Glycidic Acid Methyl Ester Research Articles

Refolding with Simultaneous Purification of Recombinant Serratia marcescens Lipase by One-Step Ultrasonication Process.

A new lipase from Serratia marcescens SRICI-01 (Trx-SmL) was successfully overexpressed in Escherichia coli with thioredoxin (Trx) fusion tag. Intriguingly, the concentration of potassium phosphate buffer (KPB) showed significant impact on the aggregation state of Trx-SmL during ultrasonic disruption. The proportion of inclusion bodies increased dramatically with the increase of KPB concentration from almost completely soluble in 10mM KPB to insoluble in 200mM KPB. Based on this new finding, a novel method for refolding and purification of recombinant Trx-SmL was developed by one-step ultrasonication. The Trx-SmL was firstly precipitated in 200mM KPB, washed for three times, and subsequently subjected to ultrasonic process in 10mM KPB where refolding and purification occurred simultaneously. This established method was proved to be a straightforward, economical, and efficient purification approach to facilely obtain recombinant Trx-SmL protein with high purity (> 90%) and activity recovery yield (> 80%) from cell lysates. The application potential of the purified fusion Trx-SmL was further demonstrated by kinetic bioresolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] producing optically pure (-)-MPGM, a key intermediate for diltiazem, with an overall yield of 41.5% and ee of 99%.

Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester.

The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipAL315S and LipAS271F, were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipAL315S, LipAS271F, and their combination LipAL315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipAS271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipAS271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m. Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.

High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli.

Serratia marcescens lipase (SmL) is an important biocatalyst used to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphynyl) glycidic acid methyl ester. However, the economically justified level recombinant soluble expression of SmL in Escherichia coli has not been established. Thus, fusion genes of lipase from S. marcescens H30 with different fusion tags were constructed and expressed in E. coli. The effects of fusion tags were revealed. A significant increase in recombinant lipase solubility showed that E. coli BL21 (DE3)/pET32a-SmL was a suitable choice for SmL production. To optimize the performance of recombinant SmL production, changes in culture medium compositions and induction conditions were systematically tested. Finally, the recombinant SmL activity and productivity reached approximately 23,000U/L and 1,278U/L/H in shake flasks, respectively. This value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. Lipase activity and productivity reached 19,650U/L and 1,228U/L/H, respectively, by scaling up SmL production in a 7.0L fermenter. The existence of the Trx tag did not influence the chiral selectivity of recombinant SmL. These findings indicate a possibility for soluble and economical SmL expression in E. coli to meet industrial needs.

Production, Characterization, and Application of an Organic Solvent-Tolerant Lipase Present in Active Inclusion Bodies

An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry.

Refolding, purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010

Abstract Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion bodies can be successfully refolded. In this study, the different parameters were investigated and optimized on the refolding of denatured lipase. The maximum lipase activity of 5000 U/L was obtained after incubation of denatured enzyme in a refolding buffer containing 20 mM Tris–HCl (pH 7.0), 1 mM Ca2+ at 20 °C. Then, the refolded lipase was purified to homogeneity by anion exchange chromatography. The purified refolded lipase was stable in broad ranges of temperatures and pH values, as well as in a series of water-miscible organic solvents. In addition, some water-immiscible organic solvents, such as petroleum ether and isopropyl ether, could reduce the polarity and increase the nonpolarity of the refolding system. The results of Fourier transform infrared (FT-IR) microspectroscopy were the first to confirm that lipase refolding could be further improved in the presence of organic solvents. The purified refolded lipase could enantioselectively hydrolyze trans-3-(4-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM]. These features render the lipase attraction for biotechnological applications in the field of organic synthesis and pharmaceutical industry.

Crosslinking of enzyme coaggregate with polyethyleneimine: A simple and promising method for preparing stable biocatalyst of Serratia marcescens lipase

Abstract Crosslinking of enzyme aggregates is a promising method for enzyme immobilization. In this work, crosslinked enzyme coaggregates of Serratia marcescens lipase with polyethyleneimine (CLECAs-SML-PEI) were prepared using polyethyleneimine (PEI) as coprecipitant and glutaraldehyde as crosslinking reagent. The crude lipase solution at a low protein concentration (0.1 mg/ml), with PEI at a mass ratio of 3:1 (PEI/protein, w/w), was found to be most adequate for the coprecipitation of SML. After crosslinking of the coaggregate of SML-PEI with 0.2% (w/v) glutaraldehyde under ambient temperature, over 70% of the total lipase activity was recovered. Compared with the free SML, the optimum temperature of the CLECAs-SML-PEI was enhanced from 50 °C to 60 °C and its thermal stability was also significantly improved. CLECAs-SML-PEI showed excellent operational stability in repeated use in aqueous–toluene biphasic system for asymmetric hydrolysis of trans -3-(4′-methoxyphenyl)glycidic acid methyl ester (MPGM), without significant inactivation after 10 rounds of repeated use.

Efficient production of diltiazem chiral intermediate using immobilized lipase from Serratia marcescens

The lipase from Serratia marcescence ECU1010 (Sml) was capable of enantioselectively catalyzing the synthesis of many chiral drug precursors. This paper investigated the immobilization of Sml on appropriate supporting materials and its performance in bioreactor. Chitosan, Celite 545, and DEAE-cellulose were found to be the ideal supports among 8 carriers tested with respect to enzyme load and activity recovery of lipase. When Sml was immobilized, significant improvements of stability against pH, thermal, and operational deactivation were observed with all the 3 better supports, and the best stability was observed when the lipase was immobilized on glutaraldehyde activated chitosan. As for the effect of organic solvent in the biphasic reaction system, the hydrolytic activity of the immobilized lipase on trans-3-(4′-methoxyphenyl)glycidic acid methyl ester ((±)-MPGM) observed in isopropyl ether was 6 and 3 times higher than those in toluene and methyl tert-butyl ether. The lipasecatalyzed production of (−)-MPGM by enzymatic resolution of (±)-MPGM with chitosan-Sml in isopropyl etherwater biphasic system was carried out in a 2 L stirred-tank reactor. The batch operation was more efficient operation mode for the enantioselective hydrolysis of (±)-MPGM, affording enantiopure (−)-MPGM in 44.3% overall yield, in contrast to 29.3% in a continuous reactor.

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe 3O 4 nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g −1 support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)- trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.

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 p I 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 (C 14). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca 2+. 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).

Immobilization of Serratia marcescens Lipase and Catalytic Resolution of Trans-3-(4′-methoxyphenyl)glycidic Acid Methyl Ester

Abstract Seven carriers were investigated for immobilization of the lipase from Serratia marcescens ECU1010, and diatomite and Eupergit C were found to be ideal supports. The thermal and storage stability of the lipase was significantly improved after being immobilized onto these two supports. After heating at 50$^{o}$C for 6 h, only a small decrease in the activity was observed for the diatomite-adsorbed lipase. About 50% of the initial activity was retained when the two immobilized enzyme samples were stored at 4$^{o}$C for 180 days. Operational stability tests indicated that the half-life of the lipase immobilized on Eupergit C was about 2-fold longer than that of the diatomite-immobilized lipase. The crosslinking with glutaraldehyde had no significant effect on the activity of the Eupergit C-immobilized lipase, whereas the reusability of the crosslinked diatomite-immobilized enzyme was slightly improved. Using the crosslinked diatomite-immobilized lipase, an up-scaled bioresolution of (±)-3-(4'-methoxyphenyl)glycidic acid methyl ester ((±)-MPGM) was performed in a 500-ml emulsion reactor at 30$^{o}$C with 200 ml of biphasic solution (toluene:water = 1:1) and 0.5 mol/L of (±)-MPGM in toluene. After 5 batches of reaction, crystalline (2 R , 3 S )-(–)-MPGM with a chemical purity of 100% and an optical purity of > 99% enantiomeric excess was obtained with a yield of 37.2%.

Enantioselective hydrolysis of methoxyphenyl glycidic acid methyl ester [(±)-MPGM] by a thermostable and alkalostable lipase from Pseudomonas aeruginosa

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).

Optimization of Serratia marcescens lipase production for enantioselective hydrolysis of 3-phenylglycidic acid ester

Lipase production and cell growth of Serratia marcescens ECU1010 were optimized in shake flasks, with lipase production being enhanced 9.5-fold (4,780 U/l) compared with the initial activity (500 U/l). Optimal carbon and nitrogen sources were Tween-80 and peptone, and the optimal ratio of Tween-80 to peptone was 1:3. The optimized cultivation conditions were 25 degrees C and pH 6.5. Lipase activity, particularly specific activity, could be improved by decreasing the cultivation temperature from 35 to 25 degrees C. Enzyme stability was significantly improved by simple immobilization with synthetic adsorption resin no. 8244. After five reaction cycles, enzyme activity decreased only very slightly, while enantioselectivity of the preparation remained constant, and the ees (enantiomeric excess of the remaining substrate) achieved in all cases was higher than 97%. The resin-8244-lipase preparation can be used for efficient enantioselective hydrolysis of trans-3-(4'-methoxyphenyl)glycidic acid methyl ester [(+/-)-MPGM], a key intermediate in the synthesis of Diltiazem.

Determination of absolute configuration using ab initio calculation of optical rotation.

Ab initio Density Functional Theory (DFT) calculations of transparent spectral region, discrete frequency specific rotations were used to assign the absolute configurations (ACs) of: 1, 2H-naphtho[1,8-bc]thiophene 1-oxide; 2, m-F-phenyl glycidic acid methyl ester; 3, o-Br-phenyl glycidic acid methyl ester; 4, p-CH(3)-phenyl glycidic acid methyl ester; 5, 2-(1-hydroxyethyl)-chromen-4-one; and 6, 6-Br-2-(1-hydroxyethyl)-chromen-4-one. The ACs of 5 and 6 were previously determined via X-ray crystallography to be: 5, R(-)/S(+); 6, R(+)/S(-). The ACs obtained using [alpha](D) are the same for both 5 and 6: R(+)/S(-). We conclude that the previously reported AC of 5 is incorrect.

Stereospecific hydrolysis of 3-(4-methoxyphenyl)glycidic ester in supercritical carbon dioxide by immobilized lipase

In the hydrolysis of racemic 3-(4-methoxyphenyl)glycidic acid methyl ester by immobilized Mucor miehei lipase in supercritical CO 2 the initial hydrolysis rate of the (2S,3R)-form was faster than the rate of the (2R,3S) -form. The stereoisomeric excess of the (2R,3S)-form reached 87 % at 53 % total conversion level. The water content of the reaction mixture and the initial concentration of the 3-(4-methoxyphenyl) glycidic acid methyl ester had no effect on isomeric purity. The reaction rate in supercritical CO 2 was considerably faster than in toluene/water -mixture.

Production of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester using an emulsion bioreactor containing lipase fromSerratia marcescens

An emulsion bioreactor for production of (2R,3S)-3-(4-methoxyphenyl) glycidic acid methyl ester ([-]MPGM) from a racemic mixture ([±]MPGM) using the lipase fromSerratia marcescens has been proposed. Kinetics of hydrolyzing reaction and purification of (-)MPGM from the reaction mixture were investigated to provide a basis for industrial application of this bioreactor. The hydrolyzing reaction in the bioreactor proceeded at a rate that was first order in substrate concentration. The reaction rate was affected by a stirring speed and the ratio of the aqueous phase containing lipase to the toluene phase containing substrate. Phase separation after the enzymatic reaction was accomplished by addition of surfactant to the reaction mixture, and crystalline (-)MPGM with a chemical purity of 100% and optical purity of 100% enantiomeric excess was obtained in a high yield of 40–43% by concentration of the toluene solution.

A membrane bioreactor combined with crystallizer for production of optically active ( 2R, 3S)-3-(4-methoxyphenyl)-glycidic acid methyl ester

The combination of a membrane bioreactor using lipase from Serratia marcescens and a crystallizer has been proposed for the production of optically active ( 2R, 3S)-3-(4-methoxyphenyl) glycidic acid methyl ester ((−)MPGM) from racemic compounds ((±)MPGM). The reaction kinetics were investigated with a view toward industrial application of this newly devised bioreactor system. The following results were obtained. (i) The hydrolysis of (±)MPGM in the bioreactor system proceeded as a first-order reaction with respect to the substrate amount. (ii) The reaction rate depended on the amount of enzyme loaded onto a membrane module and the initial amount of substrate in the crystallizer. (iii) The reaction rate was not influenced by the rate of substrate circulation between the crystallizer and membrane bioreactor. (iv) Although the level of performance of the bioreactor system with respect to hydrolysis decreased with increasing operation time, it recovered to upon loading of fresh enzyme onto the membrane. This bioreactor system in which an enzymatic reaction and product crystallization occurred simultaneously had a high level of productivity compared with that of the conventional membrane bioreactor using a solubilized (±)MPGM substrate.

バイオテクノロジーへの膜の応用 光学活性グリシッド酸エステル工業製造用膜バイオリアクターの開発

An optically active (2R, 3S) -3- (4-methoxyphenyl) glycidic acid methyl ester ((-) MPGM) was an important intermediate for the synthesis of diltiazem hydrochloride which is a typical calcium channel blocker. To establish a practical production system of this useful chiral compound, a hollow-fiber ultrafiltration membrane bioreactor has been developed. In the bioreactor system, the lipase from Serratia marcescens was immobilized on the spongy layer of the shell side of a membrane by means of physical adsorption. Asymmetric hydrolysis was carried out semi-continuously in a membrane bioreactor circulating both toluence solution containing racemate ((±) MPGM) in the shell loop and aqueous solution cotaining NaHSO3 in the lumen loop. The membrane bioreactor permitting the simultaneous achievement of enzyme reaction and phase separation was highly efficient for industrial production of (-) MPGM. This paper briefly describes the process of research development of the industrial-scale membrane bioreactor. In addition, our recent works relating to membrane bioreactors were presented.

Purification and characterization of the lipase from Serratia marcescens Sr41 8000 responsible for asymmetric hydrolysis of 3-phenylglycidic acid esters

A new lipase which enantioselectively hydrolyzes (±)- trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride, was purified from the culture supernatant of Serratia marcescens Sr41 8000. The apparent kinetic constants ( K m, V max) for hydrolysis of (2 S,3 R)-MPGM [(+)-MPGM] were 350 mM and 1.7 × 10 −3 mol/min/mg protein in a toluene-water (1:1) emulsion system. The lipase did not attack (2 R,3 S)-MPGM [(−)-MPGM], and (−)-MPGM acted as a competitive inhibitor. The molecular mass was estimated to be 62,000 ± 2,000 from SDS-PAGE. The lipase preferentially hydrolyzed (2 S,3 R)-3-phenylglycidic acid esters, but did not hydrolyze cinnamic acid esters. The lipase released glycerol and fatty acid from olive oil, and the optimum pH and temperature for hydrolysis of olive oil were pH 8 and 45°C, respectively. The lipase was inhibited by Co 2+, Ni 2+, Fe 2+, Fe 3+ and EDTA, and activated by Ca 2+, Li + and SDS. It was presumed that the lipase was a metalloenzyme containing approximately one gram atom of calcium per molecular mass of 62,000. The lipase selectively hydrolyzed the 1,3 ester of triglycerides. Sequencing of the N-terminal amino acids revealed that this lipase was distinct from other known lipases.

Production of optically active 3-phenylglycidic acid ester by the lipase from Serratia marcescens on a hollow-fiber membrane reactor

(±)- trans-3-(4-Methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride, was efficiently hydrolyzed by lipase from Serratia marcescens Sr41 8000 immobilized on a hollow-fiber ultrafiltration membrane. The lipase was immobilized on a spongy layer of the shell side of the membrane by means of physical adsorption. Asymmetric hydrolysis was carried out semi-continuously at 22°C in a membrane reactor circulating both toluene solution containing (±)-MPGM in the shell loop and aqueous solution (pH 8.5) in the lumen loop. (+)-MPGM was hydrolyzed to (+)-(2 S, 3 R)-3-(4-methoxyphenyl)glycidic acid and methanol. p-Methoxyphenylacetaldehyde derived from the glycidic acid was accumulated in the toluene phase and inhibited the enzyme reaction. The inhibition, however, was suppressed by the addition of sodium hydrogen sulfite to the aqueous phase, because the aldehyde and sodium hydrogen sulfite formed an adduct and the adduct was transferred to the aqueous phase with the progress of enzyme reaction. The lipase immobilized on the hydrophilic membrane exhibited a high stability: the half-life of enzyme activity at 22°C was 127 h, which was about 30-fold that in the conventional emulsion reactor. The maximum value of the velocity constant ( k (+)) for the hydrolysis of (+)-MPGM was 0.25 h −1 under the condition of immobilization of 1.6 × 10 5 units of lipase per m 2. Since reaction and product separation were achieved simultaneously, crystalline (−)-MPGM with a high yield of 40–43% and optical purity of 100% enantiomeric excess [e.e.] was obtained through six runs by concentration of the toluene phase after the reaction.

Lipase-catalyzed asymmetric hydrolysis of 3-phenylglycidic acid ester, the key intermediate in the synthesis of diltiazem hydrochloride

Asymmetric hydrolytic enzymes for trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride that is useful as a coronary vasodilator, were screened from 730 microorganisms. Among the microbial enzymes tested, Serratia marcescens lipase had the highest enantioselectivity (E=135) for hydrolysis of (±)-MPGM in a two-phase system using water (pH 8) and a water-immiscible solvent such as toluene. Resolution of (±)-MPGM by S. marcescens lipase gave (2 R, 3 S)-3-(4-methoxyphenyl)glycidic acid methyl ester [(−)-MPGM] with a reaction yield of 48% and optical purity of >99.9% e.e. After the reaction, the emulsion of toluene and water was separated into two clear layers by the addition of sodium dodecyl sulfate. The crystalline (−)-MPGM was isolated with a yield of over 43% and optical purity of 100% e.e. without column treatment. Diltiazem hydrochloride synthesis using asymmetric hydrolysis of (±)-MPGM was found to be a more efficient process compared to the conventional chemical synthetic process. Some enzymatic characterizations on asymmetric hydrolysis in two-phases of organic solvent-water by S. marcescens lipase were investigated.