Activity Of Free Lipase Research Articles

The preparation of Diacylglycerol-rich soybean oil by acetylated modification of arachin nanoparticles for W/O Pickering emulsion system.

Pickering emulsion catalytic system (PEC) stabilized by nanoparticles is an efficient catalytic platform. Herein, a high-performance PEC was constructed by acetylated modification of arachin nanoparticles (AAPs). The results showed the pI of arachin was decreased from pH 5.5 to pH 3.5. The surface hydrophobicity index was significantly increased (from 56.28±4.23 to 120.77±0.79) after acetylated modification. The three-phase contact angle of AAPs was 91.20±0.98°. AAPs were used as lipase immobilization carriers to increase the activity of free lipase fabricating lipase-AAPs. The immobilization efficiency and activity of lipase-AAPs were 12.95±0.03% and 1.74±0.07 U/mg, respectively. Enzymatic reaction kinetics showed that Vm of lipase-AAPs was twice of free lipase. Km was 1/5 of free lipase. The catalytic efficiency of PEC to prepare DAG was 2.36 times of biphasic catalytic system (BCS). This work provided a promising way to promote the efficiency of DAG preparation.

Silica@lipase hybrid biocatalysts with superior activity by mimetic biomineralization in oil/water two-phase system for hydrolysis of soybean oil

Enzymatic hydrolysis of vegetable oils is crucial for its potential application in foods and drugs. However, the poor activity of free lipase hinders the reaction. Herein, inspired by lipase interface activation, a novel silica@lipase hybrid biocatalyst with a superior catalytic activity for hydrolysis of vegetable oils was developed though the sol-gel biosilicification approach in the oleic acid/water two-phase system. The resultant silica@lipase hybrid biocatalyst exhibited 13.5-fold higher activity than free lipase, and excellent storage stability. After 100 days of storage, the free lipase absolutely loss hydrolytic activity whereas the silica@lipase hybrid biocatalyst still retained 197.4% of its initial activity. In particular, hydrolysis yield of soybean oil by the hybrid biocatalyst reached 98.8% within only 4 h, which is the fastest value recorded in the current literature. After 5 consecutive cycles, hydrolysis yield still retained up to 63%. Therefore, this strategy provides a facile and efficient method for preparing lipase hybrid biocatalysts with high hydrolysis activity.

Improved synthesis of dietary triglycerides by using lipase supported on clay carriers.

In the search for healthier and more functional foods, dietary triglycerides (TAGs) have played a prominent role in the food industry. The objective of this work was to evaluate new clay supports to immobilize lipase from Rhizopus oryzae and use it in the synthesis of TAGs. The immobilization of lipase by physical adsorption was carried out, determining the hydrolytic activity, esterification, immobilization yield, thermal stability, and kinetic and thermodynamic parameters. Afterwards, acidolysis was carried out and the product characterized as to composition, acidity index (AI), peroxide index (PI), and analysis of the sn-2 position. The mean hydrolytic activity of free lipase was 23,568.43±559.20 Ug-1 and for lipase immobilized in acidified kaolin and diatomite, 1409.04±51.07 and 1235.67±9.56Ug-1 ; immobilization yield of 23.78% and 20.21% and esterification activity of 1827.99 and 1722.66Ug-1 , respectively, maintaining more than 60% of the relative activity after 8 h of incubation at different temperatures. The reaction using the derivative immobilized in acidified diatomite resulted in the degree of incorporation (DI) 41.53%±3.99%. From the results, the potential of diatomite and kaolin as support in the immobilization of lipase for the synthesis of dietary TAGs was verified.

Fe3O4-PDA-Lipase as Surface Functionalized Nano Biocatalyst for the Production of Biodiesel Using Waste Cooking Oil as Feedstock: Characterization and Process Optimization

Synthesis of surface modified/multi-functional nanoparticles has become a vital research area of material science. In the present work, iron oxide (Fe3O4) nanoparticles prepared by solvo-thermal method were functionalized by polydopamine. The catechol groups of polydopamine at the surface of nanoparticles provided the sites for the attachment of Aspergillus terreus AH-F2 lipase through adsorption, Schiff base and Michael addition mechanisms. The strategy was revealed to be facile and efficacious, as lipase immobilized on magnetic nanoparticles grant the edge of ease in recovery with utilizing external magnet and reusability of lipase. Maximum activity of free lipase was estimated to be 18.32 U/mg/min while activity of Fe3O4-PDA-Lipase was 17.82 U/mg/min (showing 97.27% residual activity). The lipase immobilized on polydopamine coated iron oxide (Fe3O4_PDA_Lipase) revealed better adoptability towards higher levels of temperature/pH comparative to free lipase. The synthesized (Fe3O4_PDA_Lipase) catalyst was employed for the preparation of biodiesel from waste cooking oil by enzymatic transesterification. Five factors response surface methodology was adopted for optimizing reaction conditions. The highest yield of biodiesel (92%) was achieved at 10% Fe3O4_PDA_Lipase percentage concentration, 6:1 CH3OH to oil ratio, 37 °C temperature, 0.6% water content and 30 h of reaction time. The Fe3O4-PDA-Lipase activity was not very affected after first four cycles and retained 25.79% of its initial activity after seven cycles. The nanoparticles were characterized by FTIR (Fourier transfer infrared) Spectroscopy, XRD (X-ray diffraction) and TEM (transmission electron microscopy), grafting of polydopamine on nanoparticles was confirmed by FTIR and formation of biodiesel was evaluated by FTIR and GC-MS (gas chromatography-mass spectrometry) analysis.

Study of Catalytic Activity of Lipase and Lipase-Chitosan Complexes in Dynamics

Pancreatic porcine lipase (PPL) is the unique enzyme in numerous biochemical processes for human and animals. Stability in time is the critical point for many enzymes in relation to their further applications. The effect of chitosan on the activity of PPL during 2 months was investigated. Materials and Methods: Potentiometric method was used to study the catalytic activity of enzymes that based on measuring of the potential an electrode immersed in a triacetin (substrate) solution by titration with 0.01 M NaOH. A laboratory pH-stat with combined pH electrode was used for the measurements of this potential in the presence of PPL or PPL-chitosan complexes. Standard experimental conditions: 40 ºC, pH 7.0. Results: The following PPL activity values (data given in % to the activity of free lipase) vs. time (in 10 min. intervals) were obtained: 100%, 97.4%, 89.9%, 82.9%, 77.9% and 75.9% after 10, 20, 30, 40, 50 and 60 min., respectively. The PPL catalytic activity decreased at about ¼ to ½ of the initial values after 7 or 14 days, 1 or 2 months storage, although there were differences in the decline dynamics. The complex PPL:Chit=50:1 has better stabilizing properties as compared to other complexes; does not strongly inhibit lipase and requires a small amount of carrier (chitosan) for its formation. Conclusions: The complex PPL:Chit=25:1 is less effective as the PPL:Chit=50:1 complex, but both can be applicable in some industrial processes.

Thermal stability and reusability of home-made co-immobilized lipase from Mucor miehei in polyurethane foam for the production of bio-flavor

In this study, polyurethane foam (PUF) was used as a matrix to immobilize M. miehei lipase through a modified substance coating consisting of lecithin, gelatine, polyethylene glycol (PEG), MgCl2, known as co-immobilized. Co-immobilized lipase application was carried out on bio-flavor manufacturing through hydrolysis and esterification reactions. Fatty acid was gained from hydrolysis of glyceride coconut oil (CNO) containing main components of C8 to C16. Furthermore, esterification of fatty acids and citronellol from citronella oil was employed. The remaining triglycerides (TAGs) in hydrolysis were analysed using LC-MS/MS. While bio-flavor was analysed using GC-FID. Maximum bio-flavor production was obtained at 40 °C and fatty acids lauric acid/citronellol mole ratio of 1:5. The remaining activity of free lipase, immobilized lipase on PUF, and immobilized lipase on co-coated PUF at 50 °C for 50 min was 0 %, 34 %, and 54 %, respectively. Immobilized lipase on co-coated PUF was able to reuse up to 5 cycles. The maximum conversion of bio-flavor was attained using lipase on modified co-immobilized. The use of co-immobilized lipase of the non-commercial source used in this study could be a promising method for a zero-waste bio-flavor production in a mild and solvent-free process to support low-cost and green technology.

Mycelium-bound lipase from a locally isolated strain of Geotrichum candidum.

Mycelium-bound lipase (MBL), from a locally isolated Geotrichum candidum strain, was produced and characterized as a natural immobilized lipase. A time course study of its lipolytic activity in 1 L liquid broth revealed the maximum MBL activity at 4 h for mycelium cells harvested after 54 h. The yield and specific activity of MBL were 3.87 g/L dry weight and 508.33 U/g protein, respectively, while less than 0.2 U/mL lipase activity was detected in the culture supernatant. Prolonged incubation caused release of the bound lipase into the growth medium. The growth pattern of G. candidum, and production and properties of MBL were not affected by the scale. The stability of mycelia harboring lipase (MBL), harvested and lyophilized after 54 h, studied at 4 °C depicted a loss of 4.3% and 30% in MBL activity after 1 and 8 months, while the activity of free lipase was totally lost after 14 days of storage. The MBL from G. candidum displayed high substrate selectivity for unsaturated fatty acids containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.

The study on effective immobilization of lipase on functionalized bentonites and their properties

• The immobilized lipase on composite bentonite shows the highest activity. • Hydrophobicity and enlarged catalytic interface of the supports activate lipase. • The immobilized lipase shows excellent storage and operational stabilities. • The lower K m of immobilized lipase confirmed the improved affinity for olive oil. Three different functionalized bentonites including acid activated bentonite ( B a ), organically modified bentonite with cetyltrimethyl ammonium bromide ( B CTMAB ) and the composite by acid activation and organo-modification ( B a-CTMAB ) were prepared, and used for immobilization of lipase from bovine pancreatic lipase by adsorption. The amount of lipase adsorbed on the functionalized bentonites was in the following sequence: B a > B CTMAB > B a-CTMAB , showing the strongest affinity of B a for lipase among the three supports. However, the immobilized lipase on B a-CTMAB showed the highest activity in the hydrolysis of olive oil by 1.67 times of activity of free lipase due to the hydrophobically interfacial activation and enlarged catalytic interface. While, the activity of immobilized lipase on B a was lower than 20% of free lipase’s activity due to the absence of hydrophobic activation and negative impact of excessive hydrogen ions on the surface. The K m values for the immobilized lipase on B a-CTMAB (0.054 g/mL) and B CTMAB (0.074 g/mL) were both lower than that of free lipase (0.115 g/mL), and the V max values were higher for the immobilized lipases, exhibiting a higher affinity of the immobilized lipase toward olive oil than free lipase. In comparison to free lipase, the better resistance to heating inactivation, storage stability and reusability of the immobilized lipases on B a-CTMAB and B CTMAB were also obtained. The results show that the efficient and stable biocatalysts for industrial application can be prepared by using the low-cost bentonite mineral as the supports.

Improvement of catalytic activity and stability of lipase by immobilization on organobentonite

Organobentonites were prepared from Na-bentonite (NB) by intercalation with cetyltrimethyl ammonium bromide (CTMAB), and used to support the lipase from porcine pancreas. The immobilized lipase showed higher activity in hydrolysis of olive oil than free lipase, with the highest specific activity of 347.8Ug−1 protein for lipase immobilized on bentonite-CTMAB100 (BC100-lipase). The apparent activation energy (Ea) of catalysis declined from 29.25kJmol−1 for free lipase to 16.31kJmol−1 for BC100-lipase, resulting in a higher catalytic efficiency of lipase. The improvement of catalytic activity was attributed to the interfacial activation by hydrophobic microenvironment and highly scattered surface created by organobentonite, according to the structural and morphological characterizations by Fourier transform infrared spectroscopy (FTIR) and Transmission electron microscopy (TEM). The immobilized lipase showed better resistance to pH and heating inactivation in comparison to the free lipase. After the storage of 8 days, BC100-lipase retained 80.7% of initial activity, which was 2.7 times higher than the residual activity of free lipase. Particularly, BC100-lipase kept 82.5% of initial activity after 6 continuous recycles under vigorous agitation, showing the excellent stability of the immobilized lipase by adsorption.

Immobilization of lipase from Candida rugosa into copolymer hydrogels of poly(N-isopropylacrylamide-co-itaconic acid) synthesized in the presence of surfactants

To overcome the problems of free enzyme application as catalysts in chemical reactions, i.e. high costs of isolation and purification processes, high sensitivity to process conditions, insufficient enzyme stability etc., a different immobilization techniques are to be used. Immobilization to/within solid support improves enzyme stability decreasing its denaturation. This paper deals with hydrogels of N-isopropylacrylamide and itaconic acid with incorporated nonionic surfactants (Triton X-100, Brij 30 and Tween 80) synthesized in distilled water at room temperature by free radical polymerization. These hydrogels were used as supports for immobilization of enzyme, lipase from Candida rugosa by post-entrapment method. The aim was to investigate the effect of the nonionic surfactants on the lipase binding capacity, as well as on its hydrolytic activity. In order to characterize the obtained hydrogels FT-IR analysis has been performed. Further, the swelling behaviour of these samples in buffer solution of pH 6.80 has been investigated. The dynamic - mechanical properties of hydrogels and detailed have been studied, too. The immobilized lipase showed somewhat reduced hydrolytic activity, as compared to the activity of free lipase as well as in comparison to the lipase immobilized to the reference sample (sample synthesized under the same polymerization conditions, but in the absence of surfactants). It was concluded that the addition of surfactants increased the hydrogel mesh size. The surfactant addition did not affect the dynamic - mechanical properties of the investigated hydrogels. The largest percentage of specific activity and yield of activity were presented by the reference sample, too. It is obvious that the absence of surfactants charged groups has no influence on the lipase binding capacity, and the obtained activity yields are to be expected.

Effects of Polyelectrolyte Complex Micelles and Their Components on the Enzymatic Activity of Lipase

The enzymatic activity of Hl-lipase embedded in complexes of poly-2-methylvinylpyridinium-co-poly(ethylene oxide) (P2MVP(41)-PEO(205)) and poly(acrylic acid)(PAA(139)) is studied as a function of the PAA(139) + P2MVP(41)-PEO(205) complex composition. The measurements revealed that there are several factors that influence the enzymatic activity. When incorporated in micelles, the activity of lipase is increased, which suggests that the micelles favor the active state. The activity may further increase because the substrate tends to accumulate to the micelles. It is found that the presence of PAA(139) alone also increases the enzymatic activity somewhat. Increasing of the ionic strength decreases the enzymatic activity in all systems. However, at ionic strengths where the micelles are disintegrated (>0.5 M), the activity of lipase in the presence of both polyelectrolytes is still higher than the activity of free lipase. At 0.7 M NaCl it was found that lipase in the presence of (just) P2MVP(41)-PEO(205) is more active than lipase without this additive.

Biodiesel Production from Crude Sunflower Oil and Crude Jatropha Oil Using Immobilized Lipase

The aim of this research was to study the immobilization of lipase on chitosan by adsorption in order to examine the use of lipase as a catalyst in the transesterification of crude sunflower oil and Jatropha oil. The highest activity of immobilized lipase was found to be 857 U/g, and it corresponded to a glutaraldehyde concentration of 0.05%, pH 7, and a temperature of 37 °C. On the other hand, the activity of free lipase was 2,136 U/g. The optimum conditions for the transesterification of crude sunflower oil were as follows: oil-to-methanol molar ratio, 1:3; immobilized lipase, 100% (w/w of oil); water content, 20% (w/w of oil); shaking speed, 200 rpm; and temperature, 50 °C for 24 h. The maximum amount of methyl esters produced was 25%. When free lipase was used as the catalyst under the same conditions, the amount of methyl esters produced was 33%. The transesterification of Jatropha oil was also studied to compare the activities of the two types of lipases. Both types of lipases activate the tranesterification of sunflower oil more easily than the transesterification of Jatropha oil.

Comparative study of performances of lipase immobilized asymmetric polysulfone and polyether sulfone membranes in olive oil hydrolysis

In the present study, lipase was immobilized via glutaraldehyde crosslinking on the polysulfone and polyether sulfone asymmetric membranes. The results indicated that the overall immobilization of lipase is related to the hydrophobicity of the membrane material and thus higher immobilization is achieved for polysulfone membrane. The evidence of immobilization is done by XRD, SEM, contact angle and porometric studies. Hydrolytic activity of lipase in immobilized form is determined by hydrolyzing olive oil and compared with hydrolytic activity of free lipase. The effect of different reaction parameters viz., temperature, pH, substrate concentration, and incubation time on the lipase activity is investigated. The observed maximum reaction rate ( V max) and Michaelis–Menten constant ( K m) of polysulfone and polyether sulfone is determined.

Crosslinked enzyme aggregates in hierarchically‐ordered mesoporous silica: A simple and effective method for enzyme stabilization

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for 2 weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.

Effect of immobilization site and membrane materials on multiphasic enantiocatalytic enzyme membrane reactors.

In the experimental work reported here the optical resolution of racemic naproxen methyl ester with crude lipase immobilized in a membrane reactor was studied. The multiphasic enantiocatalytic enzyme membrane reactor consisted of an organic phase that dissolved the naproxen methyl ester, a lipase-loaded membrane, and an aqueous phase that extracted the reaction product. Lipase preferentially converted the (S)-naproxen methyl ester to (S)-naproxen acid that was simultaneously separated by a membrane. The effect of the immobilization site and membrane type on the performance of the enzyme membrane reactor was studied. Capillary polyamide membrane with 10 kDa nominal molecular weight cutoff (NMWCO) and polysulfone membrane with 30 kDa NMWCO were applied, with lipase loaded in the sponge layer or on a membrane thin layer. With various immobilization sites and membrane types, the enzyme membrane reactors showed different productivity and enantioselectivity resulting from the varying amounts of immobilized enzyme and varying microenvironment of the hydrolysis reaction. Higher amounts of immobilized enzyme led to increased productivity and generally higher enantioslectivity of the membrane reactor. It seems that the location of organic/aqueous interface on membrane, which plays an important role in a multiphasic enzyme membrane reactor, was influenced by the immobilization site and membrane type and this affected the productivity and enantioselectivity. As much as 90% enantioexcess was obtained with a polyamide membrane, and lower values with a polysulfone membrane. A sponge layer of polyamide membrane is the preferred immobilization site for its higher productivity than a thin membrane layer, and for its higher enantioselectivity than a polysulfone membrane. In comparison with the unstable hydrolysis activity of free lipase in stirred tank reactor, a stable lipase activity can be obtained with the lipase-immobilized membrane reactor, irrespective of whether a polyamide or polysulfone membrane is used.

Mycelium-Bound Lipase from a Locally Isolated Strain of Aspergillus flavusLink: Pattern and Factors Involved in its Production

Aspergillus flavus produces a lipase (EC 3.1.1.3) which is partly bound to the mycelium during growth. The production of the mycelium-bound lipase is concomitant with growth, and declines when growth ceases. Maximum productivity of the enzyme is obtained when the culture is incubated at 30°C, an initial culture pH of 6·5 and with 2% (w/v) each of corn oil and yeast extract as carbon and organic nitrogen source. Yeast extract affects not only the production of lipase but also the secretion of proteases into the culture medium. Production of the latter enzymes, which inactivate the free lipase, is enhanced by adding yeast extract (1–2%, w/v) to the culture medium. However, at 5% (w/v) yeast extract concentration, proteolytic activity is not detected and consequently, the activity of free lipase may easily be measured. Free lipase activity can easily be detected when 0·001 mol dm−3 EDTA is added to the culture medium. The presence of the chelating agent enhances the production and maintains the stability of the extracted mycelium-bound lipase.

Immobilization of lipase for effective interesterification of fats and oils in organic solvent

In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8-fold that of the original activity of free lipase when the spacer was 7-aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half-life of 97 days.