Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Immobilization of lipase from Candida rugosa on a polymer support

Lipase from Candida rugosa was immobilized by adsorption onto a macroporous copolymer support. Under optimum conditions the maximum amount of protein bound was 15.4 mg/g and the immobilization efficiency was 62%. The kinetics of lipase binding to the selected polymer carrier was assessed by using a general model of topochemical reactions. The effect of temperature on adsorption was thoroughly investigated, as was the adsorption mechanism itself. Analysis of the proposed kinetic model and the specific kinetic parameters measured suggest that surface kinetics control the adsorption process. According to the activation energy (E _a) and the rate constant, k, the enzyme has rather a high affinity for the support's active sites. The immobilized enzyme was used to catalyse the hydrolysis of palm oil in a lecithin/isooctane reaction system, in which the enzyme's activity was 70% that of the free enzyme. Kinetic parameters such as maximum velocity (V _max) and the Michaelis constant (K _m) were determined for the free and the immobilized lipase. Following repeated use, the immobilized lipase retained 56% of its initial activity after the fifth hydrolysis cycle. Received: 3 April 1998 / Received revision: 28 July 1998 / Accepted: 29 July 1998     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification

The aim of this work was to remove nitrate-N and organic pollutants from wastewater of the dairy industry by denitrification. An artificially prepared wastewater, containing 250 mg/l nitrate-N and 1.5 g/l whey powder, was completely denitrified with removal of 90%–93% of the chemical oxygen demand (COD) of the whey powder by suspended or immobilized mixed cultures and by a suspended or immobilized pure culture that was isolated from the mixed culture inoculum. For the above COD/nitrate-N ratio of 6:1, the results indicated that the organic compounds of the wastewater served as electron donors for complete denitrification and that there was no need to add an external carbon source. In batch denitrification assays the suspended or immobilized mixed cultures proved to be more active and reacted faster than the isolated pure cultures. In continuous denitrification processes with immobilized pure or mixed cultures, the alginate beads, used for immobilization, were not stable for more than 12 days of incubation. The mixed free cultures removed the nitrate-N and COD continuously with no change of their activity for at least 15 days at an optimum hydraulic retention time of 0.27 days with a loading rate of 900 mg nitrate-N l⁻¹ day⁻¹. Received: 13 October 1997 /  Received revision: 16 December 1997 / Accepted: 19 December 1997     

<Emphasis Type="Italic">Fervidobacterium changbaicum</Emphasis> Lip1: identification,cloning, and characterization of the thermophilic lipase as a new member of bacterial lipase family V

A novel lipase gene encoded 315 amino acid residues was obtained using lipase-prospecting primers and genome walking from hyperthermophilic bacterium Fervidobacterium changbaicum CBS-1. Sequence alignment and phylogenetic analysis revealed this novel lipase is a new member of bacterial lipase family V. The recombinant enzyme F. changbaicum lipase 1 (FCLip1) showed maximum activity at 78°C and pH 7.8. It displayed extreme thermostability at 70°C and was also stable across a wide pH range from 6.0 to 12.0. Kinetic study demonstrated FCLip1 preferentially hydrolyzed middle-length acyl chains, especially p-nitrophenyl caprate and tricaprylin. With p-nitrophenyl caprate as a substrate, the enzyme exhibited a K _m and k _cat of 4.67 μM and 22.7/s, respectively. In addition, FCLip1 was resistant to various detergents and organic solvents. This enzyme is the first reported thermophilic lipase from bacterial family Thermotogaceae. Its extreme stability with respect to temperature and pH, along with its triglyceride hydrolysis activity, indicate that FCLip1 has high potential for future application.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine by using microgel beads containing Pseudomonas cepacia lipase

Abstract

Pseudomonas cepacia lipase (PCL) was immobilized in alginate microgel beads by electrostatic dispersion. The high electrical potential applied in the immobilization process could significantly decrease the droplet size. The optimum conditions for lipase immobilization were 2% (w/v) alginate, 100 mM CaCl₂, 8 mg/mL enzyme, 4 kV electrical potential and 200 μm mean bead size. Under these conditions, 78.2 U/g of immobilized PCL activity was obtained with 39.1% retained activity and 57.2% immobilization efficiency. The immobilized PCL (PCL-CA) was subsequently used in the enantioselective hydrolysis of (R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester. Although PCL-CA exhibited slightly lower activity than free PCL, it preserved the high enantioselectivity (E-value >?200), which afforded enantiomerically pure (R)-acid (99% e.e._p). Furthermore, PCL-CA exhibited higher thermal stability, storage and medium stability than that of free PCL. Batch-wise operational stability studies demonstrated that PCL-CA retained its initial activity for at least 10 cycles of hydrolysis.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Purification and Characterization of a Novel Extracellular Lipase Catalyzing Hydrolysis of Oleyl Benzoate from Acinetobacter nov. sp. Strain KM109

A new lipase (OBase) which efficiently hydrolyzes oleyl benzoate (OB) was found in the culture supernatant of Acinetobacter nov. sp. strain KM109, a new isolate growing in a minimum medium containing OB as the sole carbon source. OBase was purified to homogeneity with 213-fold purification and 0.8% yield. The molecular weight was estimated to be 62,000±1,000 by SDS-PAGE under denatured-reduced conditions and to be 50,000±1,000 by gel-filtration HPLC under native conditions; these findings indicate that OBase is a monomeric enzyme. The optimum temperature and pH of OBase were about 45°C and pH 8. Temperature and pH stabilities were at or lower than 35°C and in a range of pH 6-8, respectively. Purified OBase preferentially hydrolyzed p-nitrophenyl benzoate (pNPB) over p-nitrophenyl acetate (pNPA) or p-nitrophenyl caproate (pNPC) [pNPB/pNPA=20 and pNPB/pNPC=5.4], indicating that OBase has a high affinity for benzoyl esters. Partial amino-acid sequences of OBase fragments obtained after lysyl endopeptidase treatment showed no similarity with known proteins.     

A covalent two-step immobilization technique using itaconic anhydride

β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity and the K _m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately 50% more chloramphenicol β-glucoside was obtained after 5 h. Received: 22 September 1997 / Accepted: 28 October 1997     

Enantioselective hydrolysis of β-hydroxy nitriles using the whole cell biocatalyst Rhodococcus rhodochrous ATCC BAA-870

Candida rugosa lipase was covalently immobilized onto silica gel in two different ways: via glutaraldehyde (L_GAL) and via hydrophobic spacer arm (1,6 diamino hexane) (L_SA). Free lipase, L_GAL and L_SA were used to investigate the hydrolysis of two different substrates, namely p-nitrophenyl palmytate (pNPP) and p-nitrophenyl acetate (pNPA), both in aqueous medium. In addition, these lipase samples were used to synthesize the pNPP from p-nitrophenol (pNP) and palmytic acid (PA) and pNPA from pNP and acetic acid (AA), both in hexane medium. Hydrolytic and synthetic activities of L_SA were higher than those of free lipase and L_GAL. Synthetic activities of free lipase, L_GAL and L_SA for pNPA in the presence of pNP and AA within hexane medium were higher than those of hydrolytic activities for pNPA in aqueous medium. The same tendency was also observed with pNPP. The effects of pH and temperature on hydrolytic and synthetic activities were investigated for all lipase preparations. Operational stability was the highest for L_GAL and L_SA when these enzymes were used for pNPP synthesis and in hexane medium, after 100 repeated uses, 68% and 51% of initial activities remained, respectively, at the end of 100 repeated cycles. Free lipase lost all of its activity within 15 and 20 days when stored at 25 °C and 5 °C, respectively. However, L_GAL showed 54% and 70% of initial activity at the end of 60 storage days at 25 °C and 5 °C, respectively, while these values were observed as 36% and 60% for L_SA.     

Optimized conditions forin situ immobilization of lipase in aldehyde-silica packed columns

Optimal conditions for thein situ immobilization of lipase in aldehyde-silica packed columns,via reductive amination, were investigated. A reactant mixture, containing lipase and sodium borohydride (NaCBH), was recirculated through an aldehyde-silica packed column, such that the covalent bonding of the lipase,via amination between the amine group of the enzyme and the aldehyde terminal of the silica, and the reduction of the resulting imine group by NaCBH, could occur inside the bed,in situ. Mobile phase conditions in the ranges of pH 7.0∼7.8, temperatures between 22∼28°C and flow rates from 0.8∼1.5 BV/min were found to be optimal for thein situ immobilization, which routinely resulted in an immobilization of more than 70 mg-lipase/g-silica. Also, the optimal ratio and concentration for feed reactants in thein situ immobilization: mass ratio [NaCBH]/[lipase] of 0.3, at NaCBH and lipase concentrations of 0.75 and 2.5 g/L, respectively, were found to display the best immobilization characteristics for concentrations of up to 80 mg-lipase/g-silica, which was more than a 2-fold increase in immobilization compared to that obtained by batch immobilization. For tributyrin hydrolysis, thein situ immobilized lipase displayed lower activity per unit mass of enzyme than the batch-immobilized or free lipase, while allowing more than a 45% increase in lipase activity per unit mass of silica compared to batch immobilization, because the quantity of the immobilization on silica was augmented by thein situ immobilization methodology used in this study.     

Morphological,biochemical and kinetic properties of lipase from Candida rugosa immobilized in zirconium phosphate

Zirconium phosphate (ZrP), a low-cost inorganic material with well-defined physicochemical properties, was successfully used as support for immobilizing Candida rugosa lipase by covalent bonding. The immobilized derivative showed high catalytic activity in both aqueous and non-aqueous media. Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy measurements demonstrated that the ZrP fulfilled the morphological requirements for use as a matrix for immobilizing lipases. The free and immobilized lipases were compared in terms of pH, temperature and thermal stability. The immobilized lipase had a higher pH optimum (7.5) and higher optimum temperature (50°C) than the free lipase. Immobilization also increased the thermal stability. The hydrolysis of p-nitrophenyl palmitate (pNPP) by immobilized lipase, examined at 37°C, followed Michaelis–Menten kinetics. Values for K_m=1.18 µM and V_max=325Umg^?1 indicated that the immobilized system was subject to mass transfer limitations. The immobilized derivative was also tested under repetitive reaction batches in both ester hydrolysis and synthesis.     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Production of d-amino acids using immobilized d-hydantoinase from lentil, Lens esculenta, seeds

d-Hydantoinase from the lentil, Lens esculenta, seed is quite unstable, and has been immobilized on Diethyl amino ethyl (DEAE) cellulose by an adsorption and cross-linking method. The immboilized d-hydantoinase exhibited 80% enzyme activity and contained 86% protein. The immobilization of the enzyme preparation does not change its optimum pH, temperature or affinity constant, but increases its shelf-life, thermostability and stability in various organic solvents. This immobilized d-hydantoinase can be used effectively for the production of d-amino acids from the corresponding hydantoins and may therefore be of use in the chemical and pharmaceutical industries. Received: 28 April 1998 / Received last revision: 10 July 1998 / Accepted: 10 July 1998