Showing papers on "Lipase published in 2001"

Improved Preparation and Use of Room-Temperature Ionic Liquids in Lipase-Catalyzed Enantio- and Regioselective Acylations

Abstract: Polar organic solvents such as methanol or N-methylformamide inactivate lipases. Although ionic liquids such as 3-alkyl-1-methylimidazolium tetrafluoroborates have polarities similar to these polar organic solvents, they do not inactivate lipases. To get reliable lipase-catalyzed reactions in ionic liquids, we modified their preparation by adding a wash with aqueous sodium carbonate. Lipase-catalyzed reactions that previously did not occur in untreated ionic liquids now occur at rates comparable to those in nonpolar organic solvents such as toluene. Acetylation of 1-phenylethanol catalyzed by lipase from Pseudomonas cepacia (PCL) was as fast and as enantioselective in ionic liquids as in toluene. Ionic liquids permit reactions in a more polar solvent than previously possible. Acetylation of glucose catalyzed by lipase B from Candida antarctica (CAL-B) was more regioselective in ionic liquids because glucose is up to one hundred times more soluble in ionic liquids. Acetylation of insoluble glucose in organ...

Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase

Abstract: Transesterification reaction was performed using triglycerides and short-chain alcohol by immobilized lipase in non-aqueous conditions. The long-chain fatty acid ester, which is the product of this reaction, can be used as a diesel fuel that does not produce sulfur oxide and minimize the soot particulate. Immobilized Pseudomonas fluorescens lipase showed the highest activity in this reaction. Immobilization of lipase was carried out using porous kaolinite particle as a carrier. When methanol and ethanol were used as alcohol, organic solvent like 1,4-dioxane was required. The reaction could be performed in absence of solvent when 1-propanol and 1-butanol were used as short-chain alcohol. The activity of immobilized lipase was highly increased in comparison with free lipase because its activity sites became more effective. Immobilized enzyme could be repeatedly used without troublesome method of separation and the decrease in its activity was not largely observed.

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Abstract: Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.

Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A.

Abstract: We have previously reported that a psychrotrophic bacterium,Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase fromPseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.

Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production.

Abstract: Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomvces cerevisiae MT8-1. The gene encoding lipase from R. orvzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 degrees C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 degrres C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.

Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica.

Abstract: The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated. The rate of myristyl glucose synthesized by the enzyme was increased from 3.7 to 20.2 μmol min−1 g−1 by changing the solvent from pure tert-butanol to a mixture of tert-butanol:pyridine (55:45 v/v), by increasing the temperature from 45°C to 60°C, and by optimizing the relative amounts of glucose, myristic acid, and the enzyme preparation. Addition of more than 2% DMSO to the tert-butanol:pyridine system resulted in a reduction of enzyme activity. Lowering the water content of the enzyme preparation below 0.85% (w/w) resulted in significant decreases in enzyme activity, while increasing the water content up to 2.17% (w/w) did not significantly affect the enzyme activity. The highest yields of myristyl glucose were obtained when an excess of unsolubilized glucose was present in the reaction system. In this case, all of the initially solubilized and a significant amount of the initially unsolubilized glucose was converted to the ester within 24 h of incubation, resulting in a myristyl glucose concentration of 34 mg/mL−1. Myristic acid esters of fructose (22.3 μmol min−1 g−1), α-D-methyl-glucopyranoside (26.9 μmol min−1 g−1) and maltose (1.9 μmol min−1 g−1) could also be prepared using the tert-butanol:pyridine solvent system. No synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 483–491, 2001.

Improved enantioselectivity of a lipase by rational protein engineering.

Abstract: A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.

Production of margarine fats by enzymatic interesterification with silica-granulated Thermomyces lanuginosa lipase in a large-scale study

Abstract: Interesterification of a blend of palm stearin and coconut oil (75∶25, w/w), catalyzed by an immobilized Thermomyces lanuginosa lipase by silica granulation, Lipozyme TL IM, was studied for production of margarine fats in a 1- or 300-kg pilot-scale batch-stirred tank reactor. Parameters and reusability were investigated. The comparison was carried out between enzymatic and chemical interesterified products. Experimentally, Lipozyme TL IM had similar activity to Lipozyme IM for the interesterification of the blend. Within the range of 55–80°C, temperature had little influence on the degree of interesterification for 6-h reaction, but it had slight impact on the content of free fatty acids (FFA). Drying of Lipozyme TL IM from water content 6 to 3% did not affect its activity, whereas it greatly reduced FFA and diacylglycerol contents in the products. Lipozyme TL IM was stable in the 1-kg scale reactor at least for 11 batches and the 300-kg pilot-scale reactor at least for nine batches. Due to regiospecificity of the lipase (sn-1,3 specific), enzymatically interesterified products had different fatty acid distribution at sn-2 position from the chemically randomized products, implying the potential nutritional benefits of the new technology.

Influence of water activity and water content on sugar esters lipase-catalyzed synthesis in organic media

Abstract: Lipase-catalysed ester bond synthesis in organic media was directed by thermodynamic water activity of the reaction mixture. The effects of water activity and water content were investigated with fructose palmitate synthesis catalysed by Candida antarctica lipase. Conversion yield, initial rate of synthesis and reaction selectivity were analysed. Initial water activity of the reaction medium was fixed at a desired value by pre-equilibration with saturated salt solutions. C. antarctica lipase activity depended strongly on initial water activity value. Conversion yield and initial rate decreased with the increase of water activity. Whatever the a w value, fructose monopalmitate was only synthesised. The best results were achieved for a reaction medium with an initial water activity less than 0.07. In these conditions, 28.5% of fructose was acylated in fructose monopalmitate with an initial rate of 4.9 g l −1 h −1 . To improve the process, the effect of the water was determined by drying the medium with molecular sieves. Conversion yield of fructose and initial rate of fructose monopalmitate synthesis were raised, respectively, to 73.4% (32 g l −1 ) and 10.1 g l −1 h −1 . However, the use of dried medium affected the selectivity of the reaction. In these conditions, both mono- and di-fructose palmitate were synthesised (16.7 g l −1 ).

Activity and stability of lipases from different sources in supercritical carbon dioxide and near-critical propane

Abstract: The stability and activity of lipases from Pseudomonas fluorescens, Rhizopus javanicus, Rhizopus niveus, porcine pancreas and Candida rugosa in a non-solvent system at atmospheric pressure, in supercritical carbon dioxide (SC CO2), and near-critical propane at 100 bar and 40 °C were studied. Esterification of n-butyric acid with ethanol and isoamyl alcohol was used as a model system. In supercritical carbon dioxide there was a great loss in activity of the examined lipases. Decreased relative activity of lipases in SC CO2 was attributed to the interactions between CO2 and the enzyme. The second reason for this effect was the differences in water partitioning between the enzyme and its surroundings. In contrast, the use of near-critical propane improved the activity of lipases in the comparison to the non-solvent system by four- (porcine pancreas lipase) to nine-times (Rhizopus javanicus lipase). The use of near-critical propane also improved the thermal stability of porcine pancreas lipase compared with the non-solvent system. The calculated deactivation constant for esterification between butyric acid and isoamyl alcohol, catalyzed by porcine pancreas lipase, showed that there was more than twice as much inactive as active enzyme in the non-solvent system studied whereas the ratio in propane was 1. © 2001 Society of Chemical Industry

Evaluation of amylase and lipase in the diagnosis of acute pancreatitis.

Abstract: Background: The diagnosis of acute pancreatitis relies heavily on a raised amylase. Methods: In the present study patients were prospectively categorized, without knowledge of pancreatic enzyme levels, into acute pancreatitis (AP; n = 51), disease controls (n = 35), indeterminate as to pancreatitis (n = 189) or exclusions (non-pancreatitis diseases where amylase may be elevated; n = 53). Results: Enzyme levels were analysed by receiver operator characteristics (ROC) curves, with specificity > 80%. Day 1 serum lipase gave the greatest diagnostic accuracy (area under ROC curve = 0.128; P = 0.041 vs serum amylase). At the calculated diagnostic threshold of 208 U/L, lipase gave a sensitivity of 67% and a specificity of 97%. Other diagnostic thresholds (day 1) were: serum total amylase, 176 U/L (ROC 0.104, sensitivity 45%, specificity 97%), urinary total amylase, 550 U/L (ROC 0.108, sensitivity 62%, specificity 97%) and serum pancreatic isoamylase, 41 U/L (ROC 0.107, sensitivity 63%, specificity 85%). At delayed diagnosis (3 days) no enzyme was superior to lipase. The combination of lipase and amylase did not increase diagnostic accuracy. Conclusion: Serum lipase is recommended for diagnosis of AP, both early and late in the disease. Although highly specific when elevated, all pancreatic enzymes have low sensitivity for diagnosis.