Therapeutic Ligands Antagonize Estrogen Receptor Function by Impairing Its Mobility.

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.

A novel system to determine activity of individual uridine 5'-diphospho-glucuronosyltransferase (UGT) isoforms: Recombinant UGT-beads.

Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.

Site-specific immobilization of the endosialidase reveals QSOX2 is a novel polysialylated protein.

Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.

Mitochondrial-derived microprotein MOTS-c attenuates immobilization-induced skeletal muscle atrophy by suppressing lipid infiltration.

Mitochondrial open reading frame of the 12S ribosomal RNA type-c (MOTS-c), a mitochondrial microprotein, has been described as a novel regulator of glucose and lipid metabolism. In addition to its role as a metabolic regulator, MOTS-c prevents skeletal muscle atrophy in high fat-fed mice. Here, we examined the preventive effect of MOTS-c on skeletal muscle mass, using an immobilization-induced muscle atrophy model, and explored its underlying mechanisms. Male C57BL/6J mice (10 wk old) were randomly assigned to one of the three experimental groups: nonimmobilization control group (sterilized water injection), immobilization control group (sterilized water injection), and immobilization and MOTS-c-treated group (15 mg/kg/day MOTS-c injection). We used casting tape for the immobilization experiment. After 8 days of the experimental period, skeletal muscle samples were collected and used for Western blotting, RNA sequencing, and lipid and collagen assays. Immobilization reduced ∼15% of muscle mass, whereas MOTS-c treatment attenuated muscle loss, with only a 5% reduction. MOTS-c treatment also normalized phospho-AKT, phospho-FOXO1, and phospho-FOXO3a expression levels and reduced circulating inflammatory cytokines, such as interleukin-1b (IL-1ß), interleukin-6 (IL-6), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1), in immobilized mice. Unbiased RNA sequencing and its downstream analyses demonstrated that MOTS-c modified adipogenesis-modulating gene expression within the peroxisome proliferator-activated receptor (PPAR) pathway. Supporting this observation, muscle fatty acid levels were lower in the MOTS-c-treated group than in the casted control mice. These results suggest that MOTS-c treatment inhibits skeletal muscle lipid infiltration by regulating adipogenesis-related genes and prevents immobilization-induced muscle atrophy.NEW & NOTEWORTHY MOTS-c, a mitochondrial microprotein, attenuates immobilization-induced skeletal muscle atrophy. MOTS-c treatment improves systemic inflammation and skeletal muscle AKT/FOXOs signaling pathways. Furthermore, unbiased RNA sequencing and subsequent assays revealed that MOTS-c prevents lipid infiltration in skeletal muscle. Since lipid accumulation is one of the common pathologies among other skeletal muscle atrophies induced by aging, obesity, cancer cachexia, and denervation, MOTS-c treatment could be effective in other muscle atrophy models as well.

Programmed immobilization of living cells using independent click pairs.

Therapeutic devices incorporating living cells or tissues have been intensively investigated for applications in tissue engineering and regenerative medicine. Because many biological processes are governed by spatially dependent signals, programmable immobilization of materials is crucial for manipulating multiple types of cells. In this study, click chemistry substrates were introduced onto the surfaces of cells and cover glass, and the cells were fixed on the cover glass via covalent bonds for selective cell deposition. Azide group (Az)-labeled living cells were prepared by metabolic labeling with azido sugars. Following the introduction of Az, TCO (trans-cyclooctene) was metabolically labeled into the living cells by reacting with TCO-DBCO (dibenzocyclooctyne). Az and TCO in the cells were detected using DBCO-FAM (fluorescein)and tetrazine-Cy3, respectively. The mixture of Az-labeled green fluorescent protein HeLa cells and TCO-labeled red fluorescent protein HeLa cells was reacted in a culture dish in which three different cover glasses, DBCO-, tetrazine-, or methyl-coated, were added. Az- or TCO-labeled cells could be immobilized in a functional group-dependent manner. Next, tetrazine-labeled cells were incubated on TCO- or Az-labeled cell layers instead of cover glass. Functional group-dependent immobilization was also achieved in the cell layer. Introducing substrates for the click reaction could achieve cell-selective immobilization on different patterned glass surfaces, as well as cell-cell immobilization.

A Multienzyme Cascade Pathway Immobilized in a Hydrogen-Bonded Organic Framework for the Conversion of CO2.

The reduction of carbon dioxide to valuable chemicals through enzymatic processes is regarded as a promising approach for the reduction of carbon dioxide emissions. In this study, an in vitro multi-enzyme cascade pathway is constructed for the conversion of CO2 into dihydroxyacetone (DHA). This pathway, known as FFFP, comprises formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH), formolase (FLS), and phosphite dehydrogenase (PTDH), with PTDH serving as the critical catalyst for regenerating the coenzyme NADH. Subsequently, the immobilization of the FFFP pathway within the hydrogen-bonded organic framework (HOF-101) is accomplished in situ. A 1.8-fold increase in DHA yield is observed in FFFP@HOF-101 compared to the free FFFP pathway. This enhancement can be explained by the fact that within FFFP@HOF-101, enzymes are positioned sufficiently close to one another, leading to the elevation of the local concentration of intermediates and an improvement in mass transfer efficiency. Moreover, FFFP@HOF-101 displays a high degree of stability. In addition to the establishment of an effective DHA production method, innovative concepts for the tailored synthesis of fine compounds from CO2 through the utilization of various multi-enzyme cascade developments are generated by this work.

Tungsten-Iron-Ruthenium Ternary Alloy Immobilized into the Inner Nickel Foam for High-Current-Density Water Oxidation.

The pursuit of highly-active and stable catalysts in anodic oxygen evolution reaction (OER) is desirable for high-current-density water electrolysis toward industrial hydrogen production. Herein, a straightforward yet feasible method to prepare WFeRu ternary alloying catalyst on nickel foam is demonstrated, whereby the foreign W, Fe, and Ru metal atoms diffuse into the Ni foam resulting in the formation of inner immobilized ternary alloy. Thanks to the synergistic impact of foreign metal atoms and structural robustness of inner immobilized alloying catalyst, the well-designed WFeRu@NF self-standing anode exhibits superior OER activities. It only requires overpotentials of 245 and 346 mV to attain current densities of 20 and 500 mA cm-2, respectively. Moreover, the as-prepared ternary alloying catalyst also exhibits a long-term stability at a high-current-density of 500 mA cm-2 for over 45 h, evidencing the inner-immobilization strategy is promising for the development of highly active and stable metal-based catalysts for high-density-current water oxidation process.

Comparative Study of ImmobilizedBiolipasa-R for Second Generation Biodiesel Production from an Acid Oil.

The primary objective of this work is to develop a sustainable biocatalytic transesterification process for low-grade oils, aligning with EU green technology requirements for the shift to second generation biodiesel. Thus, we investigated the immobilization and subsequent application of the lipase Biolipasa-R on transesterification processes to produce fatty acid methyl esters (FAMEs) from both a sunflower oil and an acid oil which is a bioproduct of the biodiesel industry. The lipase was immobilized on biomaterials, such as diatomaceous earth, with a yield of 60%, and commercial carriers such as methacrylic resins with a yield of 100%. The enzyme demonstrated superior activity when immobilized on diatomaceous earth, particularly in reactions involving the acid oil, outperforming the benchmark enzyme Novozym® 435 (95.1% and 35% conversion respectively). This work highlights the potential of Biolipasa-R as a cost-effective and efficient biocatalyst for biodiesel production and emphasizes the environmental benefits of utilizing industrial byproducts and eco-friendly immobilization techniques. The findings suggest that Biolipasa-R is a promising candidate for industrial applications in biodiesel production, offering a sustainable solution for waste management and energy generation.

Integrating Biocatalysts into Metal-Organic Frameworks: Disentangling the Roles of Affinity, Molecular Weight, and Size.

The integration of biocatalysts within metal-organic frameworks (MOFs) is attracting growing interest due to its potential to both enhance biocatalyst stability and sustain biocatalyst activity in organic solvents. However, the factors that facilitate the post-synthetic infiltration of such large molecules into MOF pores remain unclear. This systematic study enabled the identification of the influence of biocatalyst molecular size, molecular weight and affinity on the uptake by an archetypal MOF, NU-1000. We analyzed a range of six biocatalysts with molecular weights from 1.9 kDa to 44.4 kDa, respectively. By employing a combination of fluorescence tagging and 3D-STED confocal laser scanning microscopy, we distinguished between biocatalysts that were internalized within the MOF pores and those sterically excluded. The catalytic functions of the biocatalysts hosted within the MOF were investigated and found to show strong variations relative to the solvated case, ranging from a two-fold increase to a strong decrease.

Developing Covalent Organic Framework Biocatalysts through Enzyme Encapsulation.

Utilizing covalent organic frameworks (COFs) as porous supports to encapsulate enzyme represents an advanced strategy for constructing COFs biocatalysts, which has inspired numerous interests across various applications. As the structural advantages including ultrastable covalent-bonded linkage, tailorable pore structure, and metal-free biocompatibility, the resultant enzyme-COFs biocatalysts showcase functional enhancement in catalytic activity, chemical stability, long-term durability, and recyclability. This Concept describes the recent advances in the methodological strategies for engineering the COFs biocatalysts, with specific emphasis on the pore entrapment and in situ encapsulation strategies. The structural advantages of the COFs hybrid biocatalysts for organic synthesis, environment- and energy-associated applications are also canvassed. Additionally, the remaining challenges and the forward-looking directions in this field are also discussed. We believe that this Concept can offer useful methodological guidance for developing active and robust COFs biocatalysts.

Co-Immobilization of ADH and GDH on Metal-Organic-Framework: An Effective Biocatalyst for Asymmetric Reduction of Ketones.

Chiral alcohols are not only important building blocks of various bioactive natural compounds and pharmaceuticals, but can serve as synthetic precursors for other valuable organic chemicals, thus the synthesis of these products is of great importance. Bio-catalysis represents one effective way to obtain these molecules, however, the weak stability and high cost of enzymes often hinder its broad application. In this work, we designed a biological nanoreactor by embedding alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) in metal-organic-framework ZIF-8. The biocatalyst ADH&GDH@ZIF-8 could be applied to the asymmetric reduction of a series of ketones to give chiral alcohols in high yields (up to 99 %) and with excellent enantioselectivities (>99 %). In addition, the heterogeneous biocatalyst could be recycled and reused at least four times with slight activity decline. Moreover, E. coli containing ADH and GDH was immobilized by ZIF-8 to form biocatalyst E. coli@ZIF-8, which also exhibits good catalytic behaviours. Finally, the chiral alcohols are further converted to marketed drugs (R)-Fendiline, (S)-Rivastigmine and NPS R-568 respectively.

Biocatalysis with In-Situ Product Removal Improves p-Coumaric Acid Production.

Natural and pure p-coumaric acid has valuable applications, and it can be produced via bioprocessing. However, fermentation processes have so far been unable to provide sufficient production metrics, while a biocatalytic process decoupling growth and production historically showed much promise. This biocatalytic process is revisited in order to tackle product inhibition of the key enzyme tyrosine ammonia lyase. In situ product removal is proposed as a possible solution, and a polymer/salt aqueous two-phase system is identified as a suitable system for extraction of p-coumaric acid from an alkaline solution, with a partition coefficient of up to 13. However, a 10 % salt solution was found to reduce tyrosine ammonia lyase activity by 19 %, leading to the need for a more dilute system. The cloud points of two aqueous two-phase systems at 40 °C and pH 10 were found to be 3.8 % salt and 9.5 % polymer, and a 5 % potassium phosphate and 12.5 % poly(ethylene glycol-ran-propylene glycol) mW~2500 system was selected for in situ product removal. An immobilized tyrosine ammonia lyase biocatalyst in this aqueous two-phase system produced up to 33 g/L p-coumaric acid within 24 hours, a 1.9-fold improvement compared to biocatalysis without in situ product removal.

Enzymatic Synthesis of Indole-Based Imidazopyridine using α-Amylase.

The imidazo[1,2-a]pyridine scaffold has gained significant attention due to its presence as a lead structure in several commercially available pharmaceuticals like zolimidine, zolpidem, olprinone, soraprazan, etc. Further, indole-based imidazo[1,2-a]pyridine derivatives have been found interesting due to their anticancer and antibacterial activities. However, limited methods have been reported for the synthesis of indole-based imidazo[1,2-a]pyridines. In this study, we have successfully developed a biocatalytic process for synthesizing indole-based imidazo[1,2-a]pyridine derivatives using the α-amylase enzyme catalyzed Groebke-Blackburn-Bienayme (GBB) multicomponent reaction of 2-aminopyridine, indole-3-carboxaldehyde, and isocyanide. The generality and robustness of this protocol were shown by synthesizing differently substituted indole-based imidazo[1,2-a]pyridines in good isolated yields. Furthermore, to make α-amylase a reusable catalyst for GBB multicomponent reaction, it was immobilized onto magnetic metal-organic framework (MOF) materials [Fe3 O4 @MIL-100(Fe)] and found reusable up to four consecutive catalytic cycles without the significant loss in catalytic activity.

Immobilization by 21 days of bed rest results in type II collagen degradation in healthy individuals.

OBJECTIVE: To investigate the effects of 21 days of bed rest immobilization (with and without exercise and nutrition interventions) on type II collagen biomarker concentrations in healthy individuals. DESIGN: Twelve healthy male participants (age 34.2 ± 8.3 years; body mass index 22.4 ± 1.7 kg/m²) were exposed to 6 days ambulatory baseline data collection (BDC), 21 days head-down-tilt bed rest (HDT, CON) + interventions (HDT + resistive vibration exercise (2 times/week, 25 minutes): RVE; HDT + RVE + whey protein (0.6 g/kg body weight/day) and bicarbonate supplementation (90 mmol KHCO3/day: NeX), and 6 days of re-ambulation (R) in a cross-over designed study. The starting HDT condition was randomized (CON-RVE-NEX, RVE-NEX-CON, NEX-CON-RVE). Blood and urine samples were collected before, during, and after HDT. Serum concentrations (s) of CPII, C2C, C1,2C, and urinary concentrations (u) of CTX-II and Coll2-1NO2 were measured. RESULTS: Twenty-one days of HDT resulted in increased sCPII (p < 0.001), sC2C (p < 0.001), and sC1,2C (p = 0.001) (highest increases: sCPII (+24.2% - HDT5), sC2C (+24.4% - HDT7), sC1,2C (+13.5% - HDT2). sC2C remained elevated at R+1 (p = 0.002) and R+6 (p < 0.001) compared to baseline. NeX led to lower sCPII (p < 0.001) and sC1,2C (p = 0.003) compared to CON. uCTX-II (second void and 24-hour urine) increased during HDT (p < 0.001, highest increase on HDT21: second void +82.8% (p < 0.001); 24-hour urine + 77.8% (p < 0.001). NeX resulted in lower uCTX-II concentrations in 24-hour urine (p = 0.012) compared to CON. CONCLUSIONS: Twenty-one days of bed rest immobilization results in type II collagen degradation that does not recover within 6 days of resuming ambulation. The combination of resistive vibration exercise and protein/bicarbonate supplementation minimally counteracted this effect.

Mycorrhizal associations relate to stable convergence in plant-microbial competition for nitrogen absorption under high nitrogen conditions.

Nitrogen (N) immobilization (Nim, including microbial N assimilation) and plant N uptake (PNU) are the two most important pathways of N retention in soils. The ratio of Nim to PNU (hereafter Nim:PNU ratio) generally reflects the degree of N limitation for plant growth in terrestrial ecosystems. However, the key factors driving the pattern of Nim:PNU ratio across global ecosystems remain unclear. Here, using a global data set of 1018 observations from 184 studies, we examined the relative importance of mycorrhizal associations, climate, plant, and soil properties on the Nim:PNU ratio across terrestrial ecosystems. Our results show that mycorrhizal fungi type (arbuscular mycorrhizal (AM) or ectomycorrhizal (EM) fungi) in combination with soil inorganic N mainly explain the global variation in the Nim:PNU ratio in terrestrial ecosystems. In AM fungi-associated ecosystems, the relationship between Nim and PNU displays a weaker negative correlation (r = -.06, p < .001), whereas there is a stronger positive correlation (r = .25, p < .001) in EM fungi-associated ecosystems. Our meta-analysis thus suggests that the AM-associated plants display a weak interaction with soil microorganisms for N absorption, while EM-associated plants cooperate with soil microorganisms. Furthermore, we find that the Nim:PNU ratio for both AM- and EM-associated ecosystems gradually converge around a stable value (13.8 ± 0.5 for AM- and 12.1 ± 1.2 for EM-associated ecosystems) under high soil inorganic N conditions. Our findings highlight the dependence of plant-microbial interaction for N absorption on both plant mycorrhizal association and soil inorganic N, with the stable convergence of the Nim:PNU ratio under high soil N conditions.

Engineering industrial yeast for improved tolerance and robustness.

As an important cell factory, industrial yeast has been widely used for the production of compounds ranging from bulk chemicals to complex natural products. However, various adverse conditions including toxic products, extreme pH, and hyperosmosis etc., severely restrict microbial growth and metabolic performance, limiting the fermentation efficiency and diminishing its competitiveness. Therefore, enhancing the tolerance and robustness of yeasts is critical to ensure reliable and sustainable production of metabolites in complex industrial production processes. In this review, we provide a comprehensive review of various strategies for improving the tolerance of yeast cells, including random mutagenesis, system metabolic engineering, and material-mediated immobilization cell technology. It is expected that this review will provide a new perspective to realize the response and intelligent regulation of yeast cells to environmental stresses.

Enhancing Substrate Channeling with Multi-Enzyme Architectures in Hydrogen-Bonded Organic Frameworks.

Hydrogen-bonded organic frameworks (HOF) represent an emerging category of organic structures with high crystallinity and metal-free, which are not commonly observed in alternative porous organic frameworks. These needle-like porous structure can help in stabilizing enzymes and allow transfer of molecules between enzymes participating in cascade reactions for enhanced substrate channelling. Herein, we systematically synthesized and investigated the stability of HOF at extreme conditions followed by one-pot encapsulation of single and bi-enzyme systems. Firstly, we observed HOF to be stable at pH 1 to 14 and at high temperatures (up to 115 °C). Secondly, the encapsulated glucose oxidase enzyme (GOX) showed 80 % and 90 % of its original activity at 70 °C and pH 11, respectively. Thirdly, transient time close to 0 seconds was observed for HOF encapsulated bi-enzyme cascade reaction system demonstrating a 4.25-fold improvement in catalytic activity when compared to free enzymes with enhanced substrate channelling. Our findings showcase a facile system synthesized under ambient conditions to encapsulate and stabilize enzymes at extreme conditions.

Covalent Lysozyme Immobilization on Enzymatic Cellulose Nanocrystals.

Nanostructured materials represent promising substrates for biocatalysts immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme-based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on the enzymatic CNCs and on their TEMPO oxidized derivatives (TO-CNCs), comparing immobilization yields, materials properties, and enzymatic activities. The materials were characterized by X-ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR-FTIR), bicinchoninic acid (BCA) assay, field-emission scanning electron microscopy (FE-SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO-CNCs, based on the success of covalent bonding and on the stability of the isolated biocojugates.

Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-Enzymatic Synthesis of Rivastigmine.

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.

Termostable and effective immobilized invertase for sucrose determination in fruit juices.

In this study, immobilization of invertase enzyme was performed on a previously synthesized and characterized poly(N-vinylpyrrolidone-co-butylacrylate-co-N hydroxymethylacrylamide) terpolymer membranes by covalent bonding method. Glutaraldehyde(GA) was used as the crosslinker and Bovine Serum Albumin(BSA) was used as the binding agent. Optimum pH, temperature, amount of polymer, substrate concentration, amount of BSA and amount of GA values were determined for both free and immobilized enzyme. Optimum pH and temperature values were found as pH = 5.0, T = 55 °C, pH = 7.0 and T = 80 °C for free and immobilized enzyme, respectively. In particular, the optimum temperature of 80 °C for the immobilized enzyme provides its potential to be used commercially. The kinetic parameters of the free enzyme and the immobilized enzyme were determined using the well known Lineweaver-Burk method. The Vmax values for free (13.4 µM/min) and immobilized enzyme (12.2 µM/min) were found as close to each other, while the Km value of the immobilized enzyme (8.33 mM) was much lower than that of the free enzyme (29.41 mM). In reuse studies conducted with peach and orange juices, it was determined that the immobilized enzyme retained approximately 90% of its activity even after 30 reuses within 1 month.