Enzyme Aggregates Research Articles

Efficient decolorization and detoxification of triarylmethane and azo dyes by porous-cross-linked enzyme aggregates of Pleurotus ostreatus laccase

In this preset study, porous-cross-linked enzyme aggregates (CLEAs) of Pleurotus ostreatus laccase were utilized for the spontaneous decolorization and detoxification of triarylmethane and azo dyes, reactive blue 2 (RB) and malachite green (MG). The specific surface area and pore radius of the porous-CLEAs are 136.3 m2/g and 19.47 Ao, and the higher specific surface indicated greater biocatalytic efficiency, as increased mass transfer and dye interaction with the CLEAs laccase. CLEAs laccase decolorized 500 ppm of MG and RB with 98.12–58.33% efficiency after 120 min, at pH 5.0 and 50°C, without a mediator. Furthermore, the biotransformation of the MG and RB with immobilized laccase was confirmed with the help of UV–visible spectroscopy, high-performance liquid chromatography, and Fourier transform infrared spectroscopy. The reusability potential of CLEAs was assessed in batch mode for 10 cycles of dye decolorization. The decolorization activities for the immobilized laccase were 89% and 12% at the 6th cycle for MG and RB, respectively. This immobilized enzyme could effectively remove dyes from aqueous solution, and demonstrated significant detoxification in experimental plants (Triticum aestivum and Phaseolus mungo) and plant growth-promoting rhizobacteria (Azospirillum brasilense, Bacillus megaterium, Rhizobium leguminosarum, Bacillus subtilis, and Pseudomonas fluorescens). In conclusion, porous CLEAs laccase could be useful as a potential bioremediation tool for the detoxification and decolorization of dyeing wastewater in future.

Tailor-made active enzyme aggregates in vivo for efficient degradation of biomass polysaccharide into reducing sugar

Tailor-made active enzyme aggregates in vivo for efficient degradation of biomass polysaccharide into reducing sugar

Bio-imprinted magnetic cross-linked polyphenol oxidase aggregates for enhanced synthesis of L-dopa, a neurodegenerative therapeutic drug

Bio-imprinted magnetic cross-linked enzyme aggregates (i-m-CLEAs) of polyphenol oxidase (PPO) obtained from potato peels were prepared using amino-functionalized magnetic nanoparticles. Bio-imprinting is being used to improve the catalytic efficiency and conformational stability of enzymes. For bio-imprinting, PPO was incubated with different imprint/template molecules (catechol, 4-methyl catechol and l-3,4-dihydroxy phenylalanine) before cross-linking with glutaraldehyde. CLEAs imprinted with 4-methyl catechol showed maximum activity as compared with non-bio-imprinted magnetic CLEAs (m-CLEAs). They were further characterized by scanning electron microscopy and confocal microscopy. In bio-imprinted m-CLEAs, half-life (t1/2) of PPO significantly improved (364.74 min) as compared to free PPO (43.58 min) and non-bio-imprinted m-CLEAs (266.54 min). Bio-imprinted m-CLEAs showed excellent thermal and storage stability as well as reusability. The CLEAs preparation were used for the synthesis of l-3,4-dihydroxyphenylalanine (L-dopa, a therapeutic drug to treat neurodegenerative disorder) and a remarkable increase in L-dopa yield (23.5-fold) was obtained as compared to free enzyme. A cost effective and reusable method has been described for the production of L-dopa.

Dephytinization of wheat and rice bran by cross-linked enzyme aggregates of Mucor indicus phytase: a viable prospect for food and feed industries.

Novel feeds for improved feed intake and for enhanced nutrient bioavailability have recently attracted attention. Insoluble dietary fibers, especially rice and wheat bran, have generated much interest due to their nutritional value. Incorporating insoluble dietary fiber into diets could be a viable way to maximize the feed conversion ratio. Cross-linked phytase aggregates (CLPA) were synthesized by precipitating enzymes followed by cross-linking with 5 mmol L-1 glutaraldehyde, yielding 88.24 (U g-1 ) of enzyme load without the assistance of a proteic feeder. The epitome of the study is the dephosphorylation of wheat bran and rice bran by varying pH, enzyme concentration, and temperature. The highest inorganic phosphorus liberation by 150 U L--1 of free phytase was 23.72 (wheat bran) and 48.08 mg g-1 (rice bran) after 12 h of incubation. Furthermore, 150 U L-1 of CLPA liberated 28.72 (wheat bran) and 52.08 mg g-1 (rice bran) of inorganic phosphorus with an incubation time of 12 h. Thermostable free phytase was insolubilized to dephosphorylate the agro-residue, namely, wheat bran and rice bran, to reduce the anti-nutritional factor (the phytate content) of these insoluble dietary fibers. © 2022 Society of Chemical Industry.

Cross-linking the peroxidase: from potato peel valorization to colored effluents treatment

Despite the obvious benefits from mass production such as an increased productivity, lower product cost and rapid evolution, nowadays we are dealing with energy consumption issues and pollution. The generated waste poses a threat to the environment, so new green techniques are developed constantly. Waste valorization is one of the trending concepts that is a part of sustainability strategies. Potato exploitation due to mass production of chips, hash browns, frozen food and starch leads to a formation of high waste load. There are several available ways for potato peel valorization: as a biofertilizer, as a substrate for microbial growth, as an adsorbent, for extraction of antioxidants and for extraction of enzymes. The enzyme peroxidase is abundant in potato peel. This enzyme uses hydrogen peroxide as an activator and can be readily used for oxidation of different compounds - pollutants. In this study, peroxidase was isolated from potato peel and immobilized as cross-linked enzyme aggregates. Pectin was used as a green cross-linker. The immobilized potato peel peroxidase was used for degradation of a textile anthraquinone dye Lanaset Violet B. Under the optimal process parameters: pH 3, 0.4 mM hydrogen peroxide, 0.8 μmol/min CLEA peroxidase, 10 mg/L dye and 70 min, 85.71±1.45 % dye degradation was achieved. The operational stability, as a key parameter for immobilized enzyme systems, was also examined. After 4 cycles CLEA peroxidase kept 31.57±1.79 % of its biodegradation efficiency.

Cross-linked enzyme aggregates (combi-CLEAs) derived from levansucrase and variant inulosucrase are highly efficient catalysts for the synthesis of levan-type fructooligosaccharides

Efficient and convenient access to short to medium chain length levan-type fructooligosaccharides (LFOS) is needed in order realise the nutritional potential of this class of bioactive oligosaccharides. While LFOS are synthesised by fructansucrase enzymes, these reactions are routinely associated with high molecular weight fructan polymer formation. Recent studies have shown that FOS production can be enhanced by the combination of levansucrase and inulosucrase in a one-pot reaction. In the present study, the novel mixed enzyme cross-linked enzyme aggregates (combi-CLEAs) based on levansucrase (Lev) and N543A variant inulosucrase (Inu) were prepared by ammonium sulfate precipitation, followed by glutaraldehyde cross-linking. The effect of Lev and Inu ratio on the activity of combi-CLEAs was explored. The results showed that >70% of total sucrase activity was recovered after immobilization and that the combi-CLEAs produced high amounts of LFOS (degree of polymerisation 3 to 21), while high molecular weight polysaccharide production was much reduced. Biochemical characterisation indicated that the optimum pH and temperature of combi-CLEAs (pH 5.5 and 50ᵒC, respectively) were comparable to those of free enzyme; however, the stability of the enzyme was improved. In addition, these combi-CLEAs have operational stability for several reaction cycles, which makes them very attractive for biotechnology applications.

AFM Investigation of the Influence of Steam Flow through a Conical Coil Heat Exchanger on Enzyme Properties.

The present study is aimed at the revelation of subtle effects of steam flow through a conical coil heat exchanger on an enzyme, incubated near the heat exchanger, at the nanoscale. For this purpose, atomic force microscopy (AFM) has been employed. In our experiments, horseradish peroxidase (HRP) was used as a model enzyme. HRP is extensively employed as a model in food science in order to determine the influence of electromagnetic fields on enzymes. Adsorption properties of HRP on mica have been studied by AFM at the level of individual enzyme macromolecules, while the enzymatic activity of HRP has been studied by spectrophotometry. The solution of HRP was incubated either near the top or at the side of the conically wound aluminium pipe, through which steam flow passed. Our AFM data indicated an increase in the enzyme aggregation on mica after its incubation at either of the two points near the heat exchanger. At the same time, in the spectrophotometry experiments, a slight change in the shape of the curves, reflecting the HRP-catalyzed kinetics of ABTS oxidation by hydrogen peroxide, has also been observed after the incubation of the enzyme solution near the heat exchanger. These effects on the enzyme adsorption and kinetics can be explained by alterations in the enzyme hydration caused by the influence of the electromagnetic field, induced triboelectrically by the flow of steam through the heat exchanger. Our findings should thus be considered in the development of equipment involving conical heat exchangers, intended for either research or industrial use (including miniaturized bioreactors and biosensors). The increased aggregation of the HRP enzyme, observed after its incubation near the heat exchanger, should also be taken into account in analysis of possible adverse effects from steam-heated industrial equipment on the human body.

Durable and Versatile Immobilized Carbonic Anhydrase on Textile Structured Packing for CO2 Capture

High-performance carbon dioxide (CO2)-capture technologies with low environmental impact are necessary to combat the current climate change crisis. Durable and versatile “drop-in-ready” textile structured packings with covalently immobilized carbonic anhydrase (CA) were created as efficient, easy to handle catalysts for CO2 absorption in benign solvents. The hydrophilic textile structure itself contributed high surface area and superior liquid transport properties to promote gas-liquid reactions that were further enhanced by the presence of CA, leading to excellent CO2 absorption efficiencies in lab-scale tests. Mechanistic investigations revealed that CO2 capture efficiency depended primarily on immobilized enzymes at or near the surface, whereas polymer entrapped enzymes were more protected from external stressors than those exposed at the surface, providing strategies to optimize performance and durability. Textile packing with covalently attached enzyme aggregates retained 100% of the initial 66.7% CO2 capture efficiency over 71-day longevity testing and retained 85% of the initial capture efficiency after 1-year of ambient dry storage. Subsequent stable performance in a 500 h continuous liquid flow scrubber test emphasized the material robustness. Biocatalytic textile packings performed well with different desirable solvents and across wide CO2 concentration ranges that are critical for CO2 capture from coal and natural gas-fired power plants, from natural gas and biogas for fuel upgrading, and directly from air.

Biotransformation of 4, 4'-dihydroxybiphenyl and dienestrol by laccase from Trametes versicolor

Disemboguing of industrial and household wastes into the environment create significant challenge to human health. Both 4,4′–dihydroxybiphenyl and dienestrol are a type of environmental micro-pollutants that dreadfully act as endocrine disruptors to both humans and wildlife. In the current study, laccase from Trametes versicolor was exploited for the biotransformation of 4,4′–dihydroxybiphenyl and dienestrol. Although both the micro-contaminants possess two phenolic moieties in their structures, laccase oxidizes 4,4′–dihydroxybiphenyl with significantly higher efficiency than dienestrol. Immobilization of laccase viz., encapsulation in polyacrylamide gel (PAG-laccase) and construction of cross-linked enzyme aggregates (CLEA-laccase) led to a decrease in turnover number, and substrate affinity, which consequently resulted in a reduced catalytic activity. However, immobilization of laccase conferred substantial stability compared to free enzyme. Immobilized laccase exhibited greater thermal stability with a wider activity range of pH and temperature. Encapsulation of laccase also considerably increased its capability to withstand higher concentrations of salt, organic solvents and inhibitors than its free form. Unlike free laccase, encapsulated laccase acquired the attribute of reusability in multiple catalytic reactions, which enormously compensated its reduced activity and enhanced its applicability than native form. This study comprehensively delineates the catalytic properties of native and immobilized laccase in the removal of noxious endocrine disruptors like 4,4′–dihydroxybiphenyl and dienestrol.

Magnetic cross-linked lipase aggregates coupled with ultrasonic pretreatment for efficient synthesis of phytosterol oleate

In this study, coupling with substrate ultrasonic pretreatment technology, magnetic cross-linked enzyme aggregates (CRL-FIL-CLEAs@Fe3O4) was used as novel biocatalyst for efficient preparation of phytosterol oleate in solvent-free reaction system. The CRL loading was 0.16 mg protein/mg CRL-FIL- CLEAs@Fe3O4. The effects of ultrasonic parameters, catalyst dosage, substrate molar ratio, and reaction temperature on the synthesis of phytosterol esters were studied. The results showed that the catalytical properties of CRL-FIL-CLEAs@Fe3O4 (7.09±0.11 µmol phytosterol/mg protein. hour) was significantly improved compared with natural lipase from Candida rugosa (CRL, 5.13±0.21 µmol phytosterol/mg protein. hour). Simultaneously, ultrasonic pretreatment greatly enhanced substrate mass transfer, further reducing the esterification time. The max conversion rate 94.72% of phytosterol was obtained after 9 h reaction under optimum condition (the molar ratio of oleic acid to phytosterol was 6:1, the concentration of CRL-FIL-CLEAs@Fe3O4 was 10 wt% of the phytosterol mass). After five recycles, the activity of CRL-FIL-CLEAs@Fe3O4 (80.02%) was still 2.68 times higher than that of natural lipase system (29.91%). Meanwhile, the novel immobilized lipase (CRL-FIL-CLEAs@Fe3O4) before and after reaction was characterized by SEM, AFM, VSM, TEM, and TGA. The results showed that CRL-FIL-CLEAs@Fe3O4 was a robust and recyclable biocatalyst for phytosterol esterification.

Co-Immobilizing Two Glycosidases Based on Cross-Linked Enzyme Aggregates to Enhance Enzymatic Properties for Achieving High Titer Icaritin Biosynthesis.

Icaritin is a rare and high-value isopentane flavonoid compound with remarkable activities. Increasing yields while reducing cost has been a great challenge in icaritin production. Herein, we first reported a high titer icaritin biosynthesis strategy from epimedin C through co-immobilizing α-l-rhamnosidase (Rha1) and β-glucosidase (Glu4) using cross-linked enzyme aggregates (CLEAs). The created CLEAs exhibited excellent performances in terms of catalytic activity, thermal stability, pH stability, and reusability. Notably, Rha1-CLEAs (Ki: 1 M) and Glu4-CLEAs (Ki: 0.1 M) were more tolerant to sugars (glucose or rhamnose) than free enzymes (0.1 M for Rha1 and 0.007 M for Glu4) by immobilization, achieving the highest icaritin productivity under the highest substrate concentration ever reported. Finally, about 34.24 g/L icaritin could be obtained from 100 g/L epimedin C within 8 h, indicating the great potential for industrialization. This study also provides a promising strategy for the low-cost production of other high-value aglycone compounds by solving poor stability and sugar inhibition of glycosidase.

Robust and recyclable cross-linked enzyme aggregates of sucrose isomerase for isomaltulose production

A novel cross-linked enzyme aggregates (CLEAs) catalyst was produced by precipitation and cross-linking sucrose isomerase (SIase) for isomaltulose production. The effects of precipitants and cross-linkers on the catalytic performance of the CLEAs were first evaluated. Then, bovine serum albumin (BSA) was used as additive and two immobilized enzymes, cross-linked SIase aggregates (CLSIAs) and CLSIAs-BSA were obtained. All the immobilized preparations exhibited superior thermal stability, pH tolerance, and storage stability compared to the soluble SIase, and showed excellent reusability. These samples still retained more than 61% of their initial activity after ten reuse cycles, with CLSIAs-BSA retaining up to 91.7%. The conversion ratios of sucrose into isomaltulose using CLSIAs-BSA reached 88.4 and 81.2% with sucrose and sugar cane juice as substrate, respectively. Therefore, CLSIAs are a highly effective biocatalyst for the preparation of isomaltulose with great potential for industrial applications.

Ethanolysis of soybean oil catalyzed by magnetic CLEA of porcine pancreas lipase to produce ecodiesel. Efficient separation of ethyl esters and monoglycerides

Porcine pancreas lipase (PPL, a 1,3-specific lipase) magnetic crosslinked enzyme aggregates have been used to produce Ecodiesel, a blend of fatty acid ethyl esters (FAEE) and acylglycerides (mainly monoacylglycerides, MAG), by ethanolysis of soybean oil. An Ecodiesel containing 67 wt% FAEE and 27.7 wt% acylglycerides was obtained using 750 tributyrin activity units (TBU) of PPL per gram of oil and an oil:ethanol molar ratio of 1:7 at 37 °C after 48 h of reaction. This product was fractioned into a FAEE-rich phase and a MAG-rich phase. To this goal, Soxhlet extractor and silica gel column chromatography were used. Soxhlet extraction for 60 cycles allowed recovering 91% of the ethylic esters fed into the extractor in a nonpolar phase containing around 90 wt% of FAEE, which could be further increased to around 92% by caustic or to 94% by enzymatic polishing treatments (using Lipozyme® CALB L), this product being close to the requirements of biodiesel. On the other hand, the separation in the silica gel column chromatography was more efficient to purify MAG, allowing the recovery of the 93% acylglycerides with emulsifying properties, with a final purity of 94 wt% MAG.

Preparation, characterization and stability of cross linked nitrilase aggregates (nitrilase-CLEAs) for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid.

Nitrilases capable of performing hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid were screened, and ES-NIT-102 was the best nitrilase for said biotransformation. Nitrilase was immobilized as cross linked enzyme aggregates (nitrilase-CLEAs) by fractional precipitation with iso-propanol, and cross linked with glutaraldehyde. The nitrilase-CLEAs prepared with optimized 35mM glutaraldehyde for 120min cross linking time had 82.36 ± 4.45% residual activity, and displayed type-II structural CLEAs formation as confirmed by particle size, SEM, FTIR, and SDS-PAGE analysis. Nitrilase-CLEAs had superior pH and temperature stability, showed a shift in optimal temperature by 5°C, and retained nearly 1.5 to 1.7 folds activity over free nitrilase at 50°C and 55°C after more than 9h incubation. Nitrilase-CLEAs showed reduced affinity and decreased conversion of substrate as indicated by slightly higher Km values by 5.19% and reduced Vmax by 17%. Furthermore, these nitrilase-CLEAs showed 98% conversion, 94.72g/L product formation, and 83.30% recovery after 24h when used for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid. Nitrilase-CLEAs were catalytically active for 3 cycles showcasing 81% conversion, 75.53g/L product formation and 66.42% yield. The recovered product was confirmed by HPLC, FTIR, LC-MS, and 1H NMR, and displayed > 99% purity.

An improved process for the preparation of ethyl-(R)-2-hydroxy-4-phenylbutyrate, (R)-HPB ester by lipase from Thermomyces lanuginosus

Kinetic resolution of rac-ethyl-2-hydroxy-4-phenylbutyrate was carried out using lipase from Thermomyces lanuginosus immobilized in form of cross-linked enzyme aggregates (CLEAs). The reaction was performed in phosphate buffer (50 mM, pH 7.5) at 27 °C and 200 rpm with 10 % w/w substrate and catalyst loading in a moving basket reactor. The immobilized enzyme was recycled up to 5 times without any loss of activity, followed by marginal loss of activity (6 %) up to 10 cycles. The required pure ethyl-(R)-2-hydroxy-4-phenylbutyrate was isolated with 48 % yield and > 99 % e.e. The estimated kinetic parameters were Vmax,app = 1623 ± 81.15 mM/h/g and Km,app = 0.13 ± 0.07 M (correlation coeff. r = 0.94).

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid.

Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into the N-terminal and C-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of the TAL-CLEs was measured via a synthesis sample of p-Coumaric acid. Our TAL-CLEs showed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product—which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes.

Strategies to Reuse of Biocatalysts in the Hydrolysis and Esterification Reactions of Licuri (Syagrus coronata (Mart.) Becc.) Oil

AbstractThe industrial limitations for the use of enzymes are mainly related to their catalytic capacity and their high cost. Herein, it is proposed to evaluate the productive capacity of Burkholderia cepacia lipase (free enzyme and cross‐linked enzyme aggregates – CLEAs) for the hydrolysis and esterification reactions of licuri (Syagrus coronata (Mart.) Becc.) oil, also addressing different strategies to optimize the processes for the reuse of this valuable biocatalyst. For hydrolysis reactions, it was obtained an increase of 178 % in fatty acids productivity using CLEAS compared to free enzymes. By submitting the system to liquid‐liquid separation, it was possible to reuse the free enzymes, which maintained a relative conversion of approximately 75 % of the initial cycle. For the esterification reaction, CLEAs showed a productivity increase of 50 times with lauric acid and 15 times with palmitic acid; in addition to being recovered and reused for 5 cycles, with little loss of efficiency, showing good resistance to the organic environment.

Concentration‐dependent response of soil parameters and functions to trifluoroacetic acid

AbstractDespite growing environmental concerns about perfluoroalkyl and polyfluoroalkyl substances (PFAS) worldwide, the ultra‐short‐chain PFAS, for example, trifluoroacetic acid (TFA), have been largely neglected in the context of soil pollution. Given the persistence and increasing occurrence of TFA in the soil environment, accumulated TFA could impact soil properties and functions. Therefore, we investigated the effects of a wide range of concentrations of TFA (0.001–100 μg g−1) on chemical, physical and biological indicators of soil health, using a six‐week microcosm experiment. Our results showed that TFA treatments decreased soil pH, sulphate content, soil respiration, litter decomposition and bacterial abundance, and increased phosphate content (p < 0.05). These effects were observed for TFA concentrations ≥1 μg g−1. As expected, because of its strong acidity, TFA decreased soil pH, and this change likely contributed to effects on other soil parameters and functions, for example, reducing bacterial abundance and soluble nutrients. However, importantly, TFA clearly also affected soil parameters at concentrations at which soil pH was not changed. Soil aggregation, fungal abundance, and enzyme activities were not affected by TFA in this study. Considering the current reported environmental levels of TFA of <2.4 μg g−1, only litter decomposition would be affected under current field conditions. Our data also show that future accumulation of higher concentrations, or hotspots of TFA, will likely negatively affect soils with similar properties as our test soil. Thus, we demonstrated that microbial processes can be impacted by the accumulation of the ‘forever chemical’ TFA in the tested sandy, low organic matter soil.Highlights Trifluoroacetic acid (TFA) decreased soil pH, respiration, litter decomposition and bacterial abundance. TFA affected soil soluble nutrients. TFA affected litter decomposition at 1 μg g−1 with no change in soil pH. Effects of TFA on soil enzyme activity and aggregation and fungal abundance were negligible.

Application of laccase and hydrolases for trace organic contaminants removal from contaminated water

• 14 out of 22 TrOCs monitored were effectively removed (> 50 %) after 24 h by the laccase of Trametes hirsuta. • 16 out of 22 TrOCs monitored were effectively removed (> 50 %) after 24 h by the mixture of hydrolases. • Both laccase and the hydrolase mixture achieved high TrOC removal under the operational conditions encountered in real wastewater. The potential of a laccase from Trametes hirsuta , a hydrolase mixture of amylase, lipase cellulase and protease, was studied for the removal of a set of trace organic contaminants (TrOCs) at 10 µg/L each in a contaminated water. An attempt was also made to develop a combination of cross-linked enzyme aggregates (combi-CLEA) from the two groups of enzymes. After a 24-hour in batch treatments at 22°C and pH 7, compounds such as acetaminophen and levothyroxine showed a decrease in concentration of 97% and 99% respectively, in presence of laccase, while amlodipine, levothyroxine, and trimethoprim were removed at 98%, 99% and 99% respectively, when the contaminated water was treated with the hydrolase mixture. The study of the impact of pH, temperature and contact time revealed that the most favorable temperatures were 22°C and 30°C, but no clear correlation was found between pH and removal in the pH range of 6-8, although high removals were achieved at these pHs. Finally, except few compounds such as acetaminophen, the TrOC removal after 24 h was higher than that obtained in 4 h.

HYDROGEN PEROXIDASE ENZYME ACTIVITY AT DIFFERENT CONCENTRATIONS OF BSA

Cross-linked enzymes are more stable at a certain temperature than free enzymes. Bovine serum albumin (BSA), which has a large number of amine groups, provides a strong binding between enzyme aggregates, thus leading to increased activity and stability of the bioactive layer. The enzyme-BSA mixture becomes more stable after covalent bonding with a crosslinking agent such as glutaraldehyde. The HRP enzyme was immobilized on the gold electrode together with BSA, gelatin and glutaraldehyde with the help of UV light. Four different electrodes were prepared using different amounts of BSA at 7.5 mg, 15 mg, 30 mg and 60 mg concentrations for each electrode Using four different electrodes prepared, 16 electrochemical measurements were performed with four different ferrous iron analytes. Glutaraldehyde binds to the BSA polymer, which has a higher affinity than the enzyme, reducing the tight covalent cross-links caused by BSA on the HRP and ensuring the continuation of the enzyme activity. It was observed that different BSA concentrations significantly affected the enzyme activity. The concentration of polymers to be used for crosslinking enzymes is an important factor in electrochemical studies.