Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design

Candida rugosa lipase was covalently immobilized on silanized controlled poresilica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized from in long-term operation. Proteins (albumin and lecithin) and organic molecules (β-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150–450 U/g of dry support) was performed by experimental design. Two 2²full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better, stabilizing effect was found when small amounts of albumin or PEG-1500, were added simul-taneou sly to the lipase on to the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-150 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).     

Synthesis of monobutyrylglycerol by transesterification with soluble and immobilized lipases

Transesterification between ethyl butyrate and glycerol using very different lipase preparations (native, modified, and immobilized) in a two-phase system—no extra solvent added—has been investigated. Optimal conversion was obtained with the presence of 5% water in the reaction mixture. Only monobutyrin was produced in all conditions tested. The best enzyme preparations were nativeCandida cylindracea lipase,Mucor miehei lipase immobilized on a phenol-formaldehyde exchange resin (Lipozyme?), andC. cylindracea lipase immobilized on Celite.     

Intensification of lipase performance for long-term operation by immobilization on controlled pore silica in presence of polyethylene glycol

In agreement with previous studies, promising results were obtained when lipase was immobilized on controlled pore silica (CPS) in the presence of polyethylene glycol (PEG 1500). This methodology rendered immobilized derivatives with higher operational stability than those lacking PEG 1500. This article extends the scope of this approach by evaluating the combined effects of PEG concentration and lipase loading employing a multivariate statistical approach. A 2² factorial design with center point was adopted for a full understanding of these effects and their interactions. Conditions that maximize the immobilization yield were different from those attained for the biocatalyst’s operational stability. Possible reasons for the increase in both activity and stability of lipase immobilized on CPS in the presence of PEG 1500 are discussed in light of the influence of surface hydrophilic/hydrophobic balance.     

Calix[n]arene Carboxylic Acid Derivatives as Regulators of Enzymatic Reactions: Enhanced Enantioselectivity in Lipase-Catalyzed Hydrolysis of (R/S)-Naproxen Methyl Ester

Candida rugosa lipase was immobilized with a sol–gel encapsulation procedure in the presence and absence of a calix[n]arene carboxylic acid derivative grafted onto magnetic nanoparticles or in the presence of the calix[n]arene carboxylic acid derivative with Fe₃O₄ magnetic nanoparticles as an additive. Through the enantioselective hydrolysis of racemic naproxen methyl ester and the hydrolysis of p-nitrophenylpalmitate, the relative enzyme activity was evaluated and tested. These results show that the encapsulated lipase without supports has lower conversion and enantioselectivity compared to the Calix[n]COOH-based encapsulated lipase. It has also been observed that the Calix[4]COOH-based encapsulated lipase has excellent enantioselectivity (enantiomeric ratio (E)?>?400) as compared to encapsulated-free lipase enantioselectivity (E?=?137), and it also has an enantiomeric excess value of ~98 % for S-naproxen.     

Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica

Candida rugosa lipase was immobilized by covalent binding on controlled poresilica (CPS) using glutaraldehyde ascross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5–8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10°C higher than that for the free lipase. The thermal stability of the CPS lipase was alsogreater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.     

Properties of a recombinant β-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis

A β-glucosidase (BglA, EC 3.2.1.21) gene from the polycentric anaerobic fungus Orpinomyces PC-2 was cloned and sequenced. The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and bacterial β-glucosidases but lacked significant similarity to those from aerobic fungi. Neither cellulose- nor protein-binding domains were found in BglA. When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast cells. K _m and V _max values of the secreted BglA were 0.762 mM and 8.20 μmol/(min·mg), respectively, with p-nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate and 0.310 mM and 6.45 μmol/(min·mg), respectively, for the hydrolysis of cellobiose. Glucose competitively inhibited the hydrolysis of pNPG with a K _i of 3.6 mM. β-Glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential for use in biofuel and feedstock chemical production. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may be suitable.     

Lipase immobilized on poly(VP-co-HEMA) hydrogel for esterification reaction

Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate)(poly[VP-co-HEMA]) hydrogel, and divinylbenzene was the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases was also determined. Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90∶10 exhibited the highest activity. Lipase immobilized on VP(%):HEMA(%) 50∶50 showed the highest thermal, solvent, storage, and operational stability compared to lipase immobilized on other compositions of hydrogels as well as the native lipase.     

A versatile synthesis of poly(lauryl acrylate) using N-(n-octyl)-2-pyridylmethanimine in copper mediated living radical polymerization

A facile synthesis of poly(lauryl acrylate) has been achieved by atom transfer radical polymerization using benzyl-2-bromoisobutyrate, copper (I) bromide, and N-(n-octyl)-2-pyridylmethanimine (OPMI). The latter was of great interest as its synthesis was very easy to carry out and as it allowed the reaction mixture to be homogeneous, which was essential for the control of the reaction. The polymerization was controlled under these conditions and was optimized with the addition of copper (II) bromide as deactivator. We proved that the synthesis of poly(lauryl acrylates) with well defined molecular weights and narrow polydispersities was possible using a ligand which does not require difficult synthesis and purification. We also showed the ability of pyridylmethanimine ligands to control ATRP of an acrylate derivative. Best results were obtained at 130 °C in xylene for [Initiator]₀/[Cu(I)Br]₀/[Cu(II)Br₂]₀/[OPMI]/[lauryl acrylate] equal to 1/1/0.05/2.2/181, respectively (M_n = 19,942, DPI = 1.28).     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Poly(Methyl Methacrylate) as a matrix for immobilization of lipase

Poly(methyl methacrylate) (PMMA) was found to be suitable for the immobilization of lipase fromCandida rugosa. The best result based on hydrolytic activity was obtained by adsorption of the purified unbuffered enzyme solution onto PMMA beads without any modification of the beads. Prolonged exposure of the protein to the beads increased its adsorption but the expressed activity decreased after 1 h of exposure. The magnitude of the immobilized activity also varied with the size of the beads. Immobilization of the lipase shifted its optimal reaction temperature from 37 to 45°C. The immobilized enzyme is also more stable than the free enzyme in solution. The operational half-life of the immobilized lipase packed in a column and assayed in a closed system is 40 d.     

Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> Lipase onto Chitosan

Lipase from Rhizomucor miehei (RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v^?1 at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.     

Preparation and characterization of lipase immobilized on reversibly soluble-insoluble N-(2-carboxylbenzoyl) chitosan

N-(2-carboxylbenzoyl) chitosan (CBC), a reversibly soluble-insoluble polymer with pH change, was prepared by modifying chitosan backbone with phthalic anhydride and employed as carrier for lipase immobilization. The obtained CBC exhibited reversible solubility in aqueous solution; it was soluble at pH above 3.8 and precipitated at pH below 3.4. The porcine pancreatic lipase was covalently immobilized on CBC with glutaraldehyde as the crosslinking agent. Under the optimal immobilization condition, the retention activity of the immobilized lipase was found to be 69.8 %. The maximum activity of lipase immobilized on CBC was observed at 40 °C, pH 8.0, while the free lipase presented maximum activity at 37 °C, pH 7.5. The lipase immobilized on CBC exhibited improved thermal and storage stabilities and retained 58.7 % of its initial activity after 9 times of repeated use.     

Development of electrospun poly(vinyl alcohol) fibers immobilizing lipase highly activated by alkyl-silicate for flow-through reactors

Electrospun fibrous membranes composed of poly(vinyl alcohol) (PVA) fibers of approximately 1 μm in diameter, and immobilizing highly activated lipase entrapped in silicate cages with smaller dimensions than the fibers, were developed; and their feasibility as a component of flow-through reactors was studied. The electrospun PVA fibers were prepared from a mixture of PVA solution and a sol obtained from silicon alkoxide(s)—either tetramethoxysilane (TMOS) or dimethyldimethoxysilane (DMDMOS), or both, containing lipase. The fastest initial transesterification rate converting (s)-glycidol to glycidyl n-butyrate with vinyl n-butyrate in batchwise reactions was accomplished by treatment of lipase using the sol obtained from DMDMOS and TMOS together. The values were 4.5-, 21.8-, and 1.8-fold faster than those of systems using lipases that were either non-modified or modified using TMOS alone or DMDMOS alone, respectively. The higher activity of the lipase modified using both DMDMOS and TMOS and immobilized in PVA fibers resulted in a flow-through reactor having a higher degree of conversion at the same retention time compared with that using immobilized non-modified lipase. These results show the feasibility of flow-through reactors composed of electrospun PVA fibers immobilizing lipase highly activated by alkyl-silicate.     

Kinetic properties of soluble and immobilizedCandida rugosa lipase

Immobilized lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) fromCandida rugosa has been immobilized on commercially available microporous polypropylene and used for the batch hydrolysis of different animal fats. The effect of the reaction products at concentrations similar to those obtained at 90% hydrolysis, both on soluble and immobilized lipase, was studied. Glycerol showed low inhibitory effect but oleic acid caused 50% inhibition. A mixture of free fatty acids present in the complete hydrolysis of beef tallow inhibited lipase activity more than 70%. The stability of the enzyme (both soluble and immobilized) was highest in the presence of 20% isooctane. The apparent Michaelis constant for each substrate for the soluble enzyme did not change on immobilization.     

Synthesis of SBA-15 from low cost silica precursor obtained from sugarcane leaf ash and its application as a support matrix for lipase in biodiesel production

Ordered mesoporous silica material was synthesized from a low-cost precursor, sugarcane leaf ash, was used as a support matrix for lipase for the production of biodiesel. The mesoporous samples were characterized using Fourier transform infra red spectroscopy. The surface topography and morphology of the mesoporous materials were studied using scanning electron microscope. The pore diameter, pore volume, Brunauer Emmett and Teller surface area of the mesoporous material were determined by N₂ gas adsorption technique. Different pore size Santa Barbara Acid-15 (SBA-15) samples were synthesized and their lipase immobilization capacity and specific enzyme activity of immobilization lipase were determined and compared. Lipase from Candida Antarctica immobilized on SBA-15 (C) had shown maximum percentage immobilization and specific enzyme activity. The immobilized lipase mesoporous matrix was used for biodiesel production from crude non-edible Calophyllum inophyllum oil. The percentage yield of fatty acid methyl ester, 97.6 % was obtained under optimized conditions: 100 mg of lipase immobilized on SBA-15, 6:1 methanol to oil molar ratio, the reaction of 2 g C. inophyllum oil with methanol.     

Multicomponent thermosensitive systems for biocatalysts

Composite matrices based on macroporous silica modified by N-vinylcaprolactam copolymers with diallyldimethylammonium chloride and with 2-hydroxyethyl methacrylate were obtained. Lipase from Pseudomonas fluorescens was immobilized on the obtained materials. The temperature dependence of the hydrolytic activity of the immobilized lipase preparations in the triacetin hydrolysis was investigated. The hydrolytic activity of lipase immobilized on the matrix modified by the N-vinylcaprolactam copolymer with 2-hydroxyethyl methacrylate can be regulated by varying the temperature of the reaction medium. The temperature dependence of the hydrolytic activity of the immobilized enzyme has a maximum at 40 °C, the activity of the immobilized lipase being ∼3.5 times higher compared to that at 20 °C. After immobilization on these composite materials, lipase retained the activity in the acetylation of 1-(RS)-phenylethanol with vinyl acetate in Bu^tOMe.__________Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 443–448, February, 2005.     

高浓度盐系统中指肪酶的固定化及其催化活力

选择了四十多个可溶性的盐，实现了脂肪酶在高浓度盐系统中的固定化，并以 固定化脂肪酶的盐为催化剂，研究了正已烷中脂肪酶催化丁醇和乙酸乙烯酯间的转 酯化制备乙酸丁酯的反应和水溶液中橄榄油的水解反应，考察了高浓度盐系统中脂 肪酶的催化活力。     

Study on controlled/living free-radical polymerization of methyl acrylate in the presence of benzyl 9H-carbazole-9-carbodithioate under thermal condition

The polymerizations of methyl acrylate have been studied under thermal condition using benzyl 9H-carbazole-9-carbodithioate (BCCDT) as control agent. The obtained polymers were characterized by gel permeation chromatography (GPC), ¹H nuclear magnetic resonance (¹H NMR) and MALDI-TOF mass spectra. The results at 60 °C show that the molecular weight of the polymer increases linearly with monomer conversion, the molecular weight distribution is fairly narrow (even inferior to 1.10), and there exists a linear relationship between ln ([M]₀/[M]) and polymerization time. It is worthy of being noticed that the narrow polydispersities are comparable with those from living anionic polymerization. All of the evidences indicate that the polymerization is a good ‘living’ process. When the experiment is carried out at higher temperature, the polymerization rate is markedly faster with controlled polymerization characters except for a relatively broader polydispersity. The good control for free radical polymerization may be correlated with the large conjugation structure of carbazyl of BCCDT.     

A specific enzyme electrode for urea

A truly specific, simple enzyme electrode is described for the assay of urea in blood serum. The sensor used is the newly developed air-gap electrode of R??i?ka and Hansen, and has advantages of speed of response and specificity over earlier enzyme electrodes for urea. Potassium, sodium and ammonium ions and other organic and inorganic species present in blood do not interfere. Linear curves are obtained from 2 · 10^-2M to 1 · 10^-4M urea with slopes close to Nernstian (about 0.90 pH/decade). Urea in blood was assayed with an accuracy of 2.2% and a precision of 2.0% with immobilized urease; only 3–5 min is required per assay. The electrode was used for a month and almost 500 assays with excellent results. Since the sensor never touches the sample solution, problems caused by blood components which block membrane pores are avoided.     

Part I. A specific electrode for urea.

A specific simple enzyme stirrer electrode is described for the assay of urea in blood serum. The enzyme is placed directly on a magnetic stirrer and held in place with a nylon net. The enzyme stirrer both stirs the solution and effects an enzymatic transformation, permitting the direct assay of a substrate such as urea. Potassium, Na⁺ , NH₄⁺ and other organic and inorganic species present in blood do not interfere. Linear curves are obtained from 5· 10^-2M to 1· 10^-4M urea with slopes close to Nernstian, 0.95 pH/decade. Urea in blood was assayed with an accuracy of 1.8% and a precision of 2.0% with immobilized urease in the stirrer. The stirrers were used for 15 weeks and over 500 assays with excellent results.