Lipase-Catalyzed Resolution of Isopropylidene Glycerol: Effect of Co-Solvents on Enantioselectivity

The resolution of 1,2-O-isopropylidene glycerol via enzyme catalyzed hydrolysis of the corresponding benzoic ester was investigated. Using lipase PS from Pseudomonas cepacia, we determined the influence of organic co-solvents on the activity and enantioselectivity of the enzyme. The performance of the lipase was correlated to the nature (logP, ?,μ and the percentage of the organic media. The highest enzymatic activity was found in solvents completely miscible or completely immiscible in water. The enzyme stereoselectivity was inversely related to the logP of the solvent.     

微水——有机溶剂两相体系中消旋萘普生的酶法拆分

研究了微水－有机溶剂两相体系中固定化脂肪酶催化的萘甲酯的立体选择性水解反应,固定化酶活性受载体极性、水含量、有机溶剂的ｌｏｇＰ值,产物抑制的影响,据此构建了一种可以连续拆分产生（Ｓ）－（＋）－萘普生的微水－有机溶剂两相体系。反应在一个具有回路的连续流搅拌反应器中进行,反应器中添加有采用吸附法固定化的脂肪酶,截体为一种弱极性的合成载体,水相连同固定化酶颗粒一起永久保持在反应器中,有机流动相带入底物,     

Purification of a moderate thermotolerant Bacillus coagulans BTS1 lipase and its properties in a hydro-gel system

An alkaline thermotolerant lipase of Bacillus coagulans BTS1 was successively purified by ammonium sulfate precipitation and DEAE anion exchange chromatography. The purified lipase immobilized in alginate beads showed an optimal activity at pH 7.5 and 55 degrees C. A pH of 5.0 or 10.0 completely quenched the activity of immobilized lipase. The alginate-bound lipase retained its activity following exposure to most of the organic solvents including amines, alkanes and alcohols. Chloride salt of Al3+, Co2+, Mg2+ and NH4+ modulated the lipase activity of alginate-immobilized enzyme. The alginate entrapped lipase showed a preferentially high activity towards p-nitrophenyl palmitate (C: 16) and activity of matrix increased following exposure to SDS. Moreover, the immobilized lipase retained more than 50% of its activity after 3rd cycle of reuse.     

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the pI is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 °C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C₁₄). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca²⁺. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities (E = 83 >100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(4′-methoxyphenyl) glycidic acid methyl ester (MPGM).     

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.     

Studies on localization and regulation of lipase production by Aspergillus niger

The lipase from strain BTL of Aspergillus niger was studied. The enzyme, which was mainly extracellular, was produced at elevated activity levels under optimum growth conditions. De novo biosynthesis of lipase occurred only in the presence of lipids and was completely repressed by glucose and glycerol. The reaction products, oleic acid and glycerol, showed differed inhibition patterns during triolein hydrolysis. The enzyme exhibited high specificity towards middle chain triglycerides and was possibly activated by double bonds in the fatty acid chain. It exhibited a marked stability against organic solvents.     

Lipase-catalyzed alcoholysis of triglycerides for the preparation of 2-monoglycerides

Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl-tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol gave the highest yield (97%) at an optimal ethanol concentration of 200–300 mM. At higher alcohol concentrations, the enzyme activity was substantially lowered. The enzyme preparation showed high stability in repeated-batch reactions.     

Production of antibodies against the coenzyme pyrrolequinoline quinone

Rat intestinal epithelial cells were isolated and the activity of the enzyme diacylglycerol lipase (DG lipase, EC 3.1.1.3) was investigated. When cells were treated with Escherichia coli heat-stable toxin (ST) liberation of endogenous glycerol and fatty acids was observed. The enzyme responsible for this effect could be demonstrated to be a DG lipase by using specific substrates. It was found that the activity of DG lipase was increased 5–6-fold with the substrates diolein and 1,2-dioleyl-rac-glycerol and triolein being neutral lipid insensitive to DG lipase. ST had no direct effect on the DG lipase. The enzyme DG lipase was activated via a chain reaction due to the hydrolysis of phosphatidylinositol (PI) by the enzyme PI-specific phospholipase C stimulated by ST.     

Enzymatic resolution of (S)-(+)-naproxen in a trapped aqueous-organic solvent biphase continuous reactor

A trapped aqueous-organic biphase system for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen methyl ester by Candida rugosa lipase in a trapped aqueous-organic biphase system. The reaction has been carried out in a laboratory-scale continuous-flow stirred tank reactor (CSTR). The staring material has been supplied in and remaining substrate recovered by organic phase. YWG-C(6)H(5), a poorly polar synthetic support, has been employed to immobilize the lipase and to restrict the aqueous phase. Lipase immobilized on YWG-C(6)H(5) containing aqueous phase has been added into the CSTR to catalyze the hydrolysis. A dialysis membrane tube containing a continuous flow closed-loop buffer has been applied in the CSTR for the extraction of product and recruiting of the aqueous part consumed. Various reaction conditions have been studied. The activity of immobilized enzyme was effected by the polarity of support, the substrate concentration, logP value of organic phase and the product inhibition. At steady-state operating conditions, an initial conversion of 35% has been obtained. The CSTR was allowed to operate continuously for 60 days at 30 degrees C with a 30% loss of activity. The hydrolysis reaction yielded (S)-(+)-Naproxen with >90% enantiomeric excess and overall conversion of 30%.     

Expression and purification of organic solvent stable lipase from soil metagenomic library

Gene for organic solvent stable lipase was overexpressed from soil metagenomic library. The clone with maximum activity was selected, and enzyme was purified by gel-permeation chromatography with a molecular mass of approx. 40 kDa. The deduced aminoacid sequence indicated that the protein belongs to the lipase family I.3 and containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+) binding sites. The enzyme expressed maximum activity at 30 °C and pH 7.0 and found to be stable in pH and temperature ranging from 6.0-9.0 and 20-60 °C, respectively. Furthermore, the enzyme was found highly resistant to many organic solvents, especially isopropanol, DMSO, methanol, xylene and hexane. The enzyme showed enhanced activity in the presence of divalent cations (Mg(2+), Mn(2+), Ca(2+), Hg(2+), Cu(2+)), whereas the presence of trivalent cation (Fe(3+)) inhibited the activity.     

Characteristics of lipase-catalyzed hydrolysis of olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase solubilized in organic solvents in the presence of both surfactant and water could catalyze the hydrolysis of triglycerides, and kinetic analysis of the lipase-catalyzed reaction was found to be possible in this system. Among eight organic solvents tested, isooctane was most effective for the hydrolysis of olive oil in reversed micelles. Temperature effect, pH profile, K(m,app) and V(max,app) were determined. Among various chemical compounds, Cu(2+), Hg(2+), and Fe(3+) inhibited lipase severely. But the enzyme activity was restorable partially by adding histidine or glycine to the system containing these metal ions. The enzyme activity was dependent on R (molar ratio of water to surfactant) and maximum activity was obtained at R = 10.5. Upon addition of glycerol to the reversed micelles, lipase activity was affected in a different fashion depending on the R values. Stability of the lipase in reversed micelles was also dependent on R, and it was most stable at R = 5.5.     

Synthesis and secretion of triacylglycerol lipase by cultured rat hepatocytes

Rat hepatocytes isolated by collagenase perfusion were cultured for 48-72 h and examined for synthesis and secretion of hepatic triacylglycerol lipase activity. Low levels of enzyme activity found in the culture medium increased with time of incubation, and a 3-10-fold rise was encountered in the presence of optimal concentrations of heparin (5 U/ml). After interruption of enzyme synthesis by cycloheximide, plateauing of enzyme activity in the medium occurred, indicating that addition of heparin may not only stabilize but also enhance hepatic triacylglycerol lipase secretion. Synthesis and secretion of hepatic triacylglycerol lipase was not related to cell density, and enzyme secretion was encountered in subconfluent cultures. Release of enzyme activity into the medium was not sensitive to chlorpromazine, a lysosomal enzyme inhibitor, but was completely inhibited by treatment with tunicamycin, an inhibitor of glycosylation. As release of enzyme activity could be maintained for 12 h in the absence of serum, possible hormonal regulation was sought. Under the present experimental conditions, no modulation of hepatic triacylglycerol lipase was encountered by either gonadal or thyroid hormones. Addition of cyclic AMP to the culture medium resulted in a 30% decrease in enzyme activity. The dependence of hepatic triacylglycerol lipase secretion on the intactness of the Golgi apparatus and on vesicular transport was demonstrated by the treatment with monensin. The present results show that cultured rat hepatocytes provide a good model system by which the regulation of synthesis and secretion of hepatic triacylglycerol lipase can be studied.     

Stabilization of an intracellular Mucor circinelloides lipase for application in non-aqueous media

Different methods for stabilization of Mucor circinelloides lipase, facilitating its application in organic solvents were tested. Lipase was either isolated from the mycelium and immobilized on solid carriers (derivatives of cellulose, diatomaceous earth, modified porous glass) or immobilized in situ in the mycelium pellets and stabilized. The immobilized enzyme preparations were used for synthesis of sucrose, glucose, butyl and propyl oleates and caprylates, carried out in petroleum and di-n-pentyl ethers. Immobilized preparations of either crude or purified lipase isolated from the mycelium were at least 4–6 times less effective in sucrose esters synthesis than mycelium-bound lipase preparations. Lipase preparation with the highest synthetic activity was obtained by cross-linking of M. circinelloides mycelium pellets with glutardialdehyde (operational stability in sucrose caprylate synthesis was 94% after 4 runs (24 h each), and caprylic acid conversion was 91–85%). The best method for production of mechanically durable biocatalyst, which efficiently catalyzed sucrose esters synthesis, was found to be entrapment of the mycelium-bound lipase in polyvinyl pyrrolidone-containing chitosan beads solidified with hexametapolyphosphate.     

Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63

A lipase gene lipA and its chaperone gene lipB were cloned from Burkholderia cepacia strain G63. The lipA was composed of 1092 bp, encoding 363 amino acid residues, and the lipB composed of 1035 bp, corresponding to 344 amino acid residues. The significant amino acid similarity with Pseudomonas cepacia lipase revealed that this enzyme could be classified into the lipolytic subfamily I.2. The lipA and lipB genes were cloned into pBBR1Tp vector and conjugated into B. cepacia strains G63 with the help of pRK2013. The recombinant strain was fermented in 10 l bioreactor and the lipase was purified by a combination of ammonium sulfate fractionation, DEAE ion-exchange chromatography and gel filtration. The purified lipase kept stable at a temperature range of 40–70 °C. After incubated at 70 °C, the optimal temperature of this enzyme, for 10 h it remained 86.1% of its activity. The enzyme was also highly tolerant to a series of organic solution. Incubated in 50% methanol solution up to 48 h, the enzyme still kept 98.3% of its activity. The transesterification activity of soybean oil to fatty acid methyl esters (FAMEs) reached 87.8% after 72 h, indicating that it is a potential biocatalyzer for biodiesel production.     

Effects of water-miscible solvents and polyhydroxy compounds on the structure and enzymatic activity of thermolysin

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.     

Cloning and characterization of a fish microsomal epoxide hydrolase of Danio rerio and application to kinetic resolution of racemic styrene oxide

A potent bacterial strain, Pseudomonas aeruginosa, has been isolated from the soil which produces extracellular lipase that can carry out the excellent stereospecific hydrolysis of trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM)] to give [(−)-MPGM], an intermediate required in the synthesis of cardiovascular drug, diltiazem. As a preliminary experiment for enzymatic resolution, we characterized the fractionated enzyme. The enzyme had a pH and temperature optima of 8.0 and 60 °C, respectively. The enzyme showed high degree of thermostability. Also, the enzyme was found to be stable in alkaline condition and in organic solvents. The activity of the enzyme increased by the addition of magnesium ions. The small-scale hydrolysis of (±)-MPGM (250 mg) with partially purified enzyme (21,000 U) gave (−)-MPGM with good isolated yield (44%) and excellent enantiomeric excess (99.9%) in a very short time (12 h).     

Enhanced activity and stability of immobilized lipases by treatment with polar solvents prior to lyophilization

Lipases from Candida rugosa, Mucor javanicus and Rhizopus oryzae were respectively adsorbed on Amberlite XAD-7 followed by incubation in 2-propanol and then lyophilization. The activities of the immobilized enzymes were 1.6–3.4 times higher than those of the immobilized enzymes without incubation in the organic solvent before lyophilization for esterification of lauric acid (0.1 M) and 1-propanol (0.1 M) in isooctane at 37 °C. The immobilized C. rugosa lipase (Sigma) without the incubation did not show any activity but displayed considerable activity (19.8 μmol h⁻¹ mg⁻¹) after the incubation before lyophilization. Besides 2-propanol, acetone, 1-propanol and ethyl acetate were also found to be good solvents for treating M. javanicus lipase immobilized on Amberlite XAD-7 and acetone was the best among them. When incubated in isooctane at 25 °C for 120 h, the immobilized M. javanicus lipase prepared by incubation in acetone for 1 h before lyophilization retained 70% of its initial activity while the immobilized enzyme without the solvent treatment kept only 50% of its initial activity.     

Cloning,expression and characterization of a lipase gene (<Emphasis Type="Italic">lip3</Emphasis>) from<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> LST-03

A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0–35°C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.Communicated by H. Ikeda     

Esterification In Organic Solvents: Selection Of Hydrolases And Effects Of Reaction Conditions

Fifty different hydrolases were screened for retention of high esterification activity in an organic solvent with citronellol as substrate. Although 22 hydrolases were very active as catalysts in the organic solvent, lipase from Candida cylindracea (lipase OF 360) was selected for further examination of the effects of reaction conditions on enzyme activity, with regard to catalyst availability and activity retention after immobilization. When the enzyme was entrapped in hydrophobic polyurethane gels, water-saturated isooctane was found to be the most suitable solvent, whereas polar solvents caused reversible catalyst inactivation. Entrapment significantly enhanced the operational stability of the lipase in the organic solvent.     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.