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.     

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.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

A novel lipase-based stationary phase in liquid chromatography

In this paper, a new chiral stationary phase (CSP) based on Candida antarctica lipase B (CALB) bounding to the surface of macroporous silica gel was developed and its stereoselectivity in enantioseparation and asymmetrical hydrolysis was evaluated. Three CALB-based HPLC columns with different amounts of enzyme immobilized were prepared by employing the immobilization method, namely “in batch”. In this technique two chromatographic supports epoxy silica and aminopropyl silica were considered. This novel CSP was proven capable of hydrolyzing chiral esters asymmetrically as bioreactor and separating several aromatic alcohols and diniconazole enantiomers.     

Enzymatic resolution of (RS)-β-hydroxy selenides in organic media

Kinetic resolutions of a number of β-hydroxy selenides promoted by enzymes were performed using PPL (free Porcine pancreatic lipase), PSL (Amano PS—free Pseudomonas sp. lipase) and CALB (NOVOZYM 435^®—immobilized Candida Antarctica lipase type B) with (RS)-1-phenylselanyl-propan-2-ol. CALB gave the best results and provided both (R)- and (S)-enantiomers in high enantiomeric purity. A comparative study of the effect of temperature, solvent, enzyme immobilization and the structure of the substrates on the resolution is presented.     

Ring-opening polymerization of DD-lactide catalyzed by Novozyme 435

In contrast to LLA, DLA is converted in toluene solution under mild reaction conditions (50-70 degrees C) using Novozyme 435 (immobilized CALB) to form the corresponding polymer. The influence of several parameters, such as enzyme concentration, temperature and monomer concentration, on the polymerization rate and the monomer conversion was studied. In contrast to the Novozyme 435 catalyzed polymerization of epsilon-caprolactone, enzyme deactivation occurs. It is attributed to the deprivation of water from the enzyme. This work points out that by careful selection of the reaction conditions, it is possible to obtain poly(D-lactide) in reasonable molecular weights and in high yields using Novozyme 435 catalysis.     

Immobilization of Candida antarctica lipase B by covalent attachment to green coconut fiber

The objective of this study was to covalently immobilize Candida antarctica type B lipase (CALB) onto silanized green coconut fibers. Variables known to control the number of bonds between enzyme and support were evaluated including contact time, pH, and final reduction with sodium borohydride. Optimal conditions for lipase immobilization were found to be 2 h incubation at both pH 7.0 and 10.0. Thermal stability studies at 60 degrees C showed that the immobilized lipase prepared at pH 10.0 (CALB-10) was 363-fold more stable than the soluble enzyme and 5.4-fold more stable than the biocatalyst prepared at pH 7.0 (CALB-7). CALB-7 was found to have higher specific activity and better stability when stored at 5 degrees C. When sodium borohydride was used as reducing agent on CALB-10 there were no improvement in storage stability and at 60 degrees C stability was reduced for both CALB-7 and CALB-10.     

Optimization of Chemo‐Enzymatic Epoxidation of Cyclohexene Mediated by Lipases

Abstract

This work describes the lipase‐mediated epoxidation of cyclohexene. Lipases were used to generate peroxyoctanoic acid directly from octanoic acid and hydrogen peroxide and applied in situ to obtain cyclohexene oxide. Various parameters, which could affect this reaction, were studied such as lipases from different sources, organic solvents, temperature and acyl donor concentrations. Highest conversions to cyclohexene epoxide were achieved using a two‐phase system of toluene or xylene/water with ROL (Amano F‐Ap15 free Rhizopus orizae lipase) (84 and 80%) or CALB (Novozymes 435®‐immobilized Candida antarctica lipase type B) (>9 and 84%) as biocatalysts. Using PSL (Amano PS‐free Pseudomonas sp) the conversions were in the range of 12–53%, but an improvement was obtained by the use of the ionic liquid 1‐butyl‐3‐methylimidazolium tetrafluoroborate (20 to 41% in water/methyl dichloride).     

Immobilization of Candida antarctica lipase B on Polystyrene Nanoparticles

Polystyrene (PS) nanoparticles were prepared via a nanoprecipitation process. The influence of the pH of the buffer solution used during the immobilization process on the loading of Candida antarctica lipase B (Cal‐B) and on the hydrolytic activity (hydrolysis of p‐nitrophenyl acetate) of the immobilized Cal‐B was studied. The pH of the buffer solution has no influence on enzyme loading, while immobilized enzyme activity is very dependent on the pH of adsorption. Cal‐B immobilized on PS nanoparticles in buffer solution pH 6.8 performed higher hydrolytic activity than crude enzyme powder and Novozyme 435.

     

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.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

聚乙烯亚胺改性聚丙烯腈中空膜固定脂肪酶

聚丙烯腈是富腈基的高分子聚合物,易修饰改性,广泛应用于膜分离应用.我们以聚丙烯腈中空膜为载体,采用化学法交联聚乙烯亚胺并固定脂肪酶,固定过程中引入海藻酸钠,用CaCl_2进行后处理,得到固定化脂肪酶PAN-PEI-SA/E-CaCl_2载酶量为31.70(mg enzyme)/(g support),酶活为50.20 U/(g support),15次重复使用可保留58.77%的酶活,与游离酶相比耐酸性和耐温性有所提高,相同条件下与Nov 435相比,酶活更高,这表明最终得到的固定化脂肪酶有良好的工业应用前景.     

Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading

A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.     

Immobilization of Candida antarctica lipase B on the surface of modified sol–gel matrix

The use of modified sol–gel matrix to immobilize the enzyme Candida antartica lipase B (CALB) was investigated. Free hydroxyl groups on the matrix surface were exploited to covalently immobilize the enzyme. Based from the results, incorporating hydrophobic sol–gel precursor (ethyltrimethoxysilane) enhanced enzyme activity. An enzyme activity of 192.02 U/g beads with 80.88 % attachment was obtained. At alkaline pH, immobilization yield of enzyme increased. The attachment of enzyme on the surface of the matrix was confirmed by scanning electron microscope images. Covalently immobilized CALB on sol–gel supports has higher thermal stability with 2.7 times higher half-life compared to soluble enzymes at 60 °C. This enzyme immobilization system retains the enzyme residual activity even for repetitive use. Hence, the immobilization approach developed recommends its further application.     

Immobilization of Thermomyces lanuginosus Lipase by Different Techniques on Immobead 150 Support: Characterization and Applications

Thermomyces lanuginosus lipase (TLL) was immobilized on native and modified Immobead 150, with epoxy groups removed by hydrolysis and oxidized to add aldehyde on its surface. Immobilizations on both supports were performed by adsorption, adsorption and cross-linking, covalent attachment, multipoint covalent attachment, and, for the modified support, multipoint covalent attachment using ethylenediamine. Biocatalysts were evaluated for thermal and solvent stabilities, and the best biocatalyst was also tested after incubation in ionic liquids and used in the synthesis of butyl butyrate and isoamyl butyrate. Multipoint covalent immobilized TLL on the native Immobead 150 (Emulti) showed a half-life of 5.32 h at 70 °C, being approximately 30 times more stable than its soluble form; it showed high stability in acetone, hexane, and isooctane. Its enzymatic activity was up to 40 % when incubated in ionic liquids. Ester synthesis produced yields of esterification above 60 % in 24 h. Of all immobilization protocols, the Emulti performed best concerning the thermal, solvent, and ionic liquids stabilities. Emulti was successfully applied to the synthesis of butyl butyrate and isoamyl butyrate, which are very important products for the food and beverage industries.     

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.     

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme^®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme^®435 (L435) (lipase B from Candida antarctica), Lipozyme^®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

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.     

Hydrolysis of 1,2-diacetoxypropane by Immobilized Lipase on Cellulose Acetate-TiO2 Gel Fiber Derived from the Sol-Gel Method

Lipase from Rhizomucor miehei was entrap-immobilized on cellulose acetate-TiO₂ gel fiber by the sol-gel method. This fiber-immobilized lipase was stable in a phosphate buffer solution and easy to handle. The enantioselective hydrolysis of 1,2-diacetoxypropane catalyzed by this immobilized lipase could be performed in buffer solution unlike the lipase immobilized on an alginate matrices. The enantioselectivity was improved in presence of this fiber-immobilized lipase compared with the hydrolysis catalyzed by the native lipase. This finding indicates that the active site structure of lipase immobilized on fiber was retained to some extent, though the enzyme conformation may become flexible in presence of water. We also compared the properties of this fiber-immobilized lipase with native lipase and commercially available immobilized lipase from Rhizomucor miehei, viz., Lipozyme.