Initial Lipase Activity Research Articles

A carbon-based bifunctional heterogeneous enzyme: toward sustainable pollution control.

We present a study on an immobilized functional enzyme (IFE), a novel biomaterial with exceptional sustainability in enzyme utility, widely employed across various fields worldwide. However, conventional carriers are prone to eroding the active functional domain of the IFE, thereby weakening its intrinsic enzyme activity. Consequently, there is a burgeoning interest in developing next-generation IFEs. In this study, we engineered a carbon-based bifunctional heterogeneous enzyme (MIP-AMWCNTs@lipase) for the intelligent recognition of di(2-ethylhexyl)phthalate (DEHP), a common plasticizer. The heterogeneous enzyme contains a bifunctional structural domain that both enriches and degrades DEHP. We investigated its dual-response performance for the enrichment and specific removal of DEHP. The imprinting factor of the carrier for DEHP was 3.4, demonstrating selectivity for DEHP. The removal rate reached up to 94.2% over a short period. The heterogeneous enzyme exhibited robust activity, catalytic efficiency, and excellent stability under harsh environmental conditions, retaining 77.7% of its initial lipase activity after 7 cycles. Furthermore, we proposed a stepwise heterogeneous enzyme reaction kinetic model based on the Michaelis-Menten equation to enhance our understanding of enzyme reaction kinetics. Our study employs a dual-effect recognition strategy of molecular blotting and enzyme immobilization to establish a method for the removal of organic pollutants. These findings hold significant implications for the fields of biomaterials and environmental science.

Biochemical Study of Bacillus stearothermophilus Immobilized Lipase for Oily Wastewater Treatment

Traditional wastewater treatments involve expensive mechanical and physiochemical methods, so researchers have been developing cost-effective, sustainable technologies that use enzymes to produce higher quality effluents and recover more energy and nutrients from wastewater. A thermostable, alkaline, and solvent-tolerant lipase was partially purified from thermophilic Bacillus stearothermophilus. The lipase displayed maximum activity at 50 °C and pH 11.0 and catalyzed both short- and long-chain triacylglycerols at similar rates. B. stearothermophilus lipase also exhibited high stability when incubated at 40 °C for 1 h with anionic and non-ionic surfactants. Studies show that thermostable enzymes can be improved through immobilization and modification of other reaction conditions. Therefore, B. stearothermophilus lipase was immobilized through adsorption on CaCO3, Celite 545, and silica gel with the CaCO3 support producing the best adsorption rate (89.33%). The optimal initial lipase activity was approximately 4500 U.g−1 after 60 min. Interestingly, 93% of the initial lipase activity was retained after six cycles, and almost 50% of the initial activity remained after 12 cycles. Furthermore, immobilization improved storage stability with 98.85% of the initial lipase activity retained after 60 days of storage at 4 °C. The biochemical characteristics of immobilized lipase shifted toward a slightly alkaline region, reaching maximum activity at pH 12. The optimal temperature of immobilized lipase was 60 °C. Immobilization also improved enzymatic stability by widening the pH range from 5–9 (for free lipase) to 4–11, and thermostability by reaching 65 °C. The application of immobilized lipase in wastewater treatment was observed through oil layer biodegradation. Notably, treating wastewater for 10 days with immobilized lipase almost removed the chemical oxygen demand (COD) from 1950.1 down to 4.04 mg.L−1. Similarly, lipid content was almost removed from 15,500 ± 546 mg.L−1 down to 12 mg.L−1. All results highlight the potential value of CaCO3-immobilized lipase as an effective biocatalyst for hydrolyzing wastewater.

Enhancement lipase activity via immobilization onto chitosan beads used as seed particles during fluidized bed drying: Application in butyl butyrate production

We present a method for lipase immobilization via fluidized bed technology. The enzyme drying/immobilization solution consisted of phosphate buffer (50 mM pH 7.0) containing the lipase (5.0 mgprotein/gchitosan), 200 mM of NaCl and PEG-1500 (1.5 % m/v). The lipase immobilized on glutaraldehyde-activated enhanced 46.5 % of initial lipase activity. The optimum pH for the free lipase was slightly acid (pH 6.5), whereas the optimum pH for the immobilized systems shifted to the alkaline region (pH 8.0). As for the optimum temperature, it went from 45 °C for the free lipase to 50 °C for the immobilized systems. The immobilized systems had half-life of 5.81 h and stabilization factor of 10.75 at 80 °C. The kinetic data showed that the lipase immobilized on chitosan beads activated with glutaraldehyde was catalytically more efficient (KM: 0.015, Vmax: 170.4 μmol/min/mg, and Vmax/KM: 11,360) than the free lipase (KM: 0.035, Vmax: 120.3 μmol/min/mg, and Vmax/KM: 3437); displayed low moisture content and water activity (moisture: 3.84 % and AW: 0.10); and retained 85.5 % of its initial activity (vs 16.5 % retained activity for the free lipase) after storage for six months. After ten reuse cycles, said immobilized system retained 75.2 % of its initial activity. We employed this system as biocatalyst to esterify butyric acid with butanol. We developed a centered faced central composite design 22 and a mathematical model to describe how the ester content behaved as a function of temperature and amount of biocatalyst, as independent variables. Under optimized conditions (temperature of 45 °C and biocatalyst mass of 0.5 g), 18 g of butyl butyrate/L was attained within 6 h, which corresponded to an esterification yield of 98.96 %.

The optimization of glycidyl methacrylate based terpolymer monolith synthesis: an effective Candida rugosa lipase immobilization support

In this work, the optimization of synthesis of terpolymer monolith based on glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EGDMA) and additional cross-linkers: trimethylolpropanetriacrylate (TMPTA) or triethylene glycol dimethacrylate (TEGDMA) was performed. Moreover, novel vinyl functionalized cross-linkable polymers: ethanolamine (EA)/methacryloyl (MAC) modified poly (methyl methacrylate) (PMMA), and hydrolyzed poly (ethylene-co-vinyl acetate) copolymer (EVOH) modified with MAC either directly or via ethyl malonyl chloride/EA bridging group (m-EVA) were used as cross-linkable polymer to improve mechanical/elastic properties of the obtained monoliths. Optimization procedure, performed applying response surface methodology (RSM), was focused on the production of materials with improved dimensional stability/integrity and porosity with abundance of epoxide groups capable for immobilization of lipase from Candida rugosa (CRL). Structural characterization of the synthesized monoliths was determined using FTIR, Raman and 1H NMR spectroscopies, while morphology/porosity was determined by SEM technique and image analysis; and mechanical properties by diametral compression testing. The most potential monolith containing m-EVA polymeric cross-linker, i.e. GMA/EGDMA/TEGDMA/m-EVA monolith, was used as CRL carrier in a two-step immobilization process. Enzyme loading and the activity of obtained preparations for various initial enzyme concentrations were monitored after 4 and 48 h of immobilization. The resulting catalysts show high potency in biocatalytic reactions with the highest percentage of retained initial lipase activity of 64.5%.

Concentration of Lipase from Aspergillus oryzae Expressing Fusarium heterosporum by Nanofiltration to Enhance Transesterification

Nanofiltration membrane separation is an energy-saving technology that was used in this study to concentrate extracellular lipase and increase its total activity for biodiesel production. Lipase was produced by recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (FHL). A sulfonated polyethersulfone nanofiltration membrane, NTR-7410, with a molecular weight cut-off of 3 kDa was used for the separation, because recombinant lipase has a molecular weight of approximately 20 kDa, which differs from commercial lipase at around 30 kDa for CalleraTM Trans L (CalT). After concentration via nanofiltration, recombinant lipase achieved a 96.8% yield of fatty acid methyl ester (FAME) from unrefined palm oil, compared to 50.2% for CalT in 24 h. Meanwhile, the initial lipase activity (32.6 U/mL) of recombinant lipase was similar to that of CalT. The composition of FAME produced from recombinant concentrated lipase, i.e., C14:1, C16:0, C18:0, C18:1 cis, and C18:2 cis were 0.79%, 34.46%, 5.41%, 45.90%, and 12.46%, respectively, after transesterification. This FAME composition, even after being subjected to nanofiltration, was not significantly different from that produced from CalT. This study reveals the applicability of a simple and scalable nanofiltration membrane technology that can enhance enzymatic biodiesel production.

How the Triton X-100 modulates the activity/stability of the Thermomyces lanuginose lipase: Insights from experimental and molecular docking approaches

The lipase and Triton X-100 mixture is common for stabilization, immobilization and application processes of these kinds of enzymes. The objective of this article was to study the structural behavior and catalytic performance of Thermomyces lanuginose lipase in the presence of Triton X-100 at 25 °C and different pHs. The structural changes were followed by circular dichroism, correlating them with the catalytic performance, which is reported as the initial lipase activity in the hydrolysis of p‑nitro phenyl butyrate at zero time and residual activity after 48 h of incubation in the absence or presence of surfactant, at the selected pHs. Computational simulations allowed to explain the correlations between the physicochemical changes and the formation of surfactant protein complex, leading to the elucidation of the main interactions that drive activity and stability of this lipase in presence of the Triton X-100 surfactant. Main results showed the Triton X-100-enzyme complex modulates the site active geometry, favoring a better substrate-enzyme adjustment, which influences the activity and stability at evaluated pHs. This study contributes to understand the effect of some additives commonly used to improve the biocatalytic performance on several applications for different industrial fields.

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22.

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1-10 °C in Luria-Bertani (LB) broth or 1-30 °C in Reasoner's 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1-10 °C in LB medium. About 70-80% of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60% of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10-20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C2-C18). The highest hydrolytic activity was obtained with p-NP-butyrate (C4) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C12) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.

Novel nanostructured tin dioxide as promising carrier for Candida rugosa lipase

This is the first report on lipase immobilization on nanostructured tin dioxide. The material was applied as a support for Candida rugosa lipase. All biocatalytic characteristics obtained for this novel biocatalyst (nano-SnO2-CRL) were compared with those for lipase immobilized on polypropylene (PP-CRL). Nano-SnO2-CRL has shown a specific activity eight times higher than that found for PP-CRL. The tin dioxide preparation preserved up to 45% of the initial lipase activity after 1h incubation at pH 10.0, while PP-CRL was completely inactivated. The immobilization on the inorganic carrier enhanced lipase thermal stability. Upon heating for 1h at 55°C, nano-SnO2-CRL retained 77% active, while PP-CRL was half inactivated. The synthetic activity and the effect of several parameters on isoamyl acetate production for both biocatalysts were evaluated. Among four tested acyl donors, acetic anhydride was the most efficient for nano-SnO2-CRL. The reaction proceeded with a high reaction rate and resulted in 88.2% yield of the target ester for 2h. Among various solvents, n-decane proved to be the best for both catalysts. The increase in the reaction temperature from 50°C to 60°C enhanced the reaction rate 1.5 and 1.7 fold, for nano-SnO2-CRL and PP-CRL, respectively. A considerable decline in activity for both enzyme preparations was observed after the second esterification run due to prolonged heating and probably also to inhibition by substrates and product.

Interesterification of milkfat with soybean oil catalysed by Rhizopus oryzae lipase immobilised on SiO2‐PVA on packed bed reactor

SummaryThe potential of the lipase from Rhizopus oryzae immobilised on SiO2‐PVA to catalyse the interesterification of the milkfat with soybean oil in a packed bed reactor running on continuous mode was evaluated. The reactor operated continuously for 35 days at 45 °C, and during 12 days, no significant decrease in the initial lipase activity was verified. Interesterification yields were in the range from 35 to 38%wt, which gave an interesterified product having 59% lower consistency in relation to non‐interesterified blend. Results showed the potential of the lipase from Rhizopus oryzae to mediate the interesterification of milkfat with soybean oil in packed bed reactor, attaining a more spreadable product under a cool temperature. The biocatalyst operational stability was assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 34 days and 0.034 day−1, for half‐life and a deactivation coefficient, respectively.

Packed-Bed Reactor Running on Babassu Oil and Glycerol to Produce Monoglycerides by Enzymatic Route using Immobilized Burkholderia cepacia Lipase

The aim of this study was the glycerolysis of babassu oil catalyzed by immobilized lipase from Burkholderia cepacia, in a continuous packed-bed reactor. The best reaction conditions were previously established in batchwise via response surface methodology as a function of glycerol-to-oil molar ratio and reaction temperature. The reactor operated continuously for 22 days at 50 degrees C, and during the first 6 days, no significant decrease on the initial lipase activity was observed. Monoglycerides concentration was in the range from 25 to 33 wt.%. Subsequently, a progressive decrease in the activity was detected, and an inactivation profile described by Arrhenius model estimated values of 50 days and 1.37 x 10(-2) h(-1) the half-life and deactivation coefficient, respectively.

An organic solvents tolerant and thermotolerant lipase from Pseudomonas fluorescens P21

In this study, 38 organic solvents tolerant bacteria have been isolated from 21 different soil and water environments and the bacterium called P21 has been isolated among them due to its ability of surviving in 30% toluene, which is highly toxic to microorganisms and its capability of high lipase production (3.5 U/l). The lipase production medium has been optimized and the medium containing olive oil as an inducer and peptone as a nitrogen source has been selected as the best ingredients for the highest lipase production conditions (15 U/l). After this optimization, initial lipase activity has increased to 638.3%. In our study, to identify the physical and molecular properties of the lipase, the supernatant has been obtained at the highest production value in early stationer phase of the P21's growth curve and the supernatant has concentrated by ultrafiltration and ammonium sulphate precipitation. SDS-PAGE and zymogram analysis were exhibited two lipases with a molecular weight 15 and 38 kDa. Partially purified lipase is optimum active at pH 7.0 and 30 and 60 °C. The lipase activity is stable in the presence of organic solvents such as cyclohexane, styrene, hexane and heptane. The enzyme protects its 85.3% of its activity after incubating for one and half an hour at 70 °C. P21 strain has identified as Pseudomonas fluorescens by biochemical and molecular techniques and has recorded as P. fluorescens P21 in National Center for Biotechnology Information ( www.ncbi.nih.gov) with an accession number, FJ605510.

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10 3, 98 × 10 3, 147 × 10 3 and 196 × 10 3 U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters ( K m and V max ) were found using Michaelis–Menten enzyme kinetics. The K m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.

Evaluation of diatomaceous earth as a support for sol–gel immobilized lipase for transesterification

Enzymatic production of biodiesel by triglyceride transesterification is a promising alternative to chemically catalyzed biodiesel production despite the challenges involved with using enzymes. Celite ® supported lipase sol–gels were investigated as an option for solving some of the challenges associated with the use of enzymes for biodiesel production addressing such problems as activity, stability and reusability of the enzyme. Three types of Celite ® were considered (R633, R632, and R647) and compared to unsupported lipase sol–gels. Various factors were considered with regard to comparing the support materials. They included surface morphology characterized using surface area analysis and scanning electron microscopy, physical properties including adhesion of the sol–gel to the Celite ® and the protein loading on the Celite ®, and finally enzymatic properties based on the conversion of methanol to methyl oleate and the enzymatic activity of lipase. All the sol–gels showed good conversion and initial lipase activity, and all the Celite ® supports had similar sol–gel adhesion and protein loading. Sol–gel immobilized lipase supported on Celite ® R632 had an average 6-h percent conversion of approximately 60%, and an average initial lipase activity comparable to that of the unsupported sol–gel formulation.

The improvement of lipase secretion and stability by addition of inert compounds into Acinetobacter calcoaceticus cultures.

Acinetobacter calcoaceticus BD413 produces variable amounts of an exocellular lipase that becomes rapidly inactivated upon secretion. To achieve high yield and protect the enzyme, we assayed the addition of several inert compounds to cell-free supernatants, cell fractions, and whole cultures. Glass beads, poly(ethylene glycol) 600, Triton X-100, saccharose, gum arabic, and beta-cyclodextrin were among the compounds tested. beta-Cyclodextrin and gum arabic (and saccharose to a lesser extent) were effective enzyme stabilizers in cell-free supernatants, while gum arabic, glass beads, and Triton X-100 improved lipase secretion from cells, and, therefore, total lipase yield (30-50%, according to the additive). In whole cultures, beta-cyclodextrin was the most effective additive, particularly in combination with glass beads or gum arabic. Indeed, cultures containing beta-cyclodextrin plus gum arabic were able to maintain 95% (+/- 1.5%) of the initial lipase activity for more than 16 h, while control cultures with no additives maintained only 10% (+/- 4%) of the enzyme activity after the same period. In conclusion, the addition of inert compounds in cultures may be considered a useful approach for achieving increased yield and lipase stabilization, amenable for downstream processing.

Lipase‐catalyzed kinetic resolution of (R,S)‐1‐phenylethyl propionate in an enzyme membrane reactor

AbstractEnzymatic stereoselective hydrolysis of (R,S)‐1‐phenylethyl propionate was performed in a stirred tank and in a biphasic enzyme membrane reactor. Lipase from Pseudomonas sp. was proved to be a good enantioselective catalyst for this reaction. The enzyme was covalently immobilized in a porous polyamide membrane (flat sheet as well as hollow‐fibres) via glutaraldehyde. An influence of membrane hydrophobicity on reactor performance was observed. Initial lipase activity and productivity in the processes were equal to 1.05 × 10−4, 1.3 × 10−5 and 1.0 × 10−5 mole/(h × mg of enzyme) in the case of native lipase, in the aromatic polyamide hydrophobic membrane reactor and in the hydrophilic polyamide‐6 membrane reactor, respectively. The influence of some factors such as temperature, pH, buffer concentration, initial substrate concentration and addition of β‐cyclodextrin derivatives on reaction rate and enantioselectivity was investigated and discussed. In the enzyme membrane reactor both organic and aqueous phases circulated countercurrently on both sides of the membrane. At a conversion degree of under 55–60%, pure enantiomer of the remaining ester (i.e. > 98%) was obtained.

Fat digestion in the stomach: stability of lingual lipase in the gastric environment.

Digestion of dietary fat starts in the stomach, where lingual lipase hydrolyzes triglycerides to free fatty acids and partial glycerides at pH 3.0-6.0. Lingual lipase is secreted continuously from lingual serous glands and accumulates in the stomach between meals, when gastric pH is less than 3.0. We have, therefore, examined the resistance of lingual lipase to low pH and its possible protection by dietary components present in the stomach contents. Partially purified rat lingual lipase (7-15 micrograms enzyme protein) was preincubated at 37 degrees C for 10-60 min at pH 1.0-6.0 before incubation for assay of lipolytic activity, hydrolysis of tri-[3H]olein at pH 5.4. The data show that partially purified rat lingual lipase preparations are stable at 37 degrees C in the pH range of 2.5-6.0. Enzyme activity, however, is rapidly and irreversibly lost during preincubation at pH 1.0-2.4 for 10-30 min. Protein (gelatin 1% or albumin 1% or 2.5%) cannot prevent the inactivation of lingual lipase at low pH. The large molecular species (molecular weight greater than 500,000) of lingual lipase (thought to be an aggregate of enzyme with lipids) is slightly more resistant to inactivation than the 46,000 dalton preparation, suggesting that lipids might protect the enzyme from inactivation. Indeed, about 60% of the initial lipase activity is preserved during incubation at pH 2.0 in the presence of 50 mM lecithin or 10 mM triolein. The data indicate that triglycerides which are hydrolyzed by this enzyme as well as phospholipids that are not hydrolyzed can prevent the inactivation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Instability of endothelium-bound lipoprotein lipase activity in perfused rat hearts.

The enzymatic activity of endothelium-bound lipoprotein lipase was measured in rat hearts perfused for 1 h at 37 degrees C. Viability parameters such as beating rate, flow rate, aortic pressure, and oxygen consumption were all kept strictly constant during the entire perfusion time. The lipase activity was determined by input-output difference of the triacylglycerol content in the nonrecirculating heart perfusate which contained either an artificial triolein emulsion or rat lymph chylomicrons. With either substrate, the lipase activity decreased with time: approximately 2% of initial lipase activity was lost per minute. The presence of rat serum (10%) in heart perfusate enhanced the rate at which the lipase activity disappeared. Only a small portion of the lipoprotein lipase activity, which was lost from the perfused heart, was recovered in the outflow perfusate. Our data demonstrate that, under our experimental conditions, the enzymatic activity of rat heart lipoprotein lipase does not remain constant during heart perfusion. Caution should therefore be taken by users of heart perfusion technique, especially for those who need constant lipoprotein lipase activity. The results suggest that the first step in the catabolic fate of endothelium-bound cardiac lipoprotein lipase is the loss of its catalytic activity. Whether the inactivated enzyme is released from the endothelium or remains in situ is not yet known.