Synthesis of Geraniol Esters in a Continuous-Flow Packed-Bed Reactor of Immobilized Lipase: Optimization of Process Parameters and Kinetic Modeling

With increasing demand for perfumes, flavors, beverages, and pharmaceuticals, the various associated industries are resorting to different approaches to enhance yields of desired compounds. The use of fixed-bed biocatalytic reactors in some of the processes for making fine chemicals will be of great value because the reaction times could be reduced substantially as well as high conversion and yields obtained. In the current study, a continuous-flow packed-bed reactor of immobilized Candida antarctica lipase B (Novozym 435) was employed for synthesis of various geraniol esters. Optimization of process parameters such as biocatalyst screening, effect of solvent, mole ratio, temperature and acyl donors was studied in a continuous-flow packed-bed reactor. Maximum conversion of ~ 87% of geranyl propionate was achieved in 15 min residence time at 70 °C using geraniol and propionic acid with a 1:1 mol ratio. Novozym 435 was found to be the most active and stable biocatalyst among all tested. Ternary complex mechanism with propionic acid inhibition was found to fit the data.     

预聚-酶催化缩聚法合成超支化聚酯及其结构表征

以丙三醇、1,6-己二醇和己二酸为共聚单体,以固定化脂肪酶Novozym435为催化剂,尝试先进行共聚单体的预聚后在有机介质中进行酶催化直接缩聚反应合成脂肪族超支化聚酯的新途径,考察了反应介质和反应温度对酶催化缩聚反应的影响,并采用凝胶渗透色谱和核磁共振确定产物的分子量和结构.结果表明,将单体的预聚与酶催化缩聚反应相结...     

New chiral building blocks from acetovanillone using lipase A and B from Candida antarctica

Acetovanillone has been used as the starting material for the synthesis of a series of secondary alcohols, which were resolved by lipase catalyzed esterification. 1-(4-Benzyloxy-3-methoxyphenyl)ethanol was efficiently resolved using immobilized lipase B from Candida antarctica (Novozym 435, CAL-B), whereas immobilized lipase A from C. antarctica (Novozym 735, CAL-A) was the lipase of choice for the resolution of the corresponding 2-bromo- and 2-chloro-derivatives. The enantioenriched alcohols are new building blocks for potential use in the synthesis of bioactive compounds.     

Chemoenzymatic Synthesis of 2-Arachidonoylglycerol, An Endogenous Ligand for Cannabinoid Receptors

A simple and efficient synthesis of 2-arachidonoyl glycerol, an endogenous agonist for cannabinoid receptors was achieved using Novozym 435, immobilized lipase from Candida antarctica.     

One‐step synthesis of polycarbonates bearing pendant carboxyl groups by lipase‐catalyzed ring‐opening polymerization

The ring‐opening polymerization of a monomer containing a free carboxylic acid group is reported for the first time. The monomer, 5‐methyl‐5‐carboxyl‐1,3‐dioxan‐2‐one (MCC), was copolymerized with trimethylene carbonate (TMC) in an enzymatic ring‐opening polymerization conducted in bulk at 80 °C. The low‐melting TMC comonomer also solubilized the high‐melting MCC monomer, allowing for solvent‐free polymerizations. Six commercially available lipases were screened, and Candida antarctica lipase‐B (Novozym‐435) and Pseudomonas cepacia lipase were selected to catalyze the copolymerization because of their higher monomer conversions. Higher molecular weight polymers (weight‐average molecular weight = 7800–9200) were prepared when Novozym‐435 was used, with less MCC incorporated into the copolymer than used in the monomer feed. However, Pseudomonas cepacia lipase showed good agreement between the molar feed ratios and the molar composition, but the molecular weights (weight‐average molecular weight = 3600–4800) were lower than those obtained when Novozym‐435 was used. ¹³C NMR spectral data were used for microstructural analysis, which suggested the formation of random, linear, and pendant carboxylic acid groups containing polycarbonates with hydroxyl groups at both chain ends. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1267–1274, 2002     

Simple and economical CALB/polyethylene/aluminum biocatalyst for fatty acid esterification

Candida antarctica lipase B was immobilized for the first time (at the author's knowledge) onto linear low density polyethylene (LLDPE) films. Polymer films were previously bonded to a commercial aluminum sheet using a simple support preparation method. Biocatalyst performance was evaluated in penthyl oleate synthesis at room temperature. Two different catalyst geometries were tested and compared: one aluminum‐polyethylene 50 mm × 50 mm foil (50CAT) or near 5 mm × 5 mm aluminum‐polyethylene foils (5CAT). The obtained results demonstrate that the biocatalyst obtained with 50 mm × 50 mm aluminum‐polyethylene foil or 50CAT is reusable in up to 7 cycles, easy to separate from reaction products, and economical in comparison with commercial Novozym 435. Novel and economical CALB/LLDPE/Al biocatalyst is an attractive alternative for possible applications in a continuous monolithic reactor and future industrial scaling up.     

Polyesters by lipase-catalyzed polycondensation of unsaturated and epoxidized long-chain α,ω-dicarboxylic acid methyl esters with diols

Long-chain, symmetrically unsaturated α,ω-dicarboxylic acid methyl esters (C₁₈, C₂₀, C₂₆) were obtained by the catalytic metathetical condensation of 9-decenoic, 10-undecenoic, and 13-tetradecenoic acid methyl esters, respectively, with the homogeneous Grubbs catalyst bis(tricyclohexyl phosphine) benzylidene ruthenium dichloride dissolved in methylene chloride. The dicarboxylic acid esters were epoxidized chemoenzymatically with H₂O₂/methyl acetate with Novozym 435^®, an immobilized lipase B from Candida antarctica. Polyesters from symmetrically unsaturated or epoxidized α,ω-dicarboxylic acid methyl esters with 1,3-propanediol or 1,4-butanediol, respectively, were achieved by enzymatic polycondensation with the same biocatalyst applied. With 1,3-propanediol as a substrate, the linear unsaturated and epoxidized polyesters had molecular weights of 1950–3300 g/mol and melting points of 47–75 °C, whereas with 1,4-butanediol as a substrate, the resulting polyesters showed higher molecular weights, 7900–11,600 g/mol, with similar melting points of 55–74 °C. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 1601–1609, 2001     

Immobilization does not influence the enantioselectivity of CAL-B catalyzed kinetic resolution of secondary alcohols

Decreasing enantioselectivity (E-value) by increasing conversion has been observed in transesterification reactions of secondary alcohols catalyzed by a pure protein formulation of lipase B from Candida antarctica (Novozym 525 F). Addition of a range of enantiopure alcohols caused a temporary increase in selectivity of the transesterification reaction of 3-chloro-1-phenoxy-2-propanol with vinyl butanoate. The corresponding immobilized lipase B, (Novozym 435) showed a similar relationship between the E-value and degree of conversion.     

Convenient double-enzymatic synthesis of both enantiomers of 6-methyl-ε-caprolactone

Both enantiomers of 6-methyl-ε-caprolactone (6-MeCL) are obtained in high enantiomeric excess by the combination of an enzymatic ring opening of racemic 6-methyl-ε-caprolactone and subsequent enzymatic ring closure. Immobilized Candida antarctica lipase B (Novozym 435) was selected as the biocatalyst for both the ring-opening and the ring-closing reaction. This route provides ready access to enantiopure (S)-6-MeCL (ee = 99.6%) and (R)-6-MeCL (ee = 98.8%).     

Highly enantioselective enzymatic resolution of cis-fused octalols mediated by Candida antarctica lipase (Novozym 435)

Optically active cis-fused octalin acetates have been prepared by esterification of cis-fused octalols using Candida antarctica lipase (CALB—Novozym 435). The enzyme efficiently resolved the racemic octalols by kinetic resolution, to afford cis-fused octalols and cis-fused octalin acetates with high enantiomeric excesses (up to >99%) and good yields (>40%).     

CO2-expanded bio-based liquids as novel solvents for enantioselective biocatalysis

For the first time, CO₂-expanded bio-based liquids were reported as novel and sustainable solvents for biocatalysis. Herein, it was found that by expansion with CO₂, 2-methyltetrahydrofuran (MeTHF), and other bio-based liquids, which were not favorable solvents for immobilized Candida antarctica lipase B (Novozym 435) catalyzed transesterification, were tuned into excellent reaction media. Especially, for the kinetic resolution of challenging bulky secondary substrates such as rac-1-adamantylethanol, the lipase displayed very high activity with excellent enantioselectivity (E value > 200) in CO₂-expanded MeTHF (MeTHF concentration 10% v/v, 6 MPa), whereas there was almost no activity observed in conventional organic solvents.     

Enzymatic resolution of α-tetralols by CALB-catalyzed acetylation

A series of homochiral α-tetralols, as well as their respective acetates, has been obtained by esterification of racemic tetralols, using Candida antarctica lipase (CALB—Novozym^® 435) as the biocatalyst. This enzyme is shown to be highly efficient for the kinetic resolution of the substrates studied, affording the (+)-tetralols and the (+)-acetates in excellent enantiomeric excess (up to >99%) and very good yields (>40%).     

Enhanced selectivity in Novozym 435 catalyzed kinetic resolution of secondary alcohols and butanoates caused by the (R)-alcohols

In esterifications of secondary alcohols catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) the E-values decreased during the reaction. Hydrolysis of the corresponding butanoates showed the opposite effect. When an enantiopure (R)-alcohol, related but different, was added to the transesterification reaction, the E-value was significantly enhanced.     

New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel

The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation-anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym(?) 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol(?) oil 812 with methanol, catalyzed by Novozym(?) 435 in choline acetate/glycerol (1:1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel.     

脂肪酶催化缩聚法合成可完全降解的聚羟基丙酸酯(英文)

以3-羟基丙酸甲酯为聚合单体,建立了以固定化脂肪酶Novozym 435为催化剂的酶催化缩聚反应体系,合成可完全降解的高分子聚酯聚羟基丙酸酯,考察了反应条件和介质对反应性能的影响,结果表明,纯度大于95%的单体即可在温和条件下合成聚羟基羧酸酯;降低反应压力可有效提升产物产率和分子量.通过选择合适的有机溶剂介质和表面活性剂,可使产物分子量提升至13000(Mw)以上.脂肪酶催化剂重复利用能力优异,经6批次反应后,其相对活性保持在95%以上.     

The Purification and Characterization of Lipases from <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis>, and Their Immobilization and Use for Biodiesel Production from Coconut Oil

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)—purified 25.41-fold, recovery of 47.1%—and lipase B (32,000 Da)—purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca²⁺, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5–10.0 and 20–80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.     

A lipase catalyzed condensation reaction with a tricyclic diketone: yet another example of biocatalytic promiscuity

Novozym 435 (a commercially available immobilized form of Candida antarctica lipase B) was found to catalyze a condensation reaction of 5-hydroxy-endo-tricyclo[5.2.1.0^2,6]deca-4,8-dien-3-one with acetaldehyde (enzymatically produced from vinyl acetate in situ) under low water conditions, in presence of 10% organic co-solvent (N,N-dimethyl formamide or pyridine), to form a bis-adduct. Even though the condensation reaction occurred with pyridine (acting as a base catalyst) in the presence of acetaldehyde and in the absence of enzyme, the reaction was very slow as compared to the enzymatic process. Thus, while the non-enzymatic process took 4 days to achieve 100% conversion; in presence of enzyme it was possible within 4 h.     

Collagen-Immobilized Lipases Show Good Activity and Reusability for Butyl Butyrate Synthesis

Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.     

Precision enzymatic polymerization to polyesters with lipase catalysts

Ring-opening polymerization of lactones with different ring-size has been achieved via lipase catalysis. Small-size (4-membered) and medium-size lactones (6- and 7-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to the lipase-catalyzed polymerization. The polymerization behaviors strongly depended on the lipase origin and the ring-size of the lactones. In using Pseudomonas family lipases as catalyst, the polymerization of macrolides showing much lower anionic polymerizability proceeded much faster than that of ϵ-caprolactone. The enzymatic polymerizability of the lactones was evaluated by Michaelis-Menten kinetics. V_max increased as a function of the ring-size, whereas K_m values were not so different with each other. The granular immobilized lipase derived from Candida antarctica. showed the extremely efficient catalysis in the polymerization of ϵ-caprolactone. Single-step synthesis of methacryl- and ω-alkenyl-type polyester macromonomers was achieved by the lipase-catalyzed polymerization of 13-membered lactone in the presence of vinyl esters acting as terminator. Lipase also catalyzed a polycondensation of dicarboxylic acid and glycol in the aqueous medium, in which the dehydration took place in water.     

Synthesis of metal‐free poly(1,4‐dioxan‐2‐one) by enzyme‐catalyzed ring‐opening polymerization

To avoid the harmful effects of metallic residues in poly(1,4‐dioxan‐2‐one) (PPDO) for medical applications, the enzymatic polymerization of 1,4‐dioxan‐2‐one (PDO) was carried out at 60 °C for 15 h with 5 wt % immobilized lipase CA. The lipase CA, derived from Candida antarctica, exhibited especially high catalytic activity. The highest weight‐average molecular weight (M_w = 41,000) was obtained. The PDO polymerization by the lipase CA occurred because of effective enzyme catalysis. The water component appeared to act not only as a substrate of the initiation process but also as a chain cleavage agent. A slight amount of water enhanced the polymerization, but excess water depressed the polymerization. PPDO prepared by enzyme‐catalyzed polymerization is a metal‐free polyester useful for medical applications. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 1560–1567, 2000