Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Characteristics of immobilized lipase on hydrophobic superparamagnetic microspheres to catalyze esterification

A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity (a(w)) 0.5-0.8 or higher. At a(w) 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C(8)-C(12)). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application.     

Immobilization of animal cells using photo-crosslinkable resin

A photo-crosslinkable resin, BIX12, was selected from among various photo-crosslinkable resins for the immobilization of animal cells. BIX12 had no cytotoxic effect on the growth of hybridoma cells and the production of monoclonal antibody, although other photo-crosslinkable resins had significant inhibitory effects. Using BIX12-alginate hybrid gel particles, hybridoma cells could grow in the resins and produce monoclonal antibody. For the continuous production of monoclonal antibody, perfusion culture using a fluidized-bed bioreactor with direct air bubbling was carried out. By this cultivation, monoclonal antibody could be produced stably for more than 50 d. A high viable cell density of more than 10⁷ cells/ml-gel was attained, and the antibody productivity was improved 8.5-fold compared with conventional suspension culture using a spinner flask. Anchorage-dependent cells were also immobilized in the resin particles by three immobilization procedures. Among these procedures, porous BIX12 formed by adding gelatin powder provided good support strength and allowed the cells to grow on the surface inside of the support.     

Bioconversion of lipophilic compounds by immobilized microbial cells in organic solvents

Microbial cells were gel-entrapped with photo-crosslinkable resin prepolymers or urethane prepolymers, respectively. The resulting gels have different tailor-made hydrophobic or hydrophilic character. They were used for successful bioconversion of hydrophobic steroids and terpenoids in watersaturated mixtures of organic solvents. The experiments show the influence of the hydrophobicity of the gels and the polarity of the solvent mixtures, respectively. Use of hydrophobic gels and less polar solvents is preferable for bioconversion of hydrophobic compounds. The selective formation of a desired product among diverse products from a single substrate by appropriate use of hydrophobic or hydrophilic gels is possible. In each case, tests should be made to select the appropriate gel and solvent mixture. Bioconversions tested are: dehydroepiandrosterone to 4-androstene-3,17-dione; cholesterol to cholestenone; β-sitosterol to β-sitostenone; stigmasterol to stigmastenone; pregnenolone to progesterone; testosterone to Δ¹-dehydrotestosterone or 4-androstene-3,17-dione, respectively; all with immobilized cells of Nocardia rhodocrous; and stereoselective hydrolysis of dl-menthyl-succinate to yield l-menthol with immobilized cells of Rhodotorula minuta var. texensis.     

Mineralization of individual congeners of linear alkylbenzenesulfonate by defined pairs of heterotrophic bacteria

Parvibaculum lavamentivorans DS-1(T) utilized the commercial surfactant linear alkylbenzenesulfonate (LAS) (20 congeners with C(10) to C(13) side chains) as a carbon and energy source by shortening the side chain, and sulfophenylcarboxylates (SPCs) and similar compounds (e.g., alpha,beta-unsaturated SPCs [SPC-2Hs]) were excreted with quantitative recovery of the sulfophenyl moiety. 2-(4-Sulfophenyl)decane (2-C10-LAS) was converted largely to 3-(4-sulfophenyl)butyrate (3-C4-SPC), as were 2-C12-LAS and 2-C14-LAS; the other products were 5-C6-SPC (SPC+2C) and 3-C4-SPC-2H. 2-C11-LAS was converted largely to 4-C5-SPC with the corresponding SPC+2C and SPC-2H; similarly, 3-C12-LAS yielded 4-C6-SPC with the corresponding SPC+2C and SPC-2H. This pattern of products confirmed that LAS is degraded by omega-oxygenation and chain shortening through beta-oxidation. At least nine major SPCs were formed from commercial LAS. The novel isolates Comamonas testosteroni SPB-2 and KF-1 utilized 3-C4-SPC; Delftia acidovorans SPH-1 utilized 4-C6-SPC enantioselectively. The substrate-dependent oxygen uptake of whole cells of strain SPB-2 indicated that there was inducible oxygenation of 3-C4-SPC and of 4-sulfophenol in whole cells of the strains of C. testosteroni during growth with 3-C4-SPC or 4-sulfophenol. The degradative pathways apparently involved 4-sulfocatechol and 4-sulfocatechol 1,2-dioxygenase. Strain SPB-2 and strain DS-1(T) grew together in LAS-salts medium, and only seven of the nine major SPCs were recovered. Strain SPB-2 utilized 3-C4-SPC, 3-C5-SPC, and 3-C4-SPC-2H. Strain SPH-1 grew together with strain DS-1(T) in LAS-salts medium, and a different set of seven major SPCs was recovered. Strain SPH-1 utilized 4-C6-SPC, 4-C5-SPC, 4-C6-SPC-2H, and 4-C5-SPC-2H. A three-member community consisting of strains DS-1(T), SPB-2, and SPH-1 utilized four major SPCs. We inferred that this community mineralized the major SPCs derived from 8 of the 20 LAS congeners.     

Synthesis and characterization of amphiphilic monodisperse compounds and poly(ethylene imine)s: influence of their microstructures on the antimicrobial properties

Amphiphilic monodisperse compounds (series B-I and B-II) and poly(ethylene imine)s (PEI-I, PEI-II, and PEI-III) with different microstructures were prepared from primary amines or poly(ethylene imine) with functional carbonates bearing cationic, hydrophobic, or amphiphilic groups. Their inhibition potential against proliferation of E. coli , S. aureus , and B. subtilis was investigated and their hemolytic activities were determined. The influence of the microstructures, the alkyl chain length and the distribution of cationic and hydrophobic groups, on their antimicrobial efficacy was studied. Amphiphilic compounds with long alkyl chains (C14-C18) directly linked to the cationic groups (series B-I) are more effective against both Gram-positive and Gram-negative bacteria than amphiphilic compounds in which the hydrophobic and cationic groups (series B-II) are connected by a spacer. Poly(ethylene imine)s with amphiphilic grafts (B-I) called PEI-II are more effective than amphiphilic PEIs with the same alkyl chain but with randomly linked cationic and hydrophobic graft called PEI-I or with the amphiphilic grafts (B-II) called PEI-III. The influence of the inoculum size on the MIC value was investigated exemplarily with compounds of series B-I against S. aureus .     

The biological origin of ketotic dicarboxylic aciduria. In vivo and in vitro investigations of the omega-oxidation of C6-C16-monocarboxylic acids in unstarved, starved and diabetic rats

The conversion of radioactive C6-C16-monocarboxylic acids to urinary adipic, suberic, sebacic and 3-hydroxybutyric acids was investigated in vivo in unstarved, starved and diabetic ketotic rats. Hexanoic, octanoic and decanoic acids were converted to C6-, C6-C8- and C6-C10-dicarboxylic acids, respectively, in fed and 72-h-starved rats. Lauric acid was converted to C6-C8-dicarboxylic acids in starved rats but not in unstarved rats. Decanoic and lauric acids were converted to relatively high amounts of C6-C8-dicarboxylic acids compared with myristic acid in myristic acid in ketotic diabetic rats, while radioactivity from [1-14C]-and [16-(14)] palmitic acid was not incorporated into C6-C8-dicarboxylic acids in diabetic ketotic rats. C6-C12-monocarboxylic acids in hydrolysed rat adipose tissue wee determined by gas-liquid chromatography-mass spectrometry (selected ion monitoring). Decanoic and lauric acids were found in amounts of 7.6-9.1 and 85.9-137.5 micrometers/100 mg tissue, respectively, whereas the amounts of hexanoic and octanoic acids were negligible. It is concluded that the biological origin of the C6-C8-dicarboxylic aciduria seen in ketotic rats are C10-C14-monocarboxylic acids, which are initially omega-oxidised solely or partly as free acids and subsequently beta-oxidised to adipic and suberic acids. The in vitro omega-oxidation of C6-C16-monocarboxylic acids to corresponding dicarboxylic acids in the 100,000 Xg supernatant fraction of rat liver homogenate was measured by selected ion monitoring. 0.09, 0.14, 16.1, 5.8, 7.0 and -6.9% of, respectively, hexanoic, octanoic, decanoic, lauric, myristic and palmitic acid were omega-oxidised to dicarboxylic acids of corresponding chain lengths after 90 min of incubation, when correction for the production of dicarboxylic acids in control assays was made. An in vitro production of C12-C16-dicarboxylic acids was detected in all assays ()including control assays), probably formed from"endogenous' monocarboxylic acids preexistent in the homogenate. Ths "endogenous' production of dicarboxylic acids was inhibited by C10-C16-monocarboxylic acids, where palmitic acid had the strongest effect. In fact, palmitic acid inhibited its own omega-oxidation when added in concentrations above 0.6 mM. Starvation of rats for 72 h did not alter the "endogenous' in vitro production of hexadecanedioic acid.     

Characteristics of Zymomonas mobilis immobilized by photo-crosslinkable resin in ethanol fermentation

Zymomonas mobilis, an ethanol-producing bacterium, was immobilized in hydrophilic photo-crosslinked resin gels to form a biocatalyst. The molecular structure of the photo-crosslinkable resin could be modulated so as to minimize a disadvantage of this bacterium—poor-tolerance to salts in molasses. Characteristics of Z. mobilis immobilized by photo-crosslinkable resin gel, such as fermentability, cell growth in gel, the potential of gel materials, diffusion of materials, and salt distribution are discussed. ENTG-3800 photo-crosslinkable resin was selected as the most suitable entrapping material for Z. mobilis, especially in using molasses.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Synthesis of chitosan-based polymeric surfactants and their adsorption properties for heavy metals and fatty acids

Chitosan-based polymeric surfactants (CBPSs) were prepared by N-acylation of chitosans (chitosan 10 and 500) with several acid anhydrides such as hexanoic (C6), lauric (C12), and palmitic (C16) anhydrides. Among the CBPS samples, CBPSs having a good solubility at pH 4.0 were selected and observed for viscosity, surface tension, and adsorption of heavy metals (Cd2+, Co2+, Cr2O7(2-), and Pb2+) as well as the fatty acid (n-octanoic acid). The 1H NMR spectrum of chitosan 10 modified with C16 at the substitution ratio of 0.4 (CBPS10-C16,0.4) showed 85% of acylation in 1% DCl/D2O solutions. CBPS10 with the substitution ratio less than 0.4 showed a good solubility because of shorter repeating units and lesser amounts of hydrophobic substituents. The intrinsic viscosity of CBPS10 was slightly increased, while that of CBPS500 was decreased. As the substitution ratio and length of the carbon chain increased, the surface tension of CBPS10 tended to decrease. CBPS10-C16,0.2 had high adsorption ability for cationic metal ions such as Cd2+, Co2+, and Pb2+ comparable to chitosan. Interestingly, CBPS(10)-C(16,0.2) showed a unique pH optimum for the anionic metal ion such as Cr2O7(2-). In addition, CBPS10-C16,0.2 exhibited the highest adsorption ability for n-octanoic acid among the tested CBPS10 with different carbon chains.     

Mineralization of Individual Congeners of Linear Alkylbenzenesulfonate by Defined Pairs of Heterotrophic Bacteria

Parvibaculum lavamentivorans DS-1^T utilized the commercial surfactant linear alkylbenzenesulfonate (LAS) (20 congeners with C₁₀ to C₁₃ side chains) as a carbon and energy source by shortening the side chain, and sulfophenylcarboxylates (SPCs) and similar compounds (e.g., α,β-unsaturated SPCs [SPC-2Hs]) were excreted with quantitative recovery of the sulfophenyl moiety. 2-(4-Sulfophenyl)decane (2-C10-LAS) was converted largely to 3-(4-sulfophenyl)butyrate (3-C4-SPC), as were 2-C12-LAS and 2-C14-LAS; the other products were 5-C6-SPC (SPC+2C) and 3-C4-SPC-2H. 2-C11-LAS was converted largely to 4-C5-SPC with the corresponding SPC+2C and SPC-2H; similarly, 3-C12-LAS yielded 4-C6-SPC with the corresponding SPC+2C and SPC-2H. This pattern of products confirmed that LAS is degraded by ω-oxygenation and chain shortening through β-oxidation. At least nine major SPCs were formed from commercial LAS. The novel isolates Comamonas testosteroni SPB-2 and KF-1 utilized 3-C4-SPC; Delftia acidovorans SPH-1 utilized 4-C6-SPC enantioselectively. The substrate-dependent oxygen uptake of whole cells of strain SPB-2 indicated that there was inducible oxygenation of 3-C4-SPC and of 4-sulfophenol in whole cells of the strains of C. testosteroni during growth with 3-C4-SPC or 4-sulfophenol. The degradative pathways apparently involved 4-sulfocatechol and 4-sulfocatechol 1,2-dioxygenase. Strain SPB-2 and strain DS-1^T grew together in LAS-salts medium, and only seven of the nine major SPCs were recovered. Strain SPB-2 utilized 3-C4-SPC, 3-C5-SPC, and 3-C4-SPC-2H. Strain SPH-1 grew together with strain DS-1^T in LAS-salts medium, and a different set of seven major SPCs was recovered. Strain SPH-1 utilized 4-C6-SPC, 4-C5-SPC, 4-C6-SPC-2H, and 4-C5-SPC-2H. A three-member community consisting of strains DS-1^T, SPB-2, and SPH-1 utilized four major SPCs. We inferred that this community mineralized the major SPCs derived from 8 of the 20 LAS congeners.     

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.     

Microbial and reducing agents catalyze the rearrangement of taxanes

5alpha, 7beta, 9alpha, 10beta, 13alpha-Pentahydroxy-4(20),11(12)-taxadiene derivative 1 was converted to two unprecedented 1(15-->11)abeo-taxanes and a taxane derivative with a C10-C11 double bond by Absidia coerula ATCC 10738a. A similar compound was obtained from treatment with zinc of a triacetoxy-4(20),11(12)-taxadiene derivative.     

Effect of high-cell-density fermentation of Candida utilis on kinetic parameters and the shift to respiro-fermentative metabolism

In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts.     

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

Summary Acetone-dried cells of Arthrobacter simplex, whose steroid ¹ activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.     

Metabolic physiology of alcohol degradation and adaptation in Drosophila larvae as studied by means of carbon-13 nuclear magnetic resonance spectroscopy

Alcohol dehydrogenase-mediated degradation of [2-¹³C]ethanol was followed in third instar larvae of Drosophila by means of ¹³C NMR. The tricarboxylic acid (TCA) cycle intermediates, citrate-C(2),4 and succinate-C2,3; the amino acids, glutamate-C4,3,2, glutamine-C4,3,2, proline-C4, alanine-C2,3 and the carbon nuclei of the glucosyl units of the disaccharide, α,α-trehalose, were intensely labeled in perchloric acid extracts of whole larvae. A model of the intermediary metabolism of ethanol degradation in larvae was formulated from these observations. The C2 atom of ethanol enters the mitochondrial TCA cycle as C2-acetyl-CoA and is converted into the TCA cycle intermediates. The TCA cycle intermediate 2-oxoglutarate(-C4) apparently is readily converted into glutamate(-C4) and subsequently to glutamine(-C4) and proline(-C4). Dietary ethanol is also a substrate for trehalose synthesis. This may occur by an exchange of malate(-C2,3) between its mitochondrial and cytosolic pools. Part of the cytosolic malate(-C2,3) may be diverted into pyruvate then generating alanine(-C2,3) as another product. The other part may be converted into glucose and subsequently into α,α-trehalose by the gluconeogenic pathway. ¹³C natural abundance signals of stored fatty acids and glycerol were readily detectable in chloroform extracts of control larvae. De novo synthesis of fatty acids from labeled ethanol also occurred after a lag period. Our findings show the coordinated nature of metabolic pathways, and we point to its consequences in understanding the dynamics in evolutionary processes.     

Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity

Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles.     

Bioaffinity separation of trypsin using trypsin inhibitor immobilized in reverse micelles composed of a nonionic surfactant

Trypsin inhibitor was converted to hydrophobic states by covalently combining cholesteryl groups using an acylation reaction, and was immobilized in reverse micelles composed of a nonionic surfactant. Using this reverse micellar phase containing trypsin inhibitor as an affinity ligand, trypsin was selectively separated with high recoveries from a mixture of several kinds of contaminating proteins by forward and backward extraction. No loss of activity of the recovered trypsin was observed through these operations. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 406-408, 1997.     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

Use of magnetic poly(glycidyl methacrylate) monosize beads for the purification of lysozyme in batch system

The hydrophobic affinity ligand L-tryptophan immobilized magnetic poly(glycidyl methacrylate) [m-poly(GMA)] beads in monosize form (1.6 microm in diameter) were used for the affinity purification of lysozyme from chicken egg white. The m-poly(GMA) beads were prepared by dispersion polymerization in the presence of Fe3O4 nano-powder. The epoxy groups of the m-poly(GMA) beads were converted into amino groups with 1,6 diaminohexane (i.e., spacer arm). l-tryptophan was then covalently immobilized on spacer arm attached m-poly(GMA) beads. Elemental analysis of immobilised L-tryptophan for nitrogen was estimated as 42.5 micromol/g polymer. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration and temperature). Maximum lysozyme adsorption amount of m-poly(GMA) and m-poly(GMA)-L-tryptophan beads were 1.78 and 259.6 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, m-poly(GMA)-L-tryptophan beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. It was found to be successful in achieving purification of lysozyme in a high yield of 76% with a purification fold of 71 in a single step. The specific activity of the eluted lysozyme (62,580 U/mg) was higher than that obtained with a commercially available pure lysozyme (Sigma (60,000 U/mg).