Immobilization of lipase within carbon nanotube–silica composites for non-aqueous reaction systems

The immobilization of lipases within sol–gel derived silica, using multi-walled carbon nanotubes (MWNTs) as additives in order to protect the inactivation of lipase during sol–gel process and to enhance the stability of lipase, was investigated. Three sol–gel immobilized lipases (Candida rugosa, Candida antarctica type B, Thermomyces lanuginosus) with 0.33% (w/w) MWNT showed much higher activities than lipase immobilized without MWNT. The influence of MWNT content and MWNT shortened by acid treatment in the sol–gel process on the activity and stability of immobilized C. rugosa lipase was also studied. In hydrolysis reaction, immobilized lipase containing 1.1% pristine MWNT showed 7 times higher activity than lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT showed 10 times higher activity in esterification reaction, compared with lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT retained 96% of initial activity after 5 times reuse, while the lipase immobilized without MWNT was fully inactivated under the same condition.     

Combination of bioimprinting and silane precursor alkyls improved the activity of sol–gel-encapsulated lipase

Bioimprinting and sol–gel encapsulation of lipases by silane precursors are efficient methods of enhancing lipase performance in non-aqueous medium. The correlation between bioimprinting, the alkyl-chain length of silane precursors, and the catalytic activity of gel-encapsulated lipase was investigated using a series of silane precursors: methyltrimethoxysilane (MTMS), vinyltrimethoxysilane (VTMOS), vinyltriethoxysilane (VTEOS), and n-octyltrimethoxysilane (OTMOS). The optimal parameters for lipase immobilization were also determined. Both bioimprinting and increasing the chain-length of alkyl groups, apparently by increasing hydrophobicity, significantly improved the specific activity and the total activity of the immobilized lipase. Compared to a non-imprinted MTMS/TMOS gel, the specific activity of an imprinted OTMOS/TMOS gel improved 14.4-fold, and the total activity improved 6.8-fold. Nitrogen adsorption–desorption assays and gel matrix surface characterization showed that the bioimprinting molecule and the hydrophobic alkyl groups of silane triggered lipase to change from the closed to the open conformation, and contributed to creating sol–gel matrices that were more porous and with less mass transfer resistance structure, apparently improving the activity of encapsulated lipase.     

Influence of ionic liquids as additives on sol–gel immobilized lipase

The immobilization of lipase from Candida rugosa, using ionic liquids as additives to protect the inactivation of lipase by released alcohol and shrinking of gel during sol–gel process, was investigated. The influence of various factors, such as structure of ionic liquids, content of ionic liquids and types of precursor in the sol–gel process on the activity and stability of immobilized lipase was also studied. The highest hydrolytic activity of immobilized lipase was obtained when the hydrophilic ionic liquid, [C₂mim][BF₄], was used as an additive, while the highest stability of immobilized lipase was obtained by using hydrophobic ionic liquid, [C₁₆mim][Tf₂N]. Therefore, the binary mixtures of these ionic liquids as additives were used to obtain the optimal immobilized lipase, which shows both high activity and stability. The hydrolysis and esterification activities of lipase co-immobilized with the mixture of 1:1 at molar ratio of [C₂mim][BF₄] and [C₁₆mim][Tf₂N] were 10-fold and 14-fold greater than in silica gel without ionic liquids (ILs), respectively. After 5 days incubation of this immobilized lipase in n-hexane at 50 °C, 84% of initial activity was remained, while the residual activity of the lipase immobilized without ILs was 28%.     

Isolation and characterization of an extracellular lipase from Mucor sp strain isolated from palm fruit

During our screening of lipolytic fungus which may play a role in the acidification of palm oil, we have recently isolated a Mucor sp strain. Culture conditions were optimized and the highest lipase production amounting to 57 U/ml was achieved after 6 days of cultivation. The extracellular lipase was purified 1050-fold by ammonium sulfate precipitation, carboxymethyl–sephadex chromatography and Sephadex G75 gel filtration to a final specific activity of 6600 IU/mg. The molecular weight of the homogenous lipase was determined about 42 kDa by gel filtration and SDS–polyacrylamide gel electrophoresis. The purified lipase was determined as a glycoprotein with a pI of 6.2. The Nt sequence was determined as AspGluIleGluThrValGlyXPheThrMetAspLeuProProAsnProPro and showed no homology with the sequences of the known lipases suggesting that the enzyme may be a new lipase. The purified lipase hydrolyzed both synthetic and natural triglycerides with the optimal activity recorded on trioctanoin and sunflower oil, respectively. Its activity was strongly inhibited by Triton X-100 and SDS. Metal ions such as Fe³⁺, Fe²⁺ and Hg²⁺ also decreased the lipase activity.     

Selective measurement of triacylglycerol lipase activities in pig post-heparin plasma.

Immunochemical methods for the selective measurement of pig post-heparin plasma lipoprotein lipase and hepatic lipase are described and validated. A simple two step purification method for porcine hepatic lipase from hepatic perfusate based on affinity chromatography and gel filtration is reported. The activity of the post-heparin plasma lipoprotein lipase and hepatic lipase in swine is reported. It is demonstrated that fasting decreases the activity of post-heparin plasma lipoprotein lipase activity more than two-fold while it does not affect the hepatic lipase activity significantly.     

Some properties of triacylglycerol lipase in chicken erythrocytes

Membrane-bound acid lipase was found in the chicken erythrocytes ghosts, having an optimum pH of 4.5. The membrane-bound lipase showed its maximum activity at 38 degrees C, and it was stable below 45 degrees C. The bound lipase was activated by octyl glucoside and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), but it was markedly inhibited by chicken serum. The lipase was solubilized with CHAPS, but the solubilized lipase was labile. The solubilized lipase showed its maximum activity at pH 4.5, 38 degrees C, and it was stable below 40 degrees C. The solubilized lipase was activated by CHAPS and octyl glucoside. The lipase was markedly inhibited by chicken serum. The solubilized lipase have a molecular mass more than 230,000 by Sephacryl S-300 gel filtration.     

Purification of extracellular lipase from Pseudomonas aeruginosa.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.     

Purification and characterization of a lipase from Staphylococcus aureus

An extracellular lipase from Staphylococcus aureus (strain FN 37) was purified to homogeneity. A cell-free culture broth was subjected to ammonium sulphate precipitation, and the lipase was isolated from the resuspended pellet by adsorption chromatography on octyl-Sepharose. The purification was 957-fold, and the recovery of the octyl-Sepharose chromatography was about 100%. The specific activity of the purified lipase was 546 mU of lipase activity per micrograms protein. The purity of the final product was documented by SDS-polyacrylamide gel electrophoresis in which a homogeneous protein band of 43 kDa was found. In gel chromatography on Sephadex G-200 the lipase eluted as a homogeneous peak with an apparent molecular mass of 110 kDa, suggesting that the lipase may exist as an oligomer in physiological media. Analysis of the amino-acid composition revealed a predominance of polar, non-charged amino acids, with serine accounting for 24 mol% of the amino-acid residues.     

Properties of the lipid body lipase of Pinus eduUs and electrophoretic purification of its 64 kDa subunit

Lipase (triacyiglycerol acylhydrolase, EC 3.1.1.3) in oilseeds can be associated with either the lipid body or glyoxysomal membrane and can have various pH optima and substrate specificities. There is conflicting evidence for the subcellular location of lipase in gymnosperms, and little information exists on its activity characteristics. In this report, Pinus edulis (pinyon) was found to have an acid lipase, which was associated with the lipid body fraction and the activity of which increased during germination. Active lipase from the solubilized lipid body membrane was determined by gel permeation chromatography to have a molecular weight of 260 000. Further attempts to purify the active enzyme were unsuccessful. A lipid body membrane protein of 64 kDa which increased in parallel with lipase activity during germination was isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. excised, and polyclonal antibodies were made against it. Using these antibodies, active lipase was immunoprecipitated from solution, thus indicating that the 64 kDa protein is a subunit of the lipase. Pinus edulis lipase had a pH optimum of ca 4.5. and it exhibited little specificity for triacyiglycerol substrates in vitro. The lipase was specific in activity against fluorometric substrates, with the highest activity against methyl-umbelliferyl laurale. Lipase activity was inhibited by high concentrations of non-ionic detergent. This lipid body acid lipase appears to be primarily responsible for lipid hydrolysis during pinyon germination.     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Optimization of lipase pretreatment prior to lipase immobilization to prevent loss of activity

In our previous work, a method of pretreating lipase was developed to prevent loss of its activity during covalent immobilization. In this study, Rhizopus oryzae lipase was pretreated before immobilization and then immobilized on a silica gel surface. The effects of the various materials and conditions used in the pretreatment stage on the activity of immobilized lipase were investigated. Immobilized lipase pretreated with 0.1% of soybean oil had better activity than those pretreated with other materials. The optimal temperature, agitation speed, and pretreating time for lipase pretreatment were determined to be 40 degrees C, 200 rpm, and 45 min, respectively. The activity of immobilized soybean oil pretreated lipase was 630 U/g matrix, which is 20 times higher than that of immobilized non-pretreated lipase. In addition, immobilized lipase activity was maintained at levels exceeding 90% of its original activity after 10 reuses.     

Pretreatment of lipase with soybean oil before immobilization to prevent loss of activity

Lipase was pretreated with soybean oil in order to allow fatty acids to bond to the active site before immobilization. This pretreated lipase exhibited steric hindrance around the active site such that during immobilization, covalent bonds were formed between the carrier and the lipase region far from the active site. The activity of the pretreated lipase immobilized covalently on a silica gel was 530 U/g-matrix, which is 16 times higher than that of the immobilized non-pretreated lipase. In addition, the immobilized lipase activity was maintained at levels exceeding 90% of its original activity after 10 reuses.     

Lipoprotein lipase in cultured heart cells: characteristics and cellular location.

Lipase activity extracted from cultured neonatal rat heart cells was characterized and identified as lipoprotein lipase. Enzyme activity was stimulated by human apoC-II and rat serum; serum stimulation was prevented by human apoC-I and by apoC-II. Lipolysis was maximal at pH 8.0 and was inhibited by protamine sulfate, NaCl, and high concentrations of heparin. About 50% of heart cell lipase activity applied to heparin-Sepharose bound to the gel and was eluted with a NaCl gradient. A peak of lipase activity was observed at 0.84 M NaCl. Neonatal rat heart cells in culture are a mixture of muscle and non-muscle cells. To determine the cellular location of the lipoprotein lipase, enzyme activity and muscle cell content of the cultures were determined. Myosin ATPase was used as an index of muscle cell content since ATPase specific activity correlated (r = +0.97) with muscle cell content determined immunofluorescently. When muscle cell content of cultures was decreased or increased by differential plating, lipase specific activity was constant. Moreover, lipase specific activity was constant during culture growth despite a decrease in muscle cell content. It was concluded that lipoprotein lipase activity of cultured heart cells is not associated solely with either muscle or non-muslce cells.     

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system

Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 mug/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.     

Immobilization of lipase in composites of gelatin and polystyrene via an emulsion-gel pathway

Summary A new method for the immobilization of lipase within composites of polystyrene and gelatin is suggested. First, an emulsion of styrene (containing an initiator) in an aqueous solution of gelatin (containing a dispersant) is prepared with mechanical stirring at 50°C. An aqueous solution of lipase is added (at room temperature) under stirring to the gel-like emulsion previously prepared. The polymerization of the gel containing lipase was carried out at room temperature for four days. The activity of the immobilized lipase in the hydrolysis reaction of triacetin was investigated. The activity depends on the content of gelatin within the composite.     

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.     

Biosynthesis of lipase in the scutellum of maize kernel

In the scutellum of maize kernel after imbibition, lipase activity increased rapidly, concomitant with the decrease in storage triacylglycerols. The enzyme activity peaked at day 6, but remained at the same level from day 6-10 when most of the triacylglycerols had been depleted. By in vitro translation with extracted RNAs followed by immunoprecipitation, and by resolving the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lipase was found to be de novo synthesized in postgermination. The enzyme was synthesized by RNAs extracted from free polyribosomes and not from bound polyribosomes. Both in vitro and in vivo synthesized lipase had the same Mr of 65,000 as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as had the purified authentic enzyme; thus there was no appreciable co- or post-translational processing of the enzyme. Lipase-specific mRNA was present only between day 2-6 after imbibition. At day 6 when lipolysis was most active, more than 60% of the lipase activity was recovered in the lipid body fraction and specifically associated with the organelle membrane. From day 6-10, the lipase activity gradually shifted from the lipid body fraction to other subcellular fractions, including the 10,000 X g pellet, the 120,000 X g pellet, and the 120,000 X g supernatant. Lipase in these subcellular fractions was attributed to represent the enzyme associated with membrane ghosts of the lipid bodies which were fusing with the fragile cell vacuoles; such fusions were observed in situ by electron microscopy.     

Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship.

Activatable cholesterol esterase and triacylglycerol lipase of rat adrenal were 58-69% recovered in the 100 000 X g supernatant fraction. Activatable triacylglycerol lipase activity was differentiated from the activity of acid lipase and lipoprotein lipase also found in this fraction. Cholesterol esterase was activated 39.7 +/- 13.6% (S.D.) and triacylglycerol lipase 11.9 +/- 2.9% in a reaction dependent on ATP, cyclic AMP, and protein kinase. The two activities were shown by differential inhibition by an organophosphate, and by partial separation on salting out, to be largely due to separate enzymes. The two enzymes bound tightly to substrate emulsions with quantitatively similar distribution between competing emulsions, suggesting concerted binding. Coinciding gel filtration patterns reinforced, The hypothesis of a lipase complex. Cholesterol esterase comprised a major component of higher apparent Km for substrate and molecular weight 3-10(5)-6-10(5) by gel filtration and a minor component of lower apparent Km and heterogeneous molecular weight above 1 million, which was found mostly in complex and lipid.     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.     

Purification and molecular characterization of bovine pregastric lipase

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55000. The Mr on gel filtration was 44-48000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing approximately 10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase.