Selective synthesis of panthenyl esters by a kinetically controlled enzymatic process

Abstract

Panthenyl esters (panthenyl monoacetate and panthenyl diacetate) were synthesized in high yields (≈100%) by a kinetic reaction control using a commercial immobilized Candida Antarctica lipase B (Novozyme 435) in acetonitrile. The enzyme showed excellent synthetic activity, regioselectivity, and operational stability under the conditions used.     

Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper

Effects of five types of nonionic surfactant having a polyoxyethylene glycol (POG) group on enzymatic hydrolysis of used newspaper were studied. The surfactants examined in this work always enhanced the saccharification. The optimum surfactant concentration was 0.05% (wt/substrate wt) in the case of POG(21) sorbitane oleic ester. Among the surfactants, POG phenyl ether types showed the highest enhancement effect, for example, with two times higher conversion at 80 h than that without surfactant. Using POG nonylphenyl ether series, the effects of surfactant were considered from the point of the HLB (hydrophile-lypophile balance) value. The GFC (gel filtration chromatography) analysis of free enzyme quantity were also done to study the effect of surfactant on enzyme adsorption onto substrate. As the HLB value increased, the free enzyme quantity and the conversion both increased. It appears that surfactants help the enzyme to desorb from the binding site on the substrate surface after the completion of saccharification at that site.     

Continuous reaction-separation process for enzymatic esterification in supercritical carbon dioxide

The study of enzymatic esterification by an immobilized lipase in supercritical carbon dioxide (SCCO(2)) and in n-hexane, described in our previous works, was extended to continuous operation in a tubular fixed bed. The modeling of the reaction vessel operation was achieved through the use of the simple plug flow model coupled with the appropriate kinetic equation. Comparison with experiments proved to be satisfactory. The study of the postreactional separation, an important feature when using SCCO(2), was undertaken experimentally and good selectivities and product recovery were obtained. (c) 1994 John Wiley & Sons, Inc.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

Cross-linked enzyme aggregates of recombinant Candida antarctica lipase B for the efficient synthesis of olvanil,a nonpungent capsaicin analogue

Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase-catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross-linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB-CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co-feeders. The CALB-CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB-CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L⁻¹ of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L⁻¹ of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB-CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.     

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

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%.     

Ionic liquid pretreatment of cellulosic biomass: enzymatic hydrolysis and ionic liquid recycle

Ionic liquids (ILs) are promising solvents for the pretreatment of biomass as certain ILs are able to completely solubilize lignocellulose. The cellulose can readily be precipitated with an anti-solvent for further hydrolysis to glucose, but the anti-solvent must be removed for the IL to be recovered and recycled. We describe the use of aqueous kosmotropic salt solutions to form a three-phase system that precipitates the biomass, forming IL-rich and salt-rich phases. The phase behavior of [Emim][Ac] and aqueous phosphate salt systems is presented, together with a process for recycling the [Emim][Ac] and enzymatically hydrolyzing the cellulose. This process reduces the amount of water to be evaporated from recycled IL, permitting efficient recycle of the IL. Material balances on the process, with multiple recycles of the [Emim][Ac], quantify the major components from a Miscanthus feedstock through the pretreatment, separation, and enzymatic hydrolysis steps. A more rapid and higher yielding conversion of cellulose to glucose is obtained by use of the three-phase system as compared to the cellulose obtained from biomass pretreated with IL and precipitated with water. The addition of a kosmotropic salt during the precipitation results in partial delignification of the biomass, which makes the substrate more accessible, enhancing the enzymatic hydrolysis.     

Coupled-enzyme system for the determination of lipase activity

A new method for lipase activity determination is described, in which liberation of fatty acids by lipase-catalyzed hydrolysis is coupled to lipoxygenation. The second reaction forms hydroperoxy-fatty acids containing a conjugated double bond that are detected at 234 nm. The method is sensitive, cheap and easy to use when compared to a titration method.     

The biocatalytic activity of bromelain in ester synthesis reaction: difference between the intrinsic lipase activity and thermal catalysis

Biocatalytic activities of bromelain preparations were carried out in proteolytic (4500 units g^–1), lipolytic (67 units g^–1) and, more particularly, in fatty acid ester synthetic reactions. The ester synthesis reactions were studied and several thermodynamic parameters and non-biological reference reactions were also investigated. Only temperature had a strong influence on the maximum reaction yield (30% after 10 days) and revealed that thermal catalysis, which exists in esterification, raises doubts concerning the real biocatalytic activity of the plant extract. When this thermal catalysis is taken into account, the intrinsic lipase activity of the bromelain preparations in esterification reactions is nil.     

Evaluation of a silica-coated magnetic nanoparticle for the immobilization of a His-tagged lipase

Magnetic particles of size 10 nm have been coated with silica to a mean diameter of 40 nm and charged with Cu²⁺ ions via a multidentate ligand, iminodiacetic acid (IDA), for the immobilization of His-tagged Bacillus stearothermopilus L1 lipase. Microporous (average pore diameter of 60 Å) silica gel with a mean particle diameter of 115 µm has been used as a comparative support material. The molar ratio of Cu²⁺ to IDA was found to be 1:1.14 and 1:1.99 in the silica gel and the silica-coated magnetic nanoparticles (SiMNs), respectively. The specific activity of the immobilized enzyme was found to conform to the following order: Cu²⁺-charged SiMN>SiMN>Cu²⁺-charged silica gel>silica gel. When it was immobilized on the Cu²⁺-charged SiMNs, over 70% of the initial activity of the lipase remained after it had been reused five times. However, only 20% of the initial activity remained after the enzyme immobilized on the Cu²⁺-charged silica gel had been reused five times. For the enzyme immobilized on supports without Cu²⁺ cations, all activity was lost after threefold reuse. The differences in the specific activities and the efficiencies of reuse of the enzymes immobilized on the various support materials are discussed in terms of immobilization mechanisms (physical adsorption vs. coordination bonding), mass transfer of a substrate and a product of the enzyme reaction, and the status of the Cu (Cu bound to the IDA on the silica layer vs. Cu directly adsorbed on the silica layer).     

Effects of supercritical CO2 exposure and depressurization on immobilized lipase activity

Lipozyme IM20 from Novo Nordisk (Denmark) was examined after various treatments. Conditions were chosen to reflect those that would be considered in the design of an industrial process. A two-level factorial design was employed to assess the effects of pressurization/depressurization cycles, rate of depressurization and exposure length. A significant three-factor interaction was observed. Lowest residual activity was observed for runs in which the depressurization rate was 86–89 bar min^–1. Incubation for 12 h also yielded low residual activity but only when exposing the immobilized enzyme to one cycle. The highest residual activity was obtained for immobilized enzymes repeatedly exposed for periods of 12 h (5 times) with a depressurization rate of 4.3 to 4.45 bar min^–1. This effect may be due to the extraction of an inhibiting compound. Tuning process parameters can lead to a seven-fold change in residual activity.     

Comparison between two processes for the enzymatic synthesis of tri-docosahexaenoylglycerol in a solvent-free medium

The enzymatic synthesis of homogeneous tri-docosahexaenoylglycerol from glycerol and ethyl docosahexaenoate in a solvent-free medium was achieved using Novozym SP 435 (immobilized lipase from Candida antarctica). Two processes were tested: a constant stirred tank reactor with N₂ bubbling through and a constant stirred tank reactor under vacuum. The first experimental apparatus was clearly found to be better than the other, giving 100% (w/w) conversion after 10 h.     

Purification,characterization and thermostability of lipase from a thermophilic Bacillus sp. J33

A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The K_m and V_max for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 M min^-1 ml^-1 respectively. The immobilized enzyme was stable for 12 h at 60°C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70°C for 150 min in the presence of additives.     

A simple enzymatic procedure for the synthesis of a hydroxylated polyester from glycerol and adipic acid

Lipase B from Candida antarctica catalyzed regioselectively the polyesterification of glycerol and adipic acid. UV-MALDI-TOF-MS analysis of the polymers shows low molecular weight polyesters (1314-1716) with very narrow polydispersities (1.0–1.2).     

Development of a recovery process for novobiocin

A periodic countercurrent whole beer process was developed for the recovery of novobiocin to eliminate the high cost of mycelium filtration and accompanying antibiotic losses in the filter cake. In such a process screened, but unfiltered, novobiocin fermentation beer is contacted with a special grade of an anionic-exchange resin in a series of specially designed, well mixed columns. Each column is fitted with a screen sized to retain resin within the column but allow the cells to press. Periodically the lead column is isolated from the column train, washed free of beer solids, and eluted. A freshly eluted column is placed in the trail position to allow countercurrent operation. The eluate is then processed to crystalline product. A mathematical model for the sorption of novobiocin was developed based on a suitable continuity equation and mass transfer and equilibrium relationships determined in the laboratory. Digital computations of this model correlated well with laboratory and pilot plant data, and predicted well the performance of the production units. This simulation has been in continued use to predict and reoptimize plant operation as process changes (such as improved beer titers, increased production rate, and the evaluation of superior ion-exchange resins) occurred or were anticipated.     

A two-step enzymatic resolution process for large-scale production of (S)- and (R)-ethyl-3-hydroxybutyrate

An efficient two-step enzymatic process for production of (R)- and (S)-ethyl-3-hydroxybutyrate (HEB), two important chiral intermediates for the pharmaceutical market, was developed and scaled-up to a multikilogram scale. Both enantiomers were obtained at 99% chemical purity and over 96% enantiomeric excess, with a total process yield of 73%. The first reaction involved a solvent-free acetylation of racemic HEB with vinylacetate for the production of (S)-HEB. In the second reaction, (R)-enriched ethyl-3-acetoxybutyrate (AEB) was subjected to alcoholysis with ethanol to derive optically pure (R)-HEB. Immobilized Candida antarctica lipase B (CALB) was employed in both stages, with high productivity and selectivity. The type of butyric acid ester influenced the enantioselectivity of the enzyme. Thus, extending the ester alkyl chain from ethyl to octyl resulted in a decrease in enantiomeric excess, whereas using bulky groups such as benzyl or t-butyl, improved the enantioselectivity of the enzyme. A stirred reactor was found unsuitable for large-scale production due to attrition of the enzyme particles and, therefore, a batchwise loop reactor system was used for bench-scale production. The immobilized enzyme was confined to a column and the reactants were circulated through the enzyme bed until the targeted conversion was reached. The desired products were separated from the reaction mixture in each of the two stages by fractional distillation. The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps. Kilogram quantities of (S)-HEB and (R)-HEB were effectively prepared using this unit, which can be easily scaled-up to produce industrial quantities.     

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.     

Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

“大豆分离蛋白”工艺用复合酶制剂的研究

大豆分离蛋白的碱浸提酸沉淀工艺,目前普遍存在蛋白质回收率低,分离蛋白成品得率低,纯度低的问题,利用复合酶制剂的作用可改变这一状况。针对工艺需要,从十余种工业酶制剂中筛选出两种蛋白酶活低、多糖降解酶活高的酶制剂;Ａｎ－７６半纤维酶和果胶酶ｕｌｔｒａｚｙｒｍ＾ＴＭ。对两种酶制剂的酶活组分、主体酶的部分酶学性质,以及酶的用量与蛋白质浸出率的关系等方面进行了大量基础实验。经优化实验确定了复合酶制剂的复配比