Properties of poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel‐bound lipase of Pseudomonas aeruginosa MTCC‐4713 and its use in synthesis of methyl acrylate

Microbial lipases (E.C. 3.1.1.3) are preferred biocatalysts for the synthesis of esters in organic solvents. Various extracellular thermoalkaliphilic lipases have been reported from Pseudomonas sp. In the present study, a purified alkaline thermoalkalophilic extracellular lipase of Pseudomonas aeruginosa MTCC‐4713 was efficiently immobilized onto a synthetic poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel by adsorption and the bound lipase was evaluated for its hydrolytic potential towards various p‐nitrophenyl acyl esters varying in their C‐chain lengths. The bound lipase showed optimal hydrolytic activity towards p‐nitrophenyl palmitate (p‐NPP) at pH 8.5 and temperature 45°C. The hydrolytic activity of the hydrogel‐bound lipase was markedly enhanced by the presence of Hg²⁺, Fe³⁺, and NH salt ions in that order. The hydrogel‐immobilized lipase (25 mg) was used to perform esterification in various n‐alkane(s) that resulted in ～ 84.9 mM of methyl acrylate at 45°C in n‐heptane under shaking (120 rpm) after 6 h, when methanol and acrylic acid were used in a ratio of 100 mM:100 mM, respectively. Addition of a molecular sieve (3Å × 1.5 mm) to the reaction system at a concentration of 100 mg/reaction vol (1 mL) resulted in a moderate enhancement in conversion of reactants into methyl acrylate (85.6 mM). During the repetitive esterification under optimum conditions, the hydrogel‐bound lipase produced 71.3 mM of ester after 10th cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 183–191, 2007     

Characteristics of poly(AAc5‐co‐HPMA3‐cl‐EGDMA15) hydrogel‐immobilized lipase of Pseudomonas aeruginosa MTCC‐4713

Four series of noble networks were synthesized with acrylic acid (AAc) copolymerized with varying amount of 2‐hydroxy propyl methacrylate or dodecyl methacrylate (AAc/HPMA or AAc/DMA; 5:1 to 5:5, w/w) in the presence of ethylene glycol dimethacrylate (EGDMA; 1, 5, 10, 15, and 20%, w/w) as a crosslinker and ammonium per sulfate (APS) as an initiator. Each of the networks was used to immobilize a purified lipase from Pseudomonas aeruginosa MTCC‐4713. The lipase was purified by successive salting out with (NH₄)₂SO₄, dialysis, and DEAE anion exchange chromatography. Two of the matrices, E_15a, i.e. [poly (AAc₅‐co‐DMA₁‐cl‐EGDMA₁₅)] and I_15c, i.e. [poly (AAc₅‐co‐HPMA₃‐cl‐EGDMA₁₅)], that showed relatively higher binding efficiency for lipase were selected for further studies. I_15c‐hydrogel retained 58.3% of its initial activity after 10th cycle of repetitive hydrolysis of p‐NPP, and I_15c was thus catalytically more stable and efficient than the other matrix. The I_15c‐hydrogel‐immobilized enzyme showed maximum activity at 65°C and pH 9.5. The hydrolytic activity of free and I_15c‐hydrogel‐immobilized enzyme increased profoundly in the presence of 5 mM chloride salts of Hg²⁺, NH₄⁺, Al³⁺, K⁺, and Fe³⁺. The immobilized lipase was preferentially active on medium chain length p‐nitrophenyl acyl ester (C:8, p‐nitrophenyl caprylate). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4636–4644, 2006     

Application of lipase immobilized on nylon‐6 for the synthesis of butyl acetate by transesterification reaction in n‐heptane

A purified alkaline thermotolerant bacterial lipase from Bacillus coagulans BTS‐3 was immobilized on nylon‐6 matrix activated by glutaraldehyde. The matrix showed ～ 70% binding efficiency for lipase. The bound lipase was used to perform transesterification in n‐heptane. The reaction studied was conversion of vinyl acetate and butanol to butyl acetate and vinyl alcohol. Synthesis of butyl acetate was used as a parameter to study the transesterification reaction. The immobilized enzyme achieved ～ 75% conversion of vinyl acetate and butanol (100 mmol/L each) into butyl acetate in n‐heptane at 55°C in 12 h. When alkane of C‐chain lower or higher than n‐heptane was used as an organic solvent, the conversion of vinyl acetate and butanol to butyl acetate decreased. During the repetitive transesterification under optimal conditions, the nylon bound lipase produced 77.6 mmol/L of butyl acetate after third cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Synthesis of (Z)‐3‐hexen‐1‐yl acetate by lipase immobilized in polyvinyl alcohol nanofibers

Polyvinyl alcohol (PVA)‐nanofibers‐immobilized lipase were formed by electrospinning. The specific surface area of the nanofiber (5.96 m²/g) was about 250 times larger than that of PVA‐film‐immobilized lipase (0.024 m²/g). The PVA‐nanofibers‐immobilized lipase were used as the catalyst for the esterification of (Z)‐3‐hexen‐1‐ol (leaf alcohol) with acetic acid in hexane. The activity of the nanofiber is equivalent to that of commercially available immobilized lipase (Novozym‐435). The ester conversions of the nanofibers, Novozym‐435, the film and lipase powder reached 99.5% at 5 h, 100% at 5 h, 11.5% at 6 h, and 81.1% at 5.75 h, respectively. The nanofibers‐immobilized lipase showed higher activity for the esterification than the film‐immobilized lipase and lipase powder, probably because it has high specific surface area and high dispersion state of lipase molecules in PVA matrix. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Evaluation of lipase from Burkholderia cepacia immobilized in alginate beads and application in the synthesis of banana flavor (isoamyl acetate)

The alginate in bead forms was used to immobilize Burkholderia cepacia lipase. The microencapsulation technique for lipase entrapment was a 2% (w/v) of sodium alginate concentration prepared by ionic gelation using calcium chloride as the cross-linking agent in a gelling solution. The beads were tested in different solvents as acetone, chloroform, toluene, n-hexane, and n-heptane. Over a 5-day period (120?h), the n-heptane maintained the reasonable (excellent) residual activity of the immobilized lipase. Morphological studies on reused beads and new beads were performed. All beads for isoamyl acetate yield were tested. The reused bead leaches substantially, with a maximum ester yield of 92%. With modifications in the molar ratios, the synthesis of banana flavor (isoamyl acetate) was performed in both the alcohol per acid and acid per alcohol excesses.     

Catalytic potential of a poly(AAc‐co‐HPMA‐cl MBAm)‐matrix‐immobilized lipase from a thermotolerant Pseudomonas aeruginosa MTCC‐4713

A purified alkaline thermo‐tolerant lipase from Pseudomonas aeruginosa MTCC‐4713 was immobilized on a series of five noble weakly hydrophilic poly(AAc‐co‐HPMA‐cl MBAm) hydrogels. The hydrogel synthesized by copolymerizing acrylic acid and 2‐hydroxy propyl methacrylate in a ratio of 5 : 1 (HG_5:1 matrix) showed maximum binding efficiency for lipase (95.3%, specific activity 1.96 IU mg^?1 of protein). The HG_5:1 immobilized lipase was evaluated for its hydrolytic potential towards p‐NPP by studying the effect of various physical parameters and salt‐ions. The immobilized lipase was highly stable and retained ～92% of its original hydrolytic activity after fifth cycle of reuse for hydrolysis of p‐nitrophenyl palmitate at pH 7.5 and temperature 55°C. However, when the effect of pH and temperature was studied on free and bound lipase, the HG_5:1 immobilized lipase exhibited a shift in optima for pH and temperature from pH 7.5 and 55°C to 8.5 and 65°C in free and immobilized lipase, respectively. At 1 mM concentration, Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions promoted and Co²⁺ ions inhibited the hydrolytic activities of free as well as immobilized lipase. However, exposure of either free or immobilized lipase to any of these ions at 5 mM concentration strongly increased the hydrolysis of p‐NPP (by ～3–4 times) in comparison to the biocatalysts not exposed to any of the salt ions. The study concluded that HG_5:1 matrix efficiently immobilized lipase of P. aeruginosa MTCC‐4713, improved the stability of the immobilized biocatalyst towards a higher pH and temperature than the free enzyme and interacted with Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions to promote rapid hydrolysis of the substrate (p‐NPP). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4252–4259, 2006     

Biotransformation of (+)‐ and (−)‐bornyl acetate using the plant parasitic fungus Glomerella cingulata as a biocatalyst

The microbial transformations of (+)‐ and (?)‐bornyl acetate were investigated using the plant parasitic fungus, Glomerella cingulata. As a result, (+)‐ and (?)‐bornyl acetate were converted to (+)‐ and (?)‐5‐exo‐hydroxybornyl acetate, (+)‐ and (?)‐5‐oxobornyl acetate and (+)‐ and (?)‐borneol respectively. The structures of the metabolic products were determined by spectroscopic data. © 2001 Society of Chemical Industry     

Highly potent and selective 4,4'‐biphenyl‐4‐acylate‐4'‐N‐n‐butylcarbamate inhibitors of Pseudomonas species lipase

4,4'‐Biphenyl‐4‐acylate‐4'‐N‐n‐butylcarbamates ( 1–8 ) are synthesized and characterized as highly potent and selective pseudo‐substrate inhibitors of Pseudomonas species lipase. Thus, the n‐butylcarbamate moieties of the inhibitors bind to the first acyl chain binding site (ACS) of the enzyme. Therefore, the ester moieties of the inhibitors may bind to the second ACS of the enzyme, due to the linear 4,4'‐biphenyl moiety of the inhibitors. –logK_i, logk₂, and logk_i values of carbamates 1–8 are multiply linearly correlated with the Taft steric constant (E_S) and the Hansch hydrophobicity constant (π), but not with the Taft substituent constant (σ*). For –logK_i, logk₂, and logk_i correlations, values of δ are 0.8, 0.34, and 1.0, respectively, and values of ψ are 1.0, 0.4, and 1.3, respectively. Positive δ and ψ values for these correlations indicate that the second ACS of the enzyme prefers to bind to small and hydrophobic ester groups of the inhibitors. Among carbamates 1–8 , carbamate 3 , with a K_i value of 2.5 nM, is the most potent inhibitor.     

Improved activity and stability of pseudomonas capaci lipase in a novel biocompatible ionic liquid, 1‐isobutyl‐3‐methylimidazolium hexafluorophosphate

BACKGROUND: Enzymes may exhibit enhanced activity, stability and selectivity in ionic liquids, depending on the properties of the liquid. The physical–chemical properties of ionic liquids, however, may be modified by altering the anion or cation in the ionic liquid. This feature is a key factor for realizing successful reactions. In this work, a new ionic liquid, 1‐isobutyl‐3‐methylimidazolium hexafluorophosphate (abbreviated as [i‐C₄mim][PF₆]), was synthesized and investigated as a novel medium for the transesterification reaction of 2‐phenylethanol with vinyl acetate catalyzed by pseudomonas capaci lipase. As contrasts, the reaction was also carried out in two reference solvents; the isomeric ionic liquid [i‐C₄mim][PF₆], 1‐butyl‐3‐methylimidazolium hexafluorophosphate (abbreviated as [C₄mim][PF₆]), and hexanes. RESULTS: As reaction medium, [i‐C₄mim][PF₆] was best among the three solvents. The initial reaction rate, the equilibrium conversion of 2‐phenylethanol and the half‐lifetime of the lipase in [i‐C₄mim][PF₆] medium were about 1.5, 1.2 and 3‐fold that obtained in [C₄mim][PF₆] medium, respectively. The lipase in [i‐C₄mim][PF₆] medium was recycled 10 times without substantial diminution in activity. CONCLUSION: The ionic liquid [i‐C₄mim][PF₆] has good biocompatibility, and can be used widely as green media in various biocatalysis reactions to improve the activity and stability of enzymes. Besides hydrophobicity and nucleophilicity, the spatial configuration of ionic liquids is also considered a key factor effecting the behaviour of the enzyme in ionic liquids. Copyright © 2008 Society of Chemical Industry     

Immobilisation of different Candida rugosa lipases by adsorption onto polypropylene powder: application to chiral synthesis of ibuprofen and trans‐2‐phenyl‐1‐cyclohexanol esters

Candida rugosa lipases (CRLs) were immobilised by adsorption onto a commercial polypropylene powder EP100?. Two commercial CRLs from Sigma and Amano were used together with two CRLs obtained by fed‐batch fermentation using oleic acid as a carbon source (UAB‐CRL). Significant differences were observed in the isotherm adsorption patterns for the commercial and fermented lipases, probably caused by differences in their polysaccharide content. The commercial lipases showed a classical Langmuir adsorption pattern, whereas fed‐batch produced lipases with high polysaccharide content tended to conform to a BET multilayer adsorption equation. Immobilised CRLs also showed different behaviour in the resolution of two interesting pharmaceutical products: ibuprofen and trans‐2‐phenyl‐1‐cyclohexanol (TPCH) in the enantioselective esterification reaction in organic media. In the case of ibuprofen, CRLs showed important differences in terms of esterification rate, probably due to diffusional limitation effects caused by the high polysaccharide content present in UAB‐CRLs. In the case of TPCH, however, polysaccharide content did not appear to influence the esterification rate. A high enantioselective esterification was observed for all CRLs tested in the resolution of both products. © 2002 Society of Chemical Industry     

Synthesis and biodegradability evaluation of 2‐methylene‐1,3‐dioxepane and styrene copolymers

Biodegradable copolymers of 2‐methylene‐1,3‐dioxepane (MDO) and styrene (ST) were synthesized by free‐radical copolymerization using di‐t‐butyl peroxide (DTBP) as the initiator. The copolymers containing ester units were characterized by Fourier transform infrared (FTIR), ¹H‐NMR, and ¹³C‐NMR spectroscopy. Their molecular weight and polydispersity index were determined by gel permeation chromatography (GPC). In vitro enzymatic degradation of poly(MDO‐co‐ST) was performed at 37°C in phosphate buffer solution (PBS, pH = 7.4) in the presence of Pseudomonas lipase or crude enzyme extracted from earthworm. The experiment showed that incorporating ester units into C? C backbone chain of polystyrene would result in a biodegradable copolymer. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1146–1151, 2007     

Immobilization of Pseudomonas cepacia lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil

The immobilization of Lipase PS from Pseudomonas cepacia by entrapment within a chemically inert hydrophobic solgel support was studied. The gel-entrapped lipase was prepared by the hydrolysis of tetramethoxysilane (TMOS) with methyltrimethoxysilane (MTMS), isobutyltrimethoxysilane (iso-BTMS), and n-butyltrimethoxysilane. The immobilized lipase was subsequently used in the hydrolysis of soybean oil to determine its activity, recyclability, and thermostability. The biocatalyst so prepared was equal to or better than the free enzyme in its hydrolytic activity. The catalytic activity of the entrapped lipase strongly depended on the type of precursor that was used in its preparation. The lipase entrapped within TMOS/iso-BTMS showed the highest activity. The catalytic activity of the immobilized lipase was more pronounced during the earlier stages of the reaction. Thermostability of the lipase was significantly improved in the immobilized form. The immobilized lipase was stable up to 70°C, whereas for the free enzyme, moderate to severe loss of activity was observed beyond 40°C. The immobilized lipase was consistently more active and stable than the free enzyme. The immobilized lipase also proved to be very stable, as it retained more than 95% of its initial activity after twelve 1-h reactions.     

Copper‐Catalyzed Selective Synthesis of Isoindolin‐1‐ones and Isoquinolin‐1‐ones from the Three‐Component Coupling of 2‐Halobenzoic Acid,Alkynylcarboxylic Acid and Ammonium Acetate

Isoindolin‐1‐ones and isoquinolin‐1‐ones were selectively synthesized from the reaction of 2‐halobenzoic acid, arylalkynylcarboxylic acid and ammonium acetate (NH₄OAc) in the presence of cesium carbonate (Cs₂CO₃) and a copper catalyst. Conducting the reaction under one‐pot conditions provided isoindolin‐1‐ones in good yields. Changing the addition sequence of ammonium acetate after all reagents had reacted at 120 °C for 6 h selectively produced isoquinolin‐1‐ones. A variety of arylalkynylcarboxylic acids produced the corresponding isoindolin‐1‐ones and isoquinolin‐1‐ones in good yields.

     

Esterification of n‐butyric acid with n‐butyl alcohol and transesterification of (R, S)‐phenylethanol by lipase immobilized on cellulose acetate–TiO2 gel fibre

Lipase (EC 3.1.1.3) was immobilized on cellulose acetate–TiO₂ gel fibre by the sol–gel method. The immobilized lipases were used for esterification of n‐butyric acid with n‐butyl alcohol and enantioselective acylation of (R, S)‐phenylethanol using vinyl acetate as an acyl donor. Compared with native lipase, the activity of the immobilized lipase was stable and relatively unaffected by the water content of the solvent and the substrate concentration. The data indicate that the lipases are immobilized on the fibre surface and that enzyme activity is influenced by bound water. However, the thermal reactivity and enantioselectivity of the immobilized lipase were less than those of native lipase. This may not reflect thermal inactivation of the enzyme but rather significant thermal contraction of the gel fibre by cellulose crystallization, resulting in liberation of bound water and a decrease in the amount of enzyme which is available for the reaction. Copyright © 2001 Society of Chemical Industry     

Enzymatic synthesis of (R)- and (S)-2-cyclohepten-1-ol

The synthetically useful chiral synthons, (R)- and (S)-2-cyclohepten-1-ol, can be prepared by an enantioselective transesterification of racemic trans-2-(phenylseleno) cycloheptanol using lipases, followed by selenoxide elimination and hydrolysis.     

Chemoenzymatic synthesis of novel N‐(2‐hydroxyethyl)‐β‐peptoid oligomer derivatives and application to porous polycaprolactone films

Poly[N‐(2‐hydroxyethyl)‐β‐propylamide] oligomer is synthesized using a simple enzymatic procedure involving Candida antarctica lipase B. This novel compound is obtained by a green and chemoselective method from economic reactants in good yield. The β‐peptoid oligomer is characterized by spectroscopic methods showing low molecular weight and low dispersity. Two derivatives of the β‐peptoid oligomer are prepared by acetylation and by grafting polycaprolactone by ring opening polymerization from the pendant hydroxyl groups. These products are blended with polycaprolactone to make films by solvent casting. The inclusion of the acyl derivatives of the β‐peptoid to polycaprolactone affects the morphology of the film yielding microstructured and nanostructured patterns. © 2013 Society of Chemical Industry     

Lipase‐catalyzed dynamic hydrolytic resolution of (R,S)‐2,2,2‐trifluoroethyl α‐chlorophenyl acetate in water‐saturated isooctane

The hydrolytic resolution of (R,S)‐2,2,2‐trifluoroethyl α‐chlorophenylacetate in water‐saturated isooctane containing Lipase MY(I) at 35 °C is selected as the best reaction condition for producing (R)‐α‐chlorophenyl acetic acid. The kinetic constants, and hence an enantiomeric ratio of 33.6, are estimated and employed for the modeling of time‐course conversions of both substrates by considering product inhibition and enzyme deactivation effects. A successful dynamic kinetic resolution is also achieved, giving the desired (R)‐α‐chlorophenylacetic acid of 93.0% yield and ee_P = 89.5% when 80 mmol dm^?3 trioctylamine acting as the racemization catalyst and enzyme activator is initially added. Copyright © 2006 Society of Chemical Industry