Pilot-scale production of lipase using palm oil mill effluent as a basal medium and its immobilization by selected materials.

A pilot-scale production of lipase using palm oil mill effluent (POME) as a fermentation basal medium was carried out, and parameters for immobilization of the produced lipase were optimized. Lipase production in a 300-L bioreactor was performed using two proposed strategies, constant power per volume (P/V) and constant tip speed. Moreover, lipase immobilization on different materials was also investigated. Lipase production was performed using liquid-state bioconversion of POME as the medium and Candida cylindracea as the inoculum. The fermentation medium was composed of 1% total suspended solids (TSS) of POME, 0.5% (w/v) peptone, 0.7% (v/v) Tween-80, and 2.2% inoculum. The medium composition was decided on the basis of the medium optimization results of a previous study. The fermentation was carried out for 48 h at 30°C and pH 6. The maximum lipase production was 5.72U/mL and 21.34 U/mL, obtained from the scale-up strategies of constant tip speed and P/V, respectively. Four accessible support materials were screened for their potential use in immobilization. The most suitable support material was found to be activated carbon, with a maximum immobilization of 94%.

Eco-friendly combination of the immobilized PGA enzyme and the S-Phacm protecting group for the synthesis of Cys-containing peptides.

Enzyme-labile protecting groups have emerged as a green alternative to conventional protecting groups. These groups introduce a further orthogonal dimension and eco-friendliness into protection schemes for the synthesis of complex polyfunctional organic molecules. S-Phacm, a Cys-protecting group, can be easily removed by the action of a covalently immobilized PGA enzyme under very mild conditions. Herein, the versatility and reliability of an eco-friendly combination of the immobilized PGA enzyme and the S-Phacm protecting group has been evaluated for the synthesis of diverse Cys-containing peptides.

Highly active engineered-enzyme oriented monolayers: formation, characterization and sensing applications.

BACKGROUND: The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system. METHODS: Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S) bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function. RESULTS AND DISCUSSION: Water contact angle (65-70°), as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate) and the other (SH/S-S) at the ω-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity ~100 times that expected for the same amount of protein in solution. CONCLUSIONS: To the best of our knowledge, immobilization of a protein by the method presented here, with the resulting high enzymatic activity, has never been reported. There are many potential applications for selective localization of active proteins at patterned surfaces, for example, bioMEMS (MEMS--Micro-Electro-Mechanical Systems. Due to the success of the method, presented here, it was decided to continue a research project of a biosensor by transferring it to a high aspect ratio platform--nanotubes.

Co-immobilization of urokinase and thrombomodulin on islet surfaces by poly(ethylene glycol)-conjugated phospholipid.

Transplantation of islets of Langerhans is a promising method for treating patients with insulin-dependent diabetes mellitus. The major obstacle in clinical settings is early graft loss due to inflammation triggered by blood coagulation and complement activation on the surface of the islets after intraportal transplantation. We propose a versatile method for modifying the surface of islets with the fibrinolytic enzyme urokinase and the soluble domain of the anticoagulant enzyme thrombomodulin. The surfaces of islets were modified with a poly(ethylene glycol)-phospholipid conjugate bearing a maleimide group (Mal-PEG-lipid; PEG MW = 5000 kDa). The Mal-PEG-lipid anchored to the cell membranes of islets, resulting in the presentation of functional maleimide groups on the islet surface. The surface was further treated with thiolated urokinase and thrombomodulin that conjugated by thiol/maleimide bonding. No practical islet volume increase was observed after surface modification, and the modifications did not impair insulin release in response to glucose stimulation. Furthermore, the activity of the immobilized urokinase and thrombomodulin was maintained. These modifications could help to improve graft survival by preventing thrombus formation on the surface of transplanted islets.

Conversion of sunflower oil to biodiesel by alcoholysis using immobilized lipase.

Transesterification reaction was performed using sunflower oil and short-chain alcohol by immobilized lipases in organic solvents. The fatty acid ester, which is the product of this reaction, can be used as a diesel fuel that does not produce sulfur oxide and minimize the soot particulate. Immobilized porcine pancreatic lipase (PPL) and Candida rugosa lipase (CRL) showed the satisfactory activity in these reactions. Immobilization of lipases was carried out using inorganic absorbance Celit 545 particle as a carrier. Organic solvent like hexane in reactions was required when methanol and ethanol were used as alcoholic substrate. The reaction could be performed in absence of solvent when 1-propanol and 1-butanol were used as short-chain alcohol. The activities of immobilized lipases were highly increased in comparison with free lipases because its activity sites became more effective. Immobilized enzyme could be repeatedly used without difficult method of separation and the decrease in its activity was not largely observed.

Preparation of poly(ethylene glycol) hydrogels with different network structures for the application of enzyme immobilization.

In this study, poly(ethylene glycol) (PEG)-based hydrogels having different network structures were synthesized by UV-initiated photopolymerization and used for the enzyme immobilization. PEGs with different molecular weight were acrylated by derivatizing both ends with acryloyl chloride and photopolymerization of PEG-diacrylate (PEG-DA) yielded crosslinked hydrogel network within 5 seconds. Attachment of acrylate groups and gelation were confirmed by ATR/FT-IR and FT-Raman spectroscopy. Network structures of hydrogels could be easily controlled by changing the molecular weight (MW) of PEG-DA and characterized by calculating molecular weight between crosslinks and mesh size from the swelling measurement. Synthesis of hydrogels with higher MW of PEG produced less crosslinked hydrogels having higher water content, larger value of Mc and mesh size, which resulted in enhanced mass transfer but loss of mechanical properties. For the enzyme immobilization, glucose oxidase (GOX) was immobilized inside PEG hydrogels by means of physical entrapment and covalent immobilization. Encapsulated GOX were covalently bound to PEG backbone using acryloyl-PEG-N-hydroxysuccinimide and maintained their activity over a week period without leakage. Kinetic study indicated that immobilized enzyme inside hydrogel prepared from higher MW of PEG possessed lower apparent Km (Michaelis-Menten constant) and higher activity.

Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate.

A sandwich ELISA method using peptide tags showing a specific affinity to a hydrophilic polystyrene surface (PS-tags), PS 19 composed of RAFIASRRIKRP and KPS19R10 of KRAFIASRRIRRP and a hydrophilic polystyrene (phi-PS) plate was used to analyze protein-protein interactions. An Escherichia coli cysteine synthase complex, in which serine acetyltransferase (SAT) interacts with O-acetylserine sulfhydrylase-A (OASS) was used as a model system. When the interaction was detected by the conventional sandwich ELISA method using a hydrophobic polystyrene (pho-PS) plate, for the exclusive use of ELISA, the signal intensity was barely detectable due to conformational change of the ligand protein, OASS in the adsorbed state. On the contrary, when OASS, genetically fused with PS19 (OASS-PS19) or chemically conjugated with KPS19R10 (OASS-KPS19R10), was immobilized on the phi-PS plate, a high signal intensity was detected. Furthermore, by applying the two-step sandwich ELISA, in which OASS-PS19 or OASS-KPS19R10 formed a complex with SAT in the blocking solution before immobilization on the phi-PS plate, the signal intensity was further increased with a much shorter operational time, because SAT in the blocking solution formed a complex with OASS-PS19 or OASS-KPS19R10 without any steric hindrance.

Surface functionalization of polypyrrole film with glucose oxidase and viologen.

A surface modification technique was developed for the functionalization of polypyrrole (PPY) film with glucose oxidase (GOD) and viologen moieties. The PPY film was first graft copolymerized with acrylic acid (AAc) and GOD was then covalently immobilized through the amide linkage formation between the amino groups of the GOD and the carboxyl groups of the grafted AAc polymer chains in the presence of a water-soluble carbodiimide. Viologen moieties could also be attached to the PPY film via graft-copolymerization of vinyl benzyl chloride with the PPY film surface followed by reaction with 4,4'-bipyridine and alpha,alpha'-dichloro-p-xylene. X-ray photoelectron spectroscopy (XPS) was used to characterize the PPY films after each surface modification step. Increasing the AAc graft concentration would allow a greater amount of GOD to be immobilized but this would decrease the electrical conductivity of the PPY film. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also studied. The immobilized GOD was found to be less sensitive to temperature deactivation as compared to the free GOD. The results showed that the covalent immobilization technique offers advantages over the technique involving the entrapment of GOD in PPY films during electropolymerization. The presence of viologen in the vicinity of the immobilized GOD also enabled the GOD-catalyzed oxidation of glucose to proceed under UV irradiation in the absence of O(2).

Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

Covalent binding of enzymes to synthetic membranes containing acrylamide units, using formaldehyde.

Synthetic membranes containing 10% acrylamide units were subjected to activation with formaldehyde at pH 7.5 and 45 degrees C. Trypsin, invertase, and urease were bound to this activated membrane and the kinetic properties of immobilized enzymes were studied. The permeability of the membrane for distilled water manifests certain differences depending on the enzyme bound. The membranes with immobilized enzymes stored at 4 degrees C in a moist state showed no change in their activity for 6 months. The membrane with immobilized invertase has preserved its activity even after 20 operations with 2% sucrose solution at 25 degrees C. The proposed method of binding enzymes to synthetic membranes containing acrylamide groups, through the introduction of N-hydroxymethyl groups, possesses several advantages with respect to the activation of the membrane in a one-step reaction with cheap and accessible reagent, high operative stability of the immobilized enzymes, no danger of bacterial rotting, and long shelf life of the membrane.

Immobilization of enzymes on slowly soluble carriers.

The preparation and some properties of microspheres composed of oxidized polysaccharides and some vinyl polymers are described. The microspheres contain immobilized enzyme and can be slowly solubilized in water solutions, thereby releasing active a enzyme into the surrounding medium. The kinetic characteristics of the immobilized enzyme bound with a fragment of matrix after complete solubilization are unchanged, but the enzyme exhibits high thermostability. These preparations could have a wide range of medical applications, e.g., to form a drug "depot" directly in an affected organ.

The reactivity of alpha-chymotrypsin immobilized on radiation-grafted hydrogel surfaces.

The enzymatic activity of alpha-chymotrypsin (CT), immobilized on hydrogel-coated polymer film supports, has been investigated. The support was prepared by radiation-graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAAc) on silicone rubber films. The enzyme was covalently coupled to the carboxylic group of MAAc via the N-hydroxysuccinimide (NHS) ester active intermediate. Increasing MAAc contents of the hydrogel resulted in increased attachment of CT. The integrity of the CT active site after attachment was assessed by an active site titration with diisopropyl fluorophosphate (DFP). As the MAAc content of the hydrogel was increased, an increasing fraction of the attached CT retained its activity to DFP. A greater fraction of CT was active towards DFP when adsorbed than when coupled. The rates of hydrolysis of some synthetic model substrates by the immobilized CT were also measured. The negative charge on the hydrogel had a large effect on the rates of these hydrolyses. The pH optimum for the hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) by immobilized CT was higher than that of free CT. Increasing MAAc content of the hydrogel resulted in larger shifts in the pH optimum. The maximum rates of ATEE hydroylsis per mg CT declined sharply with increasing MAAc content of the hydrogel. This is probably related to the increasing repulsive force between the ATEE (negatively charged above congruent to pH 9.5) and the hydrogel with increasing MAAc content. The activity of immobilized CT to ATEE is small compared to that of free CT, partly due to this charge effect. Conversely, the rate of hydrolysis of BAEE, a positively charged substrate, by immobilized CT at pH 11, is almost fourfold greater than that by free CT at its pH optimum.