Kinetic characterization ofpenicillium citrinum lipase in AOT/lsooctane-reversed micelles

A lipase from a wild strain ofPenicillium citrinum was encapsulated in AOT/isooctane-reversed micelles, and the kinetic parameters were studied relative to triolein hydrolysis. Lipolytic activity was strongly dependent on the water amount in the system (W_o) and presented a bell-shaped curve for this parameter, with a maximum in the range of W_o 10–15. Optimum conditions for enzyme activity were pH 8.0 and 45?C. The influence of substrate concentration was also studied. The enzyme showed a Michaelis-Menten behavior and the apparent kinetics constants were calculated as beingV _max.app. - 120 U/mg and K_mapp = 49.2 mM.     

A stable lipase fromCandida lipolytica

Although Upases have been intensively studied, some aspects of enzyme production like substrate uptake, catabolite repression, and enzyme stability under long storage periods are seldom discussed in the literature. This work deals with the production of lipase by a new selected strain ofCandida lipolytica. Concerning nutrition, it was observed that inorganic nitrogen sources were not as effective as peptone, and that oleic acid or triacylglycerides (TAG) were essential carbon sources. Repression by glucose and stimulation by oleic acid and long chain TAG (triolein and olive oil) were observed. Extracellular lipase activity was only observed at high levels at late stationary phase, whereas intracellular lipase levels were constant and almost undetectable during the cultivation period, suggesting that the produced enzyme was attached to the cell wall, mainly at the beginning of cultivation. The crude lipase produced by this yeast strain shows the following optima conditions: pH 8.0–10.0, temperature of 55°C. Moreover, this preparation maintains its full activity for at least 370 d at 5°C.     

Kinetic studies of lipase from Candida rugosa

The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (K _d=0.98 h⁻¹), whereas for the immobilized lipase it was 1.10 h (K _d=0.63 h⁻¹). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The K _m was 0.15 mM and the V _max was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.     

Quantitative turbidity assay for lipolytic enzymes in microtiter plates

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

Protein-Coated Microcrystals of Pseudomonas aeruginosa PseA lipase

Highly active Pseudomonas aeruginosa lipase protein-coated microcrystals (PAL PCMC) have been prepared by immobilization of protein onto K₂SO₄ as excipient solid support carrier and n-propanol as precipitating solvent. Transmission electron micrographs confirmed the formation of PAL PCMC. These PCMC were found to be a catalytically more active and stable preparation for p-nitrophenyl palmitate hydrolysis in n-heptane, compared to free lipase. The V _max, K _m, and temperature optimum for PAL PCMC increased from 0.49 to 5.66 nmol min^?1 mg^?1, 589 to 679.8 mmol, and 40°C to 45°C, respectively. These were thermally more stable with 4.65, 2.56, and 1.24-fold improvement in half lives at 45°C, 55°C, and 60°C compared to free P. aeruginosa PseA lipase. Their catalytic efficiency was enhanced by tenfold over that of free enzyme. PAL PCMC offer a simple and effective technique for obtaining stable and efficient lipase preparation for biocatalysis in nonaqueous medium.     

Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH₂-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca²⁺ and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5–10 and at temperatures below 45 °C.     

An Evaluation of the Proteolytic and Lipolytic Potential of Penicillium spp. Isolated from Traditional Greek Sausages in Submerged Fermentation

A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.     

A potential high-throughput method for the determination of lipase activity by potentiometric flow injection titrations

Potentiometric FIA titrations were performed to determine enzyme activities of lipase type B from Candida antarctica, CAL-B. Two substrates, triacetin and tributyrin were hydrolyzed in phosphate buffer solutions, and the concentration change of the base component of the buffer was titrated in a carrier solution containing hydrochloric acid and potassium chloride. The system was calibrated with butyric acid and acetic acid, respectively. FIA titration peaks were evaluated with respect to peak height and peak area. Butyric acid and acetic acid could be titrated in the buffer solution from 3 × 10⁻³ mol L⁻¹ to 0.1 mol L⁻¹. The detection limit of enzyme activity was determined to be 0.07 U mL⁻¹ (15 min reaction time) and the minimum activity was calculated to be 0.035 units corresponding to 35 nmol min⁻¹. The specific activities of lipase B for the hydrolysis of tributyrin and triacetin were determined as 16 ± 2 U mg⁻¹ and 2 ± 0.2 U mg⁻¹ (per mg commercial lipase preparation), respectively.     

Thermostability of <Emphasis Type="Italic">Cromobacterium viscosum</Emphasis> lipase in AOT/isooctane reverse micelle

The thermostability of Cromobacterium viscosum lip ase (EC 3.1.1.3) entrapped in AOT (sodium bis-[2-ethylhexyl] sulfosuccinate) reverse micelles was in creased by the addition of short-chain polyethylene glycol (PEG 400). Two different approaches were considered: (1) the determination of half-life time and (2) the mechanistic analysis of deactivation kinetics. The half-life of lipase entrapped in AOT/isooctane reverse micelles with PEG 400 at 60°C was 28h, ninefold higher than that in reverse micelles without PEG 400. The lip ase entrapped in both reverse micellar systems followed a series-type deactivation mechanism involving two first-order steps. The deactivation constant for the first step at 60°C in PEG containing reverse micelles was 0.055 h¹¹, 11-fold lower than that in reverse micelles without PEG, whereas it remained almost constant for the second step. The inactivation energy of the lip ase entrapped in reverse micelles with and without PEG 400 was 88.12 and 21.97 kJ/mol, respectively.     

Role of Met93 and Thr96 in the Lid Hinge Region of Rhizopus chinensis Lipase

We engineered Rhizopus chinensis lipase to study its critical amino acid role in catalytic properties. Based on the amino acid sequence and three-dimensional model of the lipase, residues located in its lid hinge region (Met93 and Thr96) were replaced with corresponding amino acid residues (Ile93 and Asn96) found in the lid hinge region of Rhizopus oryzae lipase. The substitutions in the lid hinge region affected not only substrate specificity but also the thermostability of the lipase. Both lipases preferred p-nitrophenyl laurate and glyceryl trilaurate (C12). However, the variant S4-3O showed a slight decline in activity toward long-chain fatty acid (C16–C18). When enzymes activities decreased by half, the temperature of the variant (45 °C) was 22 °C lower than the parent (67 °C), probably substantially destabilized the structure of the lid region. The interfacial kinetic analysis of S4-3O suggested that the lower catalytic efficiency was due to a higher K _m* value. According to the lipase structure investigated, Ile93Met played a role of narrowing the size of the hydrophobic patch, which affected the substrate binding affinity, and Asn96Thr destabilized the structure of the lipase by disrupting the H-bond interaction in the lid region.     

Screening, Gene Sequencing and Characterising of Lipase for Methanolysis of Crude Palm Oil

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/β hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K _M of 0.75 mM and a V _max of 0.33 mM?min^?1 on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.     

Effect of the Pretreatment of Lipase with Organic Solvents on its Conformation and Activity in Reverse Micelles

The activity and conformation of Chromobacterium viscosum lipase-pretreated with various organic solvents were investigated. The pretreatment of lipase led to a substantial increase of enzyme activity in AOT (sodium bis [2-ethyl -1-hexyl] sulfosuccinate)/isooctane/water reverse micelles. Among the organic solvents used, n-hexane was found to be most effective. It was observed that higher hexane content with shorter agitation time and vice versa had almost the same effect on the initial activity of lipase. The kinetic study showed that the Michaelis constant (K _m) and the substrate adsorption equilibrium constant (K _ad) were reduced by the pretreatment of lipase with hexane, whereas the change in the maximum reaction rate (V _max) was insignificant. The two spectroscopic techniques (Fluorescence spectra of lipase encapsulated in RMs and Fourier transform infrared [FTIR] spectra of lipase powders) were performed to detect possible conformational changes in the enzyme caused by the pretreatment. A correlation between the maximum fluorescence intensity and the activity of treated lipase was found as a function of agitation time. The FTIR spectrum of lipase showed a new shape peak corresponding to 1,500 cm⁻¹ as a result of pretreatment with organic solvents.     

Protein Engineering of Bacillus thermocatenulatus Lipase via Deletion of the α5 Helix

Lipases from Bacillus thermocatenulatus are a member of superfamily of α/β hydrolase, but there are structural differences between them. In this work, we focused on the α5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical α/β hydrolase fold. In silico study showed that the α5 helix is a region that causes disorder in BTL2 protein. So, the α5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Δα5-BTL2) has been expressed in Pichia pastoris yeast. The α5 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.     

Production and Characterization of a Halo-, Solvent-, Thermo-tolerant Alkaline Lipase by Staphylococcus arlettae JPBW-1, Isolated from Rock Salt Mine

Studies on lipase production and characterization were carried out with a bacterial strain Staphylococcus arlettae JPBW-1 isolated from rock salt mine, Darang, HP, India. Higher lipase activity has been obtained using 10 % inoculum with 5 % of soybean oil as carbon source utilizing a pH 8.0 in 3 h at 35 °C and 100 rpm through submerged fermentation. Partially purified S. arlettae lipase has been found to be active over a broad range of temperature (30–90 °C), pH (7.0–12.0) and NaCl concentration (0–20 %). It has shown extreme stability with solvents such as benzene, xylene, n-hexane, methanol, ethanol and toluene up to 30 % (v/v). The lipase activity has been found to be inhibited by metal ions of K⁺, Co²⁺ and Fe ²⁺ and stimulated by Mn²⁺, Ca²⁺ and Hg²⁺. Lipase activity has been diminished with denaturants, but enhanced effect has been observed with surfactants, such as Tween 80, Tween 40 and chelator EDTA. The K _m and V _max values were found to be 7.05 mM and 2.67 mmol/min, respectively. Thus, the lipase from S. arlettae may have considerable potential for industrial application from the perspectives of its tolerance towards industrial extreme conditions of pH, temperature, salt and solvent.     

Esterification activity and stability of Candida rugosa lipase immobilized into chitosan

Microbial lipase from Candida rugosa immobilized into porous chitosan beads was tested for esterification selectivity with butanol and different organic acids (C2–C12), and butyric acid and different aliphatic alcohols (C2–C10). After 24 h, the acids tested achieved conversions of about 40–45%. Acetic acid was the only exception, and in this case butanol was not consumed. Different alcohols led to butyric acid conversions >40%, except for ethanol, in which case butyric acid was converted only 26%. The system’s butanol and butyric acid were selected for a detailed study by employing an experimental design. The influence of temperature, initial catalyst concentration, and acid:alcohol molar ratio on the formation of butyl butyrate was simultaneously investigated, employing a 2³ full factorial design. The range studied was 37–50°C for temperature (X₁), 1.25–2.5% (w/v) for the catalyst concentration (X₂), and 1 and 2 for the acid:alcohol molar ratio (X₃). Catalyst concentration (X₂) was found to be the most significant factor and its influence was positive. Maximum ester yield (83%) could be obtained when working at the lowest level for temperature (37°C), highest level for lipase concentration (2.5% [w/v]), and center level of acid:alcohol molar ratio (1.5). The immobilized lipase was also used repeatedly in batch esterification reactions of butanol with butyric acid, revealing a half-life of 86 h.     

Purification and partial characterization of a lipase from Bacillus coagulans ZJU318

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme activity was strongly inhibited by Ag⁺ and Cu²⁺, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.     

Cooperative catalysis and critical decomposition distances in water oxidation by tris(ethylenediamine)ruthenium (III) complex confined in a Nafion membrane

The activity of tris(ethylenediamine)ruthenium (III) complex, [Ru(en)₃]³⁺, as a water oxidation catalyst was studied in a homogeneous aqueous solution and a heterogeneous Nafion (Nf) membrane. In the aqueous solution, the apparent catalytic activity (k_app (s⁻¹)) decreased monotonously with the concentration due to a bimolecular decomposition of the complex. The bimolecular decomposition of the complex was remarkably suppressed by incorporating it into a Nf membrane. An optimum complex concentration for k_app in the Nf membrane was exhibited, which was explained both by a cooperative catalysis and a bimolecular decomposition of the complex. The k_app in the Nf membrane was analyzed in terms of an intrinsic catalytic activity (k_O2 (s⁻¹)) of the complex, a cooperative catalysis distance (r_co (nm)) and a critical decomposition distance (r_d (nm)) between them based on intermolecular distance distribution to obtain the k_O2=8.5×10⁻⁵ s⁻¹, r_co=1.44 nm and r_d=1.07 nm. The results in the [Ru(en)₃]³⁺ system were compared with those obtained in the [Ru(NH₃)₆]³⁺ system.     

Effective immobilisation of lipase to enhance esterification potential and reusability

A commercial lipase, “Lipolase T100”, was immobilised onto silica by means of physical adsorption. The silica-bound lipase was subsequently exposed to 1 vol. % glutaraldehyde (pentane-1,5-dial). The silica was loaded repeatedly with the Lipolase T100 in 0.05 M Tris buffer (pH 8.5) until saturation was achieved. During the 1st, 2nd, 3rd, 4th, and 5th cycles of loading of silica with the enzyme, the protein-binding on the silica achieved 51.73 %, 48.27 %, 26.92 %, 10.73 %, and 4.29 %, respectively. The synthesis of methyl salicylate (methyl 2-hydroxybenzoate) and linalyl ferulate (3,7-dimethylocta-1,6-dien-3-yl 4-hydroxy-3-methoxycinnamate) carried out at 45°C under shaking with mole ratios of 200 mM of acid and 500 mM alcohol in DMSO using 15 mg mL^?1 of hyper-activated biocatalyst resulted in yield(s) of 77.2 % of methyl salicylate and 65.3 % of linalyl ferulate in the presence of molecular sieves. The hyper-activated biocatalyst was more efficient than the previously reported silica-bound lipase with minimum leaching of the enzyme from the reaction mixture. The K _m and V _max of the free (0.142 mM and 38.31 μmol min^?1 mL^?1, respectively) and silica-bound lipase (0.043 mM and 26.32 μmol min^?1 mg^?1, respectively) were determined for the hydrolysis of p-NPP. During repeated esterification studies using silica-bound lipase, yields of 50.1 % of methyl salicylate after the 5th cycle, and 53.9 % of linalyl ferulate after the 7th cycle of esterification were recorded. In the presence of molecular sieves (30 mg mL^?1) in the reaction mixture, the maximum syntheses of methyl salicylate (77.2 %) and linalyl ferulate (65.3 %) were also observed. In a volumetric batch scale-up, when the reaction volume was increased to 50 mL, 44.9 % and 31.4 % yields of methyl salicylate and linalyl ferulate, respectively, were achieved.     

Extract from Eugenia punicifolia is an Antioxidant and Inhibits Enzymes Related to Metabolic Syndrome

The present study aimed to investigate in vitro biological activities of extract of Eugenia punicifolia leaves (EEP), emphasizing the inhibitory activity of enzymes related to metabolic syndrome and its antioxidant effects. The antioxidant activity was analyzed by free radicals scavengers in vitro assays: DPPH·, ABTS^·+, O₂ ^·?, and NO· and a cell-based assay. EEP were tested in inhibitory colorimetric assays using α-amylase, α-glucosidase, xanthine oxidase, and pancreatic lipase enzymes. The EEP exhibited activity in ABTS^·+, DPPH·, and O₂ ^·? scavenger (IC₅₀?=?10.5?±?1.2, 28.84?±?0.54, and 38.12?±?2.6 μg/mL), respectively. EEP did not show cytotoxic effects, and it showed antioxidant activity in cells in a concentration-dependent manner. EEP exhibited inhibition of α-amylase, α-glucosidase, and xanthine oxidase activities in vitro assays (IC₅₀?=?122.8?±?6.3; 2.9?±?0.1; 23.5?±?2.6), respectively; however, EEP did not inhibit the lipase activity. The findings supported that extract of E. punicifolia leaves is a natural antioxidant and inhibitor of enzymes, such as α-amylase, α-glucosidase, and xanthine oxidase, which can result in a reduction in the carbohydrate absorption rate and decrease of risks factors of cardiovascular disease, thereby providing a novel dietary opportunity for the prevention of metabolic syndrome.     

A comparison of lipase and trypsin encapsulated in mesoporous materials with varying pore sizes and pH conditions

Immobilized enzymes have an advantage over enzymes free in solution in that they are easily recovered after completed reaction. In addition, immobilization often gives enhanced stability. Entrapment of an enzyme in the pores of a mesoporous material is an attractive procedure since the enzyme is immobilized without any covalent bonding to a support which may be detrimental to the catalytic performance. The objective of this work is to compare the encapsulation and catalytic performance of lipase from Mucor miehei and trypsin from bovine pancreas, two hydrolases with rather dissimilar properties and structures. We also demonstrate the importance of the pore dimensions and the pH for proper function of the encapsulated enzyme. Mesoporous silica particles (SBA-15) with three different pore sizes (50 Å, 60 Å and 89 Å) were synthesized and hexagonal structures with narrow pore size distributions were confirmed with TEM, SAXS and N₂-adsorption. Lipase and trypsin were encapsulated separately in the silica particles and the results indicate distinct differences between the two enzymes, both in loading capacity and catalytic activity. For trypsin the encapsulation rate and the loading capacity were large with a maximum reached at pH 7.6. The largest product yield was obtained with the particles with 60 Å pores, however, the yield was significantly lower than with free trypsin. For lipase optimal encapsulation rate and loading capacity were reached with the particles with 89 Å pores at pH 6.0 but were low compared to trypsin. However, the catalytic activity of the encapsulated lipase was more than twice as large as for free lipase, which can be explained by an interfacial activation of lipase at the silica surface.