Glutaraldehyde Coupling Research Articles

Halloysite nanotubes-based hybrid silica monolithic spin tip for hydrophilic solid-phase extraction of sulbactam, cefoperazone, and cefuroxime in whole blood

In this study, we proposed a novel method utilizing polyethyleneimine (PEI)-modified halloysite nanotubes (HNTs)-based hybrid silica monolithic spin tip to analyze hydrophilic β-lactam antibiotics and β-lactamases inhibitors in whole blood samples for the first time. HNTs were incorporated directly into the hybrid silica monolith via a sol-gel method, which improved the hydrophilicity of the matrix. The as-prepared monolith was further modified with PEI by glutaraldehyde coupling reaction. It was found that the PEI-modified HNTs-based hybrid silica monolith enabled a large adsorption capacity of cefoperazone at 35.7 mg g−1. The monolithic spin tip-based purification method greatly reduced the matrix effect of whole blood samples and had a detection limit as low as 0.1 − 0.2 ng mL−1. In addition, the spiked recoveries of sulbactam, cefuroxime, and cefoperazone in blank whole blood were in the range of 89.3–105.4 % for intra-day and 90.6–103.5 % for inter-day, with low relative standard deviations of 1.3–7.2 % and 4.9–10.5 %, respectively. This study introduces a new strategy for preparing nanoparticles incorporated in a hybrid silica monolith with a high adsorption capacity. Moreover, it offers a valuable tool to monitor sulbactam, cefoperazone, and cefuroxime in whole blood from pregnant women with the final aim of guiding their administration.

Real-time monitoring of papain digestion of antibodies immobilized with various strategies by optical interferometry

Antigen binding fragments (Fabs) employed in research are typically generated by the papain digestion of monoclonal antibodies. However, the interaction between papain and antibodies at the interface remains unclear. Herein, we developed ordered porous layer interferometry for the label-free monitoring of the interaction between the antibody and papain at liquid-solid interfaces. Human immunoglobulin G (hIgG) was used as the model antibody, and different strategies were employed to immobilize it on the surface of silica colloidal crystal (SCC) films which are optical interferometric substrates. It was observed that different immobilization strategies induced different changes in the optical thickness (OT) of SCCs. The order of rate of the changes of OT from largest to smallest was IgG immobilized by protein A orientation, glutaraldehyde coupling, and physical adsorption. This phenomenon can be explained by the varied orientations of the antibodies created at the interface by the different modification procedures. The Fab-up orientation maximized the exposure of the hinge region sulfhydryl group and easily underwent conformational transitions because hIgG was immobilized by protein A. This process stimulates papain to produce the highest degree of activity, resulting in the greatest decrease in OT. This study provides insights into the catalysis of papain on antibodies.

Glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) as a generator system for production of NADPH: Development and application in metabolism studies

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) participates in several anabolic and catabolic pathways, being essential in numerous biochemical reactions involving energy release. Most of these reactions require a high amount of NADPH, which can be expensive from an industry point of view. Thus, biotechnology industries developed a great interest in NADPH production. Currently, there are different ways to obtain NADPH in situ, however, the most common is by enzymatic reactions, known as generator systems. Although this approach can be beneficial in terms of cost, the major drawback is the impossibility of reusing the enzyme. To overcome this, enzyme immobilization is a proven alternative. Herein, we report the use of glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) through glutaraldehyde coupling to produce high amounts of NADPH. The G6PDH-Mbs were kinetically characterized showing a sigmoidal curve. Besides, the stability was evaluated, and their reuse was demonstrated for a period superior to 40 days. The G6PDH-Mb was used to in situ production of the NADPH metabolism experiments, using human liver microsome solutions and either albendazole or fiscalin B as model targets. The production of in vitro metabolites from albendazole and fiscalin B was evaluated by comparing the use of NADPH generated in situ with those obtained by commercial NADPH. Moreover, the activity of the G6PDH-Mb was monitored after using it for five consecutive albendazole metabolism reactions, with only a minor decrease in the enzyme activity (3.58 ± 1.67%) after the fifth time of use. The higher concentration obtained when using the designed G6PDH-Mb generator system demonstrated proof of the concept and its applicability.

Biocatalytic Elimination of Pharmaceutics Found in Water With Hierarchical Silica Monoliths in Continuous Flow

Pharmaceutical products (PPs) are considered as emerging micropollutans in wastewaters, river and seawaters, and sediments. The biodegradation of PPs, such as ciprofloxacin, amoxicillin, sulfamethoxazole, and tetracycline by enzymes in aqueous solution was investigated. Laccase from Trametes versicolor was immobilized on silica monoliths with hierarchical meso-/macropores. Different methods of enzyme immobilization were experienced. The most efficient process was the enzyme covalent bonding through glutaraldehyde coupling on amino-grafted silica monoliths. Silica monoliths with different macropore and mesopore diameters were studied. The best support was the monolith featuring the largest macropore diameter (20 µm) leading to the highest permeability and the lowest pressure drop and the largest mesopore diameter (20 nm) ensuring high enzyme accessibility. The optimized enzymatic reactor (150 mg) was used for the degradation of a PP mixture (20 ppm each in 30 ml) in a continuous recycling configuration at a flow rate of 1 ml/min. The PP elimination efficiency after 24 h was as high as 100% for amoxicillin, 60% for sulfamethoxazole, 55% for tetracycline, and 30% for ciprofloxacin.

A novel electrochemical immunosensor for ultrasensitive detection of tumor necrosis factor α based on polystyrene - PAMAM dendritic polymer blend nanofibers

A simple, rapid, and highly sensitive label-free immunosensor based on polystyrene (PS)-polyamidoamine (PAMAM) dendritic polymer nanofibers (NFs) for detection of tumor necrosis factor-α (TNF-α) cytokine was developed. The choice of PAMAM was due to the possibility of designing the surface of NFs to create a novel interface with the high control ability of the density of amine groups at the surface by which capture antibody (anti-TNF-α) could be covalently attached to the surface of NFs through glutaraldehyde (GA) coupling chemistry. Evaluation of the electrode surface using cyclic voltammetry (CV) proved the electrode manufacturing stages were successful by decreasing the peak current values due to the blockage of electron transfer by the biomolecule layer on the surface of NFs, as well as the ability to precisely control the current peak by changing the diameter of the NFs and the concentration of dendrimer. Electrochemical impedance spectroscopy (EIS) was used to test the detection capability of the immunosensors and compare the role of the amount of PAMAM presented on the surface of NFs on their analytical performance. Increasing the concentration of dendrimer greatly increased the sensitivity of immunosensors due to the higher stabilization of the antibody on the nanofiber surface. The immunosensor had a very high affinity for TNF-α cytokines, with a linear response ranging from 10 to 200 pg/mL and a low limit of detection (LOD) of 669 fg/mL. Interference results with Interleukin-10 (IL-10) cytokine showed that the immunosensor was extremely specific and may be utilized to detect cytokines in clinical diagnosis.

Cold-Active Lipase-Based Biocatalysts for Silymarin Valorization through Biocatalytic Acylation of Silybin

Extremophilic biocatalysts represent an enhanced solution in various industrial applications. Integrating enzymes with high catalytic potential at low temperatures into production schemes such as cold-pressed silymarin processing not only brings value to the silymarin recovery from biomass residues, but also improves its solubility properties for biocatalytic modification. Therefore, a cold-active lipase-mediated biocatalytic system has been developed for silybin acylation with methyl fatty acid esters based on the extracellular protein fractions produced by the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania). The extracellular production of the lipase fraction was enhanced by 1% olive-oil-enriched culture media. Through multiple immobilization approaches of the cold-active putative lipases (using carbodiimide, aldehyde-hydrazine, or glutaraldehyde coupling), bio-composites (S1–5) with similar or even higher catalytic activity under cold-active conditions (25 °C) have been synthesized by covalent attachment to nano-/micro-sized magnetic or polymeric resin beads. Characterization methods (e.g., FTIR DRIFT, SEM, enzyme activity) strengthen the biocatalysts’ settlement and potential. Thus, the developed immobilized biocatalysts exhibited between 80 and 128% recovery of the catalytic activity for protein loading in the range 90–99% and this led to an immobilization yield up to 89%. The biocatalytic acylation performance reached a maximum of 67% silybin conversion with methyl decanoate acylating agent and nano-support immobilized lipase biocatalyst.

Graphene Bioelectronic Nose for the Detection of Odorants with Human Olfactory Receptor 2AG1

A real-time sensor for the detection of amyl butyrate (AB) utilising human olfactory receptor 2AG1 (OR2AG1), a G-protein coupled receptor (GPCR) consisting of seven transmembrane domains, immobilized onto a graphene resistor is demonstrated. Using CVD graphene as the sensor platform, allows greater potential for more sensitive detection than similar sensors based on carbon nanotubes, gold or graphene oxide platforms. A specific graphene resistor sensor was fabricated and modified via non-covalent π–π stacking of 1,5 diaminonaphthalene (DAN) onto the graphene channel, and subsequent anchoring of the OR2AG1 receptor to the DAN molecule using glutaraldehyde coupling. Binding between the target odorant, amyl butyrate, and the OR2AG1 receptor protein generated a change in resistance of the graphene resistor sensor. The functionalized graphene resistor sensors exhibited a linear sensor response between 0.1–500 pM and high selectively towards amyl butyrate, with a sensitivity as low as 500 fM, whilst control measurements using non-specific esters, produced a negligible sensor response. The approach described here provides an alternative sensing platform that can be used in bioelectronic nose applications.

Preparation of uniform polyurea microspheres at high yield by precipitation polymerization and their use for laccase immobilization

Uniform polyurea (PU) microspheres were prepared using isophorone diisocyanate as the only monomer either in binary solvents of water/acetone (H2O/AT) or water/acetonitrile (H2O/AN), or in ternary solvent of H2O/AN/DMF, by precipitation polymerization without any shaking or stirring. Through careful examination, highly uniform microspheres were obtained at monomer concentration of 28.0 wt% at 65 °C in H2O/AN at 20/80 by mass, a value much higher than the previously (13.0 wt%) achieved in H2O/AT/DMF. The microspheres were analyzed by FTIR and NMR to confirm their PU structure and used as support for covalent immobilization of laccase via glutaraldehyde coupling. The immobilized laccase showed higher storage and thermal stability than the free laccase, and exhibited higher catalytic activity and better reusability in biodegradation of Congo Red and RBBR, compared with reported studies. In addition to enhancing the productivity of uniform microspheres, this study provides a new pathway to supported catalyst for enzymatic remediation of dyes-polluted water.

Conjugation of carbon coated-iron nanoparticles with biomolecules for NMR-based assay.

In this work, we developed and optimized conjugates of carbon-coated iron nanoparticles (Fe@C) with streptavidin and monoclonal antibodies. The conjugation procedure included two stages. First, amino groups were grafted onto the carbon shell to facilitate noncovalent sorption of bovine serum albumin (BSA). Further, the covalent attachment of proteins to the BSA layer via glutaraldehyde coupling was performed. It was established and confirmed that the synthesis procedure is reproducible and allows preparation of stable conjugates. The resulting nanoparticles are clusters of Fe@C particles coated by proteins. The size of the clusters is in the range of 100-190 nm and can be controlled via the tuning of conjugation conditions, including pH, BSA-to-Fe@C ratio, etc. Conjugates of Fe@C with streptavidin and monoclonal antibodies (sizes of approximately 140-150 nm) were synthesized. Proton T2 relaxometry was used to detect these conjugates with very high sensitivity due to the magnetic markers, Fe@C. The relaxivity (r2) of different conjugates varied within the range of 290-450 1/s*mM. Conjugate applicability for relaxometry-based assay was confirmed by direct detection of streptococcal protein G and biotinylated BSA in a dot immunoassay.

Stability and repeatability improvement of horseradish peroxidase by immobilization on amino-functionalized bacterial cellulose

Abstract Bacterial cellulose (BC) is a biodegradable material with many excellent properties for enzyme immobilization. However, hydroxyl groups of low reactivity in glucose units cannot directly react with the amines of enzymatic proteins. In this work, amino-functionalized bacterial cellulose was firstly used as carrier to immobilize horseradish peroxidase (HRP) via glutaraldehyde coupling. SEM, FT-IR, XPS, BET, and TGA were used to characterize the properties of modified BC and showed that it is more suitable for enzyme immobilization. The optimum pH range for immobilized HRP (pH 5.5–8.5) was wider than that of free enzyme (pH 6–8), and the immobilized HRP exhibited good adaptability to environmental alkalinity. The relative activity of immobilized HRP at 25–40 °C was greater than 90%, significantly surpassing that of free HRP. Furthermore, the obtained kinetic constant values showed that modified BC had decreased affinity for the substrate. Additionally, modified BC-immobilized HRP was reused efficiently for 10 cycles with greater than 70% of its original activity retained. Under optimal conditions, coupling ratio and specific activity could reach 86.7% and 41.7 Ug -1 min -1 , respectively. These results show that the immobilization of HRP on amino-functionalized BC enhanced its appropriateness for a future use in various biotechnological and environmental applications.

Ficus sycomorus latex: An efficient alternative Egyptian source for horseradish peroxidase in labeling with antibodies for immunodiagnostic kits.

Aim:In view of various peroxidase applications, the searching for new sources of unique peroxidase properties is highly desirable. The present study aims to evaluate the efficiency of the peroxidase of locally grown sycamore latex (POL) for conjugation with antibodies and to study the conjugate optimal conditions, storage stability, and affinity toward different substrates as compared with commercial horseradish peroxidase (HRP).Materials and Methods:Anti-mouse antibodies were prepared in rabbits and purified by protein A sepharose affinity column chromatography. The POL and HRP conjugates were prepared by one-step glutaraldehyde coupling method. The reactivity of the prepared conjugates was examined using the enzyme-linked immunosorbent assay (ELISA). The optimal enzymatic conditions, storage stability, and affinity toward substrates were also determined for both the conjugates.Results:The POL showed higher percent recovery (98%) than HRP (78%) over the initial activity after conjugation process. The POL and HRP conjugates showed ELISA titers of 1:120 and 1:80, respectively, demonstrating high binding affinity of POL-conjugate. The POL-conjugate showed high thermal stability up to 70°C compared with HRP-conjugate up to 40°C. After conjugation, POL had wide pH stability (5.0-8.0) compared with HPR (4.5-6.0). Both of the prepared conjugates had a high affinity toward the substrates used in immunoassays with lower Km values. The POL-conjugate showed high storage stability for its enzymatic activity and ELISA titer compared with HRP-conjugate after 1 year at −20°C.Conclusion:The POL of Ficus sycomorus latex is an efficient source for labeling antibodies and could be utilized in immunodiagnostic kits.

Design of an aptamer-based magnetic adsorbent and biosensor systems for selective and sensitive separation and detection of thrombin

An aptasensor was designed for sensitive detection of thrombin using in biological fluids by integrating a magnetic aptamer-microbeads. To achieve this goal, the surface of gold plated QCM crystals was coated with L-cysteine and a thrombin binding DNA aptamer was immobilized on the L-cysteine coated QCM crystals surface via glutaraldehyde coupling. The binding interactions of thrombin to QCM crystals were characterized. Magnetic poly(2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate-vinylene carbonate), Mp(HEMA-EGDMA-VC) microbeads were synthesized and thrombin binding aptamer (TBA) was immobilized. The Mp(HEMA-EGDMA-VC)-TBA microbeads were effectively adsorbed thrombin from serum in a relatively short contact time (ca. 5.0 min), and the eluted protein from Mp(HEMA-EGDMA-VC)-TBA was transferred to the QCM aptasensor that showed a specific detection of thrombin from serum. The detection limit of thrombin using aptasensor was 1.00 nmol L−1. The calculation dissociation constant of the aptasensor was 68.5 nmol L−1. The selectivity of the aptasensor system was tested with three different proteins (i.e., elastin, immunoglobulin G (IgG) and human serum albumin (HSA)) and showed high specificity to thrombin. The aptasensor was regenerated by washing with NaOH solution, and repeatedly used until 20 cycles without a change in the performance.

Fast and Sensitive Detection of Salmonella in Milk Samples Using Aptamer-Functionalized Magnetic Silica Solid Phase and MCM-41-Aptamer Gate System.

General detection methods for S. enterica include PCR analysis, immunologic methods, solid culturing techniques, and various microscopic studies. Milk and other food samples demonstrate an especially difficult challenge for direct detection, resulting from high biological contents. In this report, we aimed for fast detection of pathogen cells through an efficient magnetic capture and subsequent quick detection based on aptamer affinity. The Fe3O4@SiO2@pGMA and MCM-41 particles were prepared separately and used for preconcentration and detection, respectively. Aptamer oligonucleotide sequences against S. enterica were fixed on both amine-functionalized MCM-41 and Fe3O4@SiO2@pGMA particles via glutaraldehyde coupling. The captured Salmonella cells were determined by a fluorescent homogeneous assay in the samples by aptamer-gated MCM-41 silica particles. Our method achieved a sensitive assay to detect Salmonella cells in milk samples as low as 103 CFU/ml without any culturing. Hence, the proposed sensing strategy might be an efficient platform for pathogen detection in a food matrix.

Covalently Immobilized Lipase on a Thermoresponsive Polymer with an Upper Critical Solution Temperature as an Efficient and Recyclable Asymmetric Catalyst in Aqueous Media

AbstractA thermoresponsive lipase catalyst with an upper critical solution temperature (UCST) of about 26 °C was exploited by covalent immobilization of an enzyme, Pseudomonas cepacia lipase (PSL), onto poly(acrylamide‐co‐acrylonitrile) by glutaraldehyde coupling. The experimental conditions for the PSL immobilization were optimized. The immobilized PSL was much more stable for wide ranges of temperature and pH than free PSL. The material was also evaluated as an asymmetric catalyst in the kinetic resolution of racemic α‐methylbenzyl butyrate at 55 °C in an aqueous medium and exhibited high catalytic performance and stability. Up to 50 % conversion and 99.5 % product enantiomeric excess were achieved, thus providing highly pure enantiomers. This biocatalyst could be easily recovered by simple decantation for reuse based on temperature‐induced precipitation. It showed good reusability and retained 80.5 % of its original activity with a well reserved enantioselectivity in the 6th cycle. This work would shed light on the future development of new UCST‐type enzyme catalysts.

Lipase NS81006 immobilized on functionalized ferric-silica magnetic nanoparticles for biodiesel production

ABSTRACTLipase-mediated biodiesel production has been drawing increasing attention in recent years. In this paper, lipase NS81006 was immobilized on magnetic nanoparticles and then used for biodiesel production. The surface of the superparamagnetic nanoparticles prepared by a co-precipitation method was coated with silica by the Stöber method and functionalized with organosilane compounds APTES (3-aminopropyltriethoxysilane) and MPTMS (3-mercaptopropyltrimethoxysilane). Further, lipase (NS18006) was covalently immobilized onto the organosilane functionalized ferric-silica carrier by glutaraldehyde coupling reagent, and was employed to catalyze biodiesel production. The maximum lipase immobilization efficiency of 78.7 and 77% was obtained in APTES and MPTMS, respectively, at 10% of glutaraldehyde. The effect of reaction temperature and oil-to-methanol molar ratio, and the catalyst reusability were investigated. The maximum yield (71%) of biodiesel was obtained in both catalysts. The immobilized lipase could maintain its catalytic activity for up to five cycles.

A conductometric creatinine biosensor prepared through contact printing of polyvinyl alcohol/polyethyleneimine based enzymatic membrane

A conductometric creatinine biosensor was prepared through creatinine deaminase entrapped into a polyvinyl alcohol/polyethyleneimine/gold nanoparticles composite film, previously deposited on the surface of a PDMS stamp and then, after glutaraldehyde coupling, transferred on interdigitated microelectrodes by contact printing method. The detection of creatinine in a sucrose background solution, revealed a wide linear calibration plot (10–600μM creatinine concentration range), with a limit of detection of 2μM (S/N=3), a good repeatability. The resulting creatinine biosensor exhibits a drastic increase of detection range compared to that of the biosensor prepared by simple dropping: 10–300μM creatinine concentration range. The developed biosensor was validated by determining creatinine in spiked artificial blood serum.

Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor

The nanoparticles (NPs) aggregates of commercial urease from jack beans (Canavalia ensiformis) were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine dihydrochloride. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM), UV and Fourier transform infrared (FTIR) spectroscopy. The TEM images of urease NPs showed their size in the range, 18–100nm with an average of 51.2nm. The ENPs were more active and stable with a longer shelf life than native enzyme molecules. The ENPs were immobilized onto chitosan (CHIT) activated nitrocellulose (NC) membrane via glutaraldehyde coupling with 32.22% retention of initial activity of free ureaseNPs with a conjugation yield of 1.63mg/cm2. This NC membrane was mounted at the lower/sensitive end of the ammonium ion selective electrode (AISE) with O-ring and then electrode was connected to a digital pH meter to construct a potentiometric urea biosensor. The biosensor exhibited optimum response within 10s at pH 5.5and 40°C. The biosensor was employed for measurement of potentiometric determination of urea in sera of apparently healthy and persons suffering from kidney disorders. The biosensor displayed a low detection limit of 1µM/L with a wide working range of 2–80µM/L (0.002–0.08mM) and sensitivity of 23mV/decade. The analytical recovery of added urea in serum was 106.33%. The within and between-batch coefficient of variations (CVs) of present biosensor were 0.18% and 0.32% respectively. There was a good correlation (r = 0.99) between sera urea values obtained by reference method (Enzymic colorimetric kit method) and the present biosensor. The biosensor had negligible interference from Na+,K+,NH+4 and Ca2+ but Mg2+,Cu2+ and ascorbic acid but had slight interference, which was overcome by specific ion selective electrode. The ENPs bound NC membrane was used maximally 8–9 times per day over a period of 180 days, when stored in 0.01M sodium acetate buffer pH 5.5 at 4°C.

High yield preparation of uniform polyurea microspheres through precipitation polymerization and their application as laccase immobilization support

Uniform polyurea (PU) microspheres were prepared through precipitation polymerization using isophorone diisocyanate (IPDI) as the only monomer in mixed solvent of water-acetonitrile. The highest IPDI loading allowed to have uniform PU microspheres went up to 23wt%, with a high yield of about 95% for the microspheres. The productivity of microspheres thus obtained was about 22 times higher than that by free radical precipitation polymerization, and practically doubled that previously reported on the same polymerization in the mixed solvent of water-acetone. In addition, the polymerization was done under quiescent process with the reactor standing still without any stirring or shaking. TGA and FTIR tests indicated that the microspheres were of high thermal stability with abundant amine group on the surface. As a model enzyme, laccase was immobilized on the surface of the PU microspheres via glutaraldehyde coupling. Compared with the free laccase, the immobilized one exhibited higher activity in alkaline environment and higher thermal stability. The immobilized laccase was used as biocatalyst for degradation of Remazol Brilliant Blue R and showed good decolorization capacity with good reusability. About 65% of its initial catalytic activity was retained after 7 cycles of reuse. This work provides a practically operational protocol for large scale production of uniform PU microspheres, and also demonstrates that these microspheres have promising applications as enzyme immobilization carrier and as biocatalyst in organic dyes degradation.

Effective synthesis of theaflavin-3,3′-digallate with epigallocatechin-3-O-gallate and epicatechin gallate as substrates by using immobilized pear polyphenol oxidase

In the present study, pear polyphenol oxidase (PPO) was purified, immobilized and applied for the synthesis of theaflavin-3,3′-digallate (TF3). Firstly, PPO of pear (Pyrus bretschneideri Rehd cv. Huangguan) was purified 24.36-fold in specific activity with a recovery of 6.77% by sequential use of precipitation with 70% saturated ammonium sulfate and chromatography of DEAE-Sepharose Fast Flow column. The purified pear PPO, found to be a dimer of identical subunits of molecular mass 35kDa, was then successfully immobilized onto Fe3O4/chitosan nanoparticles via glutaraldehyde coupling reaction. Finally, TF3 was effectively synthesized by applying the immobilized PPO as biocatalyst with epicatechin gallate (ECG) and epigallocatechin-3-O-gallate (EGCG) as substrates, and the maximum yield of TF3 was 42.23% based on the amount of ECG added. In addition, the immobilized enzyme still remained 85% of its initial ability after successive usage of 8 cycles and could be stored for 30days with less loss of activity. All the results suggest that the developed procedure is quite useful and has great potential for synthesis of TF3 and other theaflavins.

A KINETIC STUDY OF ENZYMATIC HYDROLYSIS OF OIL PALM BIOMASS FOR FERMENTABLE SUGAR USING POLYETHYLENE GLYCOL (PEG) IMMOBILIZED CELLULASE

In this work, enzymatic hydrolysis by cellulase in a soluble and an immobilized form was studied to convert lignocellulosic oil palm empty fruit bunch (EFB) biomass into fermentable sugars as a feedstock for bioethanol production. The cellulase was covalently immobilized with activated and functionalized polyethylene glycol (PEG) via glutaraldehyde coupling method. As a whole, the immobilized cellulase displayed 50% higher efficiency over free cellulase, in reducing sugar recovery during hydrolysis reactions at pH of 4.8 and temperature of 50°C. From the kinetic study, it showed that Michaelis constant (Km) and limiting velocity (Vmax) of immobilized cellulase were 179.2 mg/ml and 33.5 mg/ml.min respectively, comparable with the value for free cellulose, 171.8 mg/ml and 34.5 mg/ml.min respectively. This result could be attributed to the effect of PEG on the binding cellulase to substrate desorb substrates, and enables free interaction of cellulase to hydrolyse cellulose maximally