construct gelatin coating onto the 3-D porous PDL-LA scaffold. Scanning electronic microscopy showed that there is no notable difference between modified and unmodified PLA scaffolds, with regard to the porosity, pore diameter, and scaffold integration. The dual-tunnel confocal laser scanning microscopy indicated uniform gelatin distribution on the inner surface of the 3-D porous scaffold. The gradual build-up of protein layer on scaffold was investigated by radioiodination technique. Chondrocyte was chosen to test the cell behavior on modified and unmodified PDL-LA scaffolds. The results of the cell viability, total intracellular protein content, and cell morphology on the PEI/gelatin multilayers modified PDL-LA scaffold showed to promote chondrocyte growth. Comparing conventional coating methods, polyelectrolyte multilayers are easy and stable to prepare. It may be a promising choice for the surface modification of complex biomedical devices. These very flexible systems allow broad medical applications for drug delivery and tissue engineering.     

Enzyme distribution and matrix characteristics in biocatalytic particles

In a study of Assemblase, an industrial immobilized penicillin-G acylase, various electron microscopic techniques were used to relate intra-particle enzyme heterogeneity with the morphological heterogeneity of the support material at various levels of detail. Transmission electron microscopy was used for the study of intra-particle penicillin-G acylase distribution in Assemblase particles of various sizes; it revealed an abrupt increase in enzyme loading at the particle surface (1.4-fold) and in the areas (designated halo's) surrounding internal macro-voids (7.7-fold). Cryogenic field-emission scanning electron microscopy related these abrupt local enzyme heterogeneities to local heterogeneity of the support material by revealing the presence of dense top layers surrounding both the particle exterior and the internal macro-voids. Furthermore, it showed a very distinct morphological appearance of the halo. Most probably, all these regions contained relatively more chitosan than gelatin (the polymers Assemblase was constructed of), which suggested local polymer demixing during particle production. A basic thermodynamic line of reasoning suggested that a difference in hydrophilicity between the two polymers induced local demixing. In the future, thermodynamic knowledge on such polymer interactions resulting in matrix heterogeneity may be used as a tool for biocatalyst design.     

测序级胰蛋白酶的制备工艺与质量检测简

目的：制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶,应用于蛋白组学研究的蛋白质鉴定与分析中。方法：取实验室自制的猪胰蛋白酶粗酶,经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解,采用高效液相色谱仪15RPC反相柱纯化,收集对应的甲基化胰蛋白酶峰组分,冷冻干燥;甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮（TPCK）修饰,以抑制糜蛋白酶等非特异性酶切活性,并经反相色谱柱再纯化,获得终产物即质谱测序级胰蛋白酶;自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定,并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等,检测其纯度、酶水解效率及酶切位点特异性。结果：自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95％,酶比活力为200U/mgP（TAME）以上,质谱分析酶切特异性好;且酶的制备工艺流程稳定,可应用于测序级胰蛋白酶产品的生产与开发中。结论：制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强,可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。     

ULTRATHIN FROZEN SECTIONS : II. Demonstration of Enzymic Activity

Endogenous enzyme activity can be readily and routinely demonstrated in ultrathin, frozen sections for electron microscopy. The procedure employed to obtain the best structural preservation as well as enzyme activity in thin sections involved fixation in glutaraldehyde, embedding in thiolated gelatin or pure gelatin, partial dehydration in glycerol, and sectioning in a cryostat at -35°C with a slightly modified Porter-Blum microtome on which the tissue is maintained at -70°C and the knife at -23°C. Kidney cortex was used as test tissue, but a few other organs were occasionally used. Thin sections were floated on the surface of several incubation media routinely employed for enzyme cytochemistry. Positive, specific reactions were obtained for alkaline phosphatase in kidney brush border, for adenosine triphosphatase in brush border and in basal membranes of distal tubules, for acid phosphatase and esterase in lysosomes, and for NADH diaphorase in mitochondria. Mitochondrial ATPase was sporadically evident only in the distal tubule of the kidney. Localizations of enzyme activity reported by other technical approaches were confirmed and in some cases somewhat improved.     

明胶酶谱实验方法的优化简

目的：优化明胶酶谱实验方法,并检测不同浓度凝血酶刺激人皮肤成纤维细胞分泌的明胶酶B（MMP-9）活性的变化。方法：用含明胶的聚丙烯酰胺凝胶电泳分开上清中各蛋白条带,经2．5％TritonX-100洗脱、明胶酶缓冲液孵育、考马斯亮蓝染色液染色及脱色液脱色后,经光密度扫描记录,应用ImageJ软件进行密度计量分析。结果：凝血酶刺激人皮肤成纤维细胞分泌MMP一9具有浓度依赖性。结论：利用优化的明胶酶谱实验方法可做出高质量的明胶酶谱图片,对检测明胶酶活性有重要意义。     

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA_app) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g⁻¹ of gel for GLU, 7.76 mg g⁻¹ of gel for GLY, and 7.65 mg g⁻¹ of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g⁻¹ of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.