Non-immobilized Enzyme Research Articles

Enhanced pollutant degradation via green-synthesized core-shell mesoporous Si@Fe magnetic nanoparticles immobilized with metagenomic laccase

With rapid industrial expansion, environmental pollution from emerging contaminants has increased, posing severe ecosystem threats. Laccases offer an eco-friendly solution for degrading hazardous substances, but their use as free-form biocatalysts face challenges. This study immobilized laccase (PersiLac1) on green-synthesized Si@Fe nanoparticles (MSFM NPs) to remove pollutants like Malachite Green-containing wastewater and degrade plastic films. Characterization techniques (FTIR, VSM, XRD, SEM, EDS, BET) confirmed the properties and structure of MSFM NPs, revealing a surface area of 31.297 m2.g−1 and a pore diameter of 12.267 nm. The immobilized PersiLac1 showed enhanced activity across various temperatures and pH levels, retaining over 82 % activity after 15 cycles at 80°C with minimal leaching. It demonstrated higher stability, half-life, and decimal reduction time than free laccase. Under 1 M NaCl, its activity was 1.8 times higher than the non-immobilized enzyme. The immobilized laccase removed 98.11 % of Malachite Green-containing wastewater and retained 82.92 % activity over twenty cycles of dye removal. Additionally, FTIR and SEM confirmed superior plastic degradation under saline conditions. These findings suggest that immobilizing PersiLac1 on magnetic nanoparticles enhances its function and potential for contaminant removal. Future research should focus on scalable, cost-effective laccase immobilization methods for large-scale environmental applications.

Tyrosinase Magnetic Cross-Linked Enzyme Aggregates: Biocatalytic Study in Deep Eutectic Solvent Aqueous Solutions.

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free immobilization towards the preparation of both non-magnetic and magnetic cross-linked enzyme aggregates (CLEAs). The prepared biocatalyst was characterized and the biocatalytic and structural traits of free tyrosinase and tyrosinase magnetic CLEAs (mCLEAs) were evaluated in numerous DES aqueous solutions. The results showed that the nature and the concentration of the DESs used as co-solvents significantly affected the catalytic activity and stability of tyrosinase, while the immobilization enhanced the activity of the enzyme in comparison with the non-immobilized enzyme up to 3.6-fold. The biocatalyst retained the 100% of its initial activity after storage at -20 °C for 1 year and the 90% of its activity after 5 repeated cycles. Tyrosinase mCLEAs were further applied in the homogeneous modification of chitosan with caffeic acid in the presence of DES. The biocatalyst demonstrated great ability in the functionalization of chitosan with caffeic acid in the presence of 10% v/v DES [Bet:Gly (1:3)], enhancing the antioxidant activity of the films.

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst.

Cross-linked enzyme aggregates (CLEAs) represent an effective tool for carrier-free immobilization of enzymes. The present study promotes a successful application of functionalized magnetic nanoparticles (MNPs) for stabilization of cellulase CLEAs. Catalytically active CLEAs and magnetic cross-linked enzyme aggregates (mCLEAs) of cellulase from Trichoderma reesei were prepared using glutaraldehyde (GA) as a cross-linking agent and the catalytic activity and stability of the CLEAs/mCLEAs were investigated. The influence of precipitation agents, cross-linker concentration, concentration of enzyme, addition of bovine serum albumin (BSA), and addition of sodium cyanoborohydride (NaBH3CN) on expressed activity and immobilization yield of CLEAs/mCLEAs was studied. Particularly, reducing the unsaturated Schiff's base to form irreversible linkages is important and improved the activity of CLEAs (86%) and mCLEAs (91%). For increased applicability of CLEAs/mCLEAs, we enhanced the activity and stability at mild biochemical process conditions. The reusability after 10 cycles of both CLEAs and mCLEAs was investigated, which retained 72% and 65% of the initial activity, respectively. The thermal stability of CLEAs and mCLEAs in comparison with the non-immobilized enzyme was obtained at 30 °C (145.65% and 188.7%, respectively) and 50 °C (185.1% and 141.4%, respectively). Kinetic parameters were determined for CLEAs and mCLEAs, and the KM constant was found at 0.055 ± 0.0102 mM and 0.037 ± 0.0012 mM, respectively. The maximum velocity rate (Vmax) was calculated as 1.12 ± 0.0012 µmol/min for CLEA and 1.17 ± 0.0023 µmol/min for mCLEA. Structural characterization was studied using XRD, SEM, and FT-IR. Catalytical properties of immobilized enzyme were improved with the addition of reducent NaBH3CN by enhancing the activity of CLEAs and with addition of functionalized aminosilane MNPs by enhancing the activity of mCLEAs.

Immobilization of fibrinolytic protease from Mucor subtilissimus UCP 1262 in magnetic nanoparticles

This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40–60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.

A portable blood lactate sensor with a non-immobilized enzyme for early sepsis diagnosis.

Early determination of blood lactate levels may accelerate the detection of sepsis, one of the most time-sensitive illnesses. We developed and validated a portable blood lactate detection kit for clinical screening that can measure early bedside lactate levels in intensive care unit (ICU) patients suspected of having sepsis. A TiO2 sol-G nanocomposite was prepared and coated on a screen-printed carbon electrode (SPCE) integrated with non-immobilized lactate oxidase (LOx) to produce a novel lactate biosensor with high sensitivity and high storage stability for human blood lactate measurement. The detection kit was based on an electrochemical technique and showed a wide linear range of 1-20 mM (R2 = 0.9937) with a low detection limit of 0.2 mM for lactate detection. This allowed for differentiating patient groups who may have sepsis using a cut-off level of 4 mM. The device was successfully implemented for blood lactate determination in critical patients, showing an accuracy range from 75% to 112%. This device provided high-precision and rapid quantitative information validated using a blood gas analyzer. Our detection kit might help to reduce the morbidity and mortality rates in severe sepsis and septic shock patients in community hospitals.

Synthesis of a precursor of D-fagomine by immobilized fructose-6-phosphate aldolase.

Fructose-6-phosphate aldolase (FSA) is an important enzyme for the C-C bond-forming reactions in organic synthesis. The present work is focused on the synthesis of a precursor of D-fagomine catalyzed by a mutant FSA. The biocatalyst has been immobilized onto several supports: magnetic nanoparticle clusters (mNC), cobalt-chelated agarose (Co-IDA), amino-functionalized agarose (MANA-agarose) and glyoxal-agarose, obtaining a 29.0%, 93.8%, 89.7% and 53.9% of retained activity, respectively. Glyoxal-agarose FSA derivative stood up as the best option for the synthesis of the precursor of D-fagomine due to the high reaction rate, conversion, yield and operational stability achieved. FSA immobilized in glyoxal-agarose could be reused up to 6 reaction cycles reaching a 4-fold improvement in biocatalyst yield compared to the non-immobilized enzyme.

Effect of alkaline protease produced from fish waste as substrate by Bacillus clausii on destaining of blood stained fabric

Alkaline protease or peptidases are the largest groups of enzymes in the biological industry with a variety of application in manufacturing units used in the process of substrate stabilization, dehairing, diagnosis, extraction, food production, destaining, etc., where the pH of the environmental conditions remain above neutral pH. Because of these wider applications of alkaline proteases in industries, their demand is increasing to compete with their chemical counterpart. An alkaline tolerant bacterial strain Bacillus clausii wasisolated from fish waste and used for mass production of alkaline protease using fish waste homogenate as media. Preliminary study on optimization of conditions for the mass production carried out. The optimum temperature for production ranges between 25°C and 35°C and pH determined as 9. The mass production of extracellular alkaline protease carried out using mobilized and immobilized cells of B. clausii at optimized condition using production media, the mixture of production media and fish waste homogenate and in nutrient broth. The recorded results showed that the maximum enzyme production obtained with immobilized cells in nutrient broth media and followed by fish waste homogenate media of 8900 U/ml and 8600 U/ml. Purification protease enzyme yielded 0.35 g/ml from the production media . Bloodstained cloth treated with immobilized enzyme removed the stain completely compared to treatment with non-immobilized enzyme and commercially used detergent. So, the current study suggests the usage of microbial alkaline protease in household detergents to replace the usage of synthetic detergents and save the environment from chemical pollutants.

Immobilization studies of a pectinase produced by Aspergillus terreus.

Aspergillus terreus can produce different holocellulose-degrading enzymes when grown in sugarcane bagasse, with predominant pectinase activity. Thus, pectinase was selected for purification and immobilization studies. Ion exchange and molecular exclusion chromatography studies were performed, after which it was possible to semipurify the enzyme with a yield of 80%. The crude extract pectinase (PECEB) and the partially purified enzyme (PEC2) were immobilized on monoamino-N-aminoethyl (MANAE)-agarose with pectinase activity yields of 66% and 98%, respectively. After immobilization in MANAE-agarose, the pectinase showed higher activity at acidic pH (pH 4.0) when compared to the nonimmobilized enzyme. It was also found that after the immobilization process, there was a threefold improvement in the enzyme's thermostability. Also, it was possible to reuse the immobilized enzyme for up to five cycles of hydrolysis with effective production of reducing sugars (0.196 mg/g of substrate). The industrial application test revealed a significant decrease in the viscosity of guava juice when the immobilized enzyme was used. PECEB, immobilized on MANAE-agarose, was the enzyme sample that generated the highest pulp viscosity reduction (approximately 47%). Although additional studies are needed for practical industrial application, the results obtained herein reveal the potential of application of immobilized pectinase in the industry.

Immobilization of Old Yellow Enzymes via Covalent or Coordination Bonds

Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziGTM particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziGTM, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziGTM Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions.

Changes in the Activity of Lipases in the Presence of Synthetic and Natural Polymers

Abstract—Both synthetic and natural polymers are promising carriers for the immobilization of enzymes, including lipases from different sources. This research trend is of fundamental and applied importance, since immobilized lipases are widely used in various biotechnological processes. In the present work, we study how synthetic and natural polymers (polystyrene, polylysine, and chitosan) influence the catalytic properties of lipases from the hog’s pancreatic gland (LPP), yeast fungus Сandida cylindracea (LCC), and wheat germs (LWG) in the hydrolysis of triacetin (used as a substrate in all the experiments). A polystyrene-based latex (even without surface modification) is found to have a strong effect on the activity of lipases of different origins. In the presence of large particles with a size of about 1 μm (irrespective of their concentration in the range of 1–10%), the activity of all three lipases (LPP, LCC, and LWG) significantly increases compared to the nonimmobilized enzyme. A polystyrene latex with a carboxylated surface shows another effect on the catalytic properties of these lipases. The activity of LPP is found to depend on the concentration of chitosan (at ratios ranging from 100 : 1 to 1 : 1), as well as on the parameters of the medium (pH and temperature). The measurement of the catalytic activity of lipase with polylysine showed that, at ratios of 10 : 1 and 5 : 1 (LPP was in excess), the activity of enzyme increased by 17 and 9% relative to the free form, respectively. This is due to the interaction between the positively charged polylysine and the enzyme having an extra negative charge. The results obtained seem promising for biotechnological applications.

Enhanced activity of immobilized transglutaminase for cleaner production technologies

Abstract The enzyme transglutaminase (TGM) has a very important role in applications in various industries. Potentially, a substrate for microbial TGM may be any proteinaceous molecule with glutamine and lysine residues, which broadens the importance of microbial TGM for clean industrial applications or for the use of biosensors. Because stabilization and reusability of an enzyme is an economically important goal for enzymatic processes, the purpose of this study was immobilization of TGM onto magnetic nanoparticles (MNPs) synthesized by the co-precipitation of Fe2+ in Fe3+ ions and modified with carboxymethyl-dextran (CMD). Synthesized nanoparticles were firstly activated with a cross-linking agent (glutaraldehyde (GA) and/or pentaethylenehexamine (PEHA)) and then used for covalent immobilization of the enzyme. Activity of immobilized TGM was determined and compared with activity of the free enzyme. Under optimal immobilization conditions, the enzyme was hyperactivated and showed 99% immobilization efficiency and 110% residual activity. The immobilized TGM showed excellent thermal stability at 50 °C and 70 °C in comparison with non-immobilized enzyme and very good reusability and can be used for cleaner production in different real life industrial applications, such as in leather, textile and wool industry.

Enzyme Immobilization in Polymerized Ionic Liquids-based Hydrogels for Active and Reusable Biocatalysts

Enzyme immobilization in polymerized ionic liquids (PILs) promises to be a versatile tool for simple recovery and reuse of catalysts. In this study, a raw extract of CalB was encapsulated in poly(VEImBr) and assessed with respect to solvent, temperature, amount of enzyme, leaching behavior, and reusability on the example of the kinetic resolution of rac-1-phenylethanol with vinyl acetate. This immobilization method increased the enzyme activity of the CalB raw extract in comparison to the non-immobilized enzyme. The desired product was synthesized with high enantiomeric excess (ee) and no leaching of active enzyme was observed in the experiments. The immobilization method was compared to Novozyme 435 and Lipozyme RM IM, as commercially available immobilisates. Nonpolar solvents, including n-heptane and n-dodecane, proved to be the best reaction solvents, showing nearly full conversion and high catalytic activities. The encapsulated ­lipase was easily recovered from the reaction mixture and reused for ten cycles.

English

Glucose isomerase produced from Streptomyces albaduncus was purified to homogeneity by ammonium sulphate precipitation, followed by ion exchange DEAE-cellulose chromatography, and finally on DEAE-Sephadex A-50 chromatography. The molecular weight of the purified enzyme was estimated to be 54 kDa by sodium dodecyl sulphate polyacrylamide gel electrophresis (SDS-PAGE). The final preparation by the purification procedure had 10.3% final activity recovery and 13.3-fold purification. The optimum pH and temperature for GI activity were 7 and 70°C, respectively. In addition, the presence of the combination of Mg2+ and Co2+ ions (5 mM) improve the GI activity. Partially purified GI enzyme was immobilized by cross-linking with gluteraldahyde, adsorption on DEAE-Cellulose, and entrapment into polyacrylamide. Immobilization of GI enzyme caused sight decrease (3-19% reduction) in the enzymatic activity as compared to that of the non-immobilized enzyme. The maximum enzymatic activity (97%) and stability (88%) was obtained in the immobilized enzyme prepared by entrapment into polyacrylamide.   Key words: Glucose isomerase, Streptomyces albaduncus, purification, immobilization.

Hyaluronan degrading silica nanoparticles for skin cancer therapy

We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes.

Immobilization of Enzyme on Hydrophobic Room Temperature Solid-Phase Ionic Liquids and Its Use for Biocatalytic Transesterification in Organic Solvent: Remarkably Enhanced Activity of Lipase

secondary alcohol in an organic solvent, resulting in an about 2–11-fold increase in enantioselectivity compared to that of a nonimmobilized enzyme. 14 Recently, we found that an enzyme in buffer solution can be immobilized on bulk aggregates of hydrophobic RTSPILs based on alkyl imidazolium (or pyridinum) cation and PF6 anion, which have not functional moieties such as methacryloyl group, via ionic attraction with surface of enzyme. Herein, we further report the activity of an enzyme immobilized on various RTSPILs for the transesterification of sec-phenethyl alcohol with vinyl acetate in an organic solvent (Scheme 1). Interestingly, we found that the RTSPILimmobilized enzyme exhibited an around two-orders-ofmagnitude increase in catalytic activity and could be reused in an organic solvent. The ten RTSPILs with PF6 anion (1-10) used in this study were prepared according to the procedure reported previously: 1-ethyl-3-methylimidazolium ([EMIM], 1, mp 60 o C), 1-(3-phenylpropyl)-3-methyl-imidazolium ([PPMIM], 2, mp 52 o C), 1-(3-phenylpropyl)-2,3-dimethylimidazolium ([PPDMIM], 3, mp 116 o C), 1-dodecyl-3-methylimidazolium ([C12MIM], 4, mp 50 o C), 1-dodecyl-2,3-dimethylimidazolium ([C12DMIM], 5, mp 69 o C), 1-ethyl-4-methylpyridinum ([E4MPy], 6, mp 78 o C), 1-butyl-4-methylpyridinum ([B4MPy], 7, mp 43 o C), 1-(3-phenylpropyl)-4methylpyridinum ([PP4MPy], 8, mp 80 o C), and 1-(3-phenylpropyl)-3-methylpyridinum ([PP4MPy], 9, mp 74 o C).

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.

An Optical Biosensor based on Immobilization of Laccase and MBTH in Stacked Films for the Detection of Catechol.

The fabrication of an optical biosensor by using stacked films where 3-methyl-2-benzothiazolinone hydrazone (MBTH) was immobilized in a hybrid nafion/sol-gelsilicate film and laccase in a chitosan film for the detection of phenolic compounds wasdescribed. Quinone and/or phenoxy radical product from the enzymatic oxidation ofphenolic compounds was allowed to couple with MBTH to form a colored azo-dye productfor spectrophometric detection. The biosensor demonstrated a linear response to catecholconcentration range of 0.5-8.0 mM with detection limit of 0.33 mM and response time of10 min. The reproducibility of the fabricated biosensor was good with RSD value of 5.3 %(n = 8) and stable for at least 2 months. The use of the hybrid materials of nafion/sol-gelsilicate to immobilize laccase has altered the selectivity of the enzyme to various phenoliccompounds such as catechol, guaicol, o-cresol and m-cresol when compared to the non-immobilized enzyme. When immobilized in this hybrid film, the biosensor response onlyto catechol and not other phenolic compounds investigated. Immobilization in this hybridmaterial has enable the biosensor to be more selective to catechol compared with the non-immobilized enzyme. This shows that by a careful selection of different immobilizationmatrices, the selectivity of an enzyme can be modified to yield a biosensor with goodselectivity towards certain targeted analytes.

The covalent immobilization of microsomal uridine diphospho-glucuronosyltransferase (UDPGT): Initial synthesis and characterization of an UDPGT immobilized enzyme reactor for the on-line study of glucuronidation

The microsomal fraction of rat liver containing uridine diphospho-glucuronosyltransferase (UDPGT; EC 2.4.1.17) has been covalently immobilized on a high performance chromatographic support. In this study Nucleosil Si-500 silica was converted into diol-bonded silica and subsequently converted into an aldehyde form through oxidation with sodium periodate. The microsomal fraction was immobilized via Schiff base formation followed by reduction with sodium cyanoborohydride. The resulting immobilized enzyme reactor (IMER) was placed in a multi-dimensional chromatographic system which utilized a mixed mode (C 18 and anion exchange) column to trap the parent compound and glucuronide and a C 18 column to separate the substrate and product. The IMER system was used for the online glucuronidation of 4-methylumbelliferone (4Me7OHC) and acetaminophen (APAP). The Michaelis-Menten kinetic parameters ( K m and V max) associated with the formation of 4Me7OHC and APAP glucuronides demonstrated that the immobilization had not significantly affected the enzymatic activity of the UDPGT relative to the non-immobilized enzyme. The IMER retained enzymatic activity for more than 6 weeks. The results of this study demonstrate an easy and convenient way to identify compounds which may be glucuronidated and to synthesize and characterize the resulting products.

Immobilization, stability and esterification studies of a lipase from a Bacillus sp.

In the present paper a comparative account of the immobilization of a Bacillus lipase on different solid supports with different surface properties and their thermostability is presented. Immobilization enhanced the thermostability of lipase. At higher temperatures, lipase immobilized and cross-linked on a hydrophobic surface showed the maximum thermostability. The optimum temperature for immobilized lipase was 9 degrees C higher than for the free enzyme, while the pH optima were the same. The half-life of soluble lipase at 50 degrees C was calculated to be 4.5 h, while immobilized lipase did not lose any activity even after 8 h. The temperature stability (for 1 h) of immobilized enzyme was enhanced from 50 to 60 degrees C in comparison with non-immobilized enzyme. Applications of immobilized lipase for esterification are also presented.

Development of an immobilized brain glutamine synthetase liquid chromatographic stationary phase for on-line biochemical studies

Glutamine synthetase (GS) plays a key role in the regulation of glutamate availability to neurons. In the present study glutamine synthetase was immobilized on a silica-based immobilized artificial membrane liquid chromatographic stationary phase (IAM-SP) to create the GS-IAM. The stability of GS was improved by immobilization, but the enzyme’s affinity for the substrates l-glutamate and d-glutamate was significantly decreased. In contrast, immobilization significantly increased GS sensitivity to inhibition by methionine sulfoximine. The GS-IAM was packed into a chromatography column to create an immobilized enzyme reactor (GS-IMER). On-line experiments with the GS-IMER demonstrated that the immobilized enzyme was comparable to the non-immobilized enzyme with regards to retention of activity and selectivity toward substrates and inhibitors and was reusable for several weeks.