Enzyme Aggregates Research Articles

Chemotherapy and radiofrequency hyperthermia (oncothermia) in the treatment of metastatic colorectal cancer.

e15569 Background: Colorectal cancer (CRC) is the second leading cause of death in adults. Standard treatment for metastatic disease includes chemotherapy and biological agents. Although more than 50% of patients respond to multi agent chemotherapy, resistance to treatment is a significant problem and more than 85 % of patients die within five years. Radiofrequency hyperthermia (Oncothermia- OT) is a non-invasive treatment modality which utilizes 13 MHz external radiofrequency waves to selectively heat cancer cells to 45 C, contributing to apoptosis, and aggregation of DNA repair enzymes, potentially overcoming resistance and improving chemotherapy efficacy. It has been used alone or in combination with chemotherapy. Since most metastatic CRC patients have disease limited to the abdominal cavity and liver, Oncothermia can be applied with a single electrode covering the entire abdominal cavity, immediately following chemotherapy without additional side effects. Methods: We treated 25 consecutive CRC patients with disease limited to liver and/or abdominal cavity with chemotherapy and biological agents and concomitant weekly OT (Oncotherm-EHY 3010) for one hour to the whole abdomen. Chemotherapy was FOLFIRI in 2, FOLFOX in 6, FOLFIRINOX in 17. Bevacizumab was used with chemotherapy in 22 and cetuximab in 1 patient. Patients were followed by periodic CT/MRI/PET-CT. Treatment duration ranged from 4 to 60 months. Results: No unexpected or grade III-IV toxicity was observed. Complete response was observed in 3 patients, partial response in 21 patients and stable disease in one patient for an objective response rate of 96%. Median follow up is 20.1 months (range 4-82+ months). 16 patients are alive, 3 with no disease and 13 on treatment. 9 patients have died. Conclusions: Although this is a single center retrospective observation, the combination of chemotherapy with Oncothermia appears tolerable and feasible in metastatic CRC. Despite the limitations of this observation, activity of this multi-modality treatment appears promising. Prospective randomized trials are needed to confirm these findings.

Effect of Nitrogen, Air, and Oxygen on the Kinetic Stability of NAD(P)H Oxidase Exposed to a Gas-Liquid Interface.

Biocatalytic oxidation is an interesting prospect for the selective synthesis of active pharmaceutical intermediates. Bubbling air or oxygen is considered as an efficient method to increase the gas-liquid interface and thereby enhance oxygen transfer. However, the enzyme is deactivated in this process and needs to be further studied and understood to accelerate the implementation of oxidative biocatalysis in larger production processes. This paper reports data on the stability of NAD(P)H oxidase (NOX) when exposed to different gas-liquid interfaces introduced by N2 (0% oxygen), air (21% oxygen), and O2 (100% oxygen) in a bubble column. A pH increase was observed during gas bubbling, with the highest increase occurring under air bubbling from 6.28 to 7.40 after 60 h at a gas flow rate of 0.15 L min-1. The kinetic stability of NOX was studied under N2, air, and O2 bubbling by measuring the residual activity, the deactivation constants (kd1) were 0.2972, 0.0244, and 0.0346 with the corresponding half-lives of 2.2, 28.6, and 20.2 h, respectively. A decrease in protein concentration of the NOX solution was also observed and was attributed to likely enzyme aggregation at the gas-liquid interface. Most aggregation occurred at the air-water interface and decreased greatly from 100 to 14.16% after 60 h of bubbling air. Furthermore, the effect of the gas-liquid interface and the dissolved gas on the NOX deactivation process was also studied by bubbling N2 and O2 alternately. It was found that the N2-water interface and O2-water interface both had minor effects on the protein concentration decrease compared with the air-water interface, whilst the dissolved N2 in water caused serious deactivation of NOX. This was attributed not only to the NOX unfolding and aggregation at the interface but also to the N2 occupying the oxygen channel of the enzyme and the resultant inaccessibility of dissolved O2 to the active site of NOX. These results shed light on the enzyme deactivation process and might further inspire bioreactor operation and enzyme engineering to improve biocatalyst performance.

Stopped Flow of Glycerol Induces the Enhancement of Adsorption and Aggregation of HRP on Mica.

Glycerol is a usable component of heat-transfer fluids, and is thus suitable for the use in microchannel-based heat exchangers in biosensors and microelectronic devices. The flow of a fluid can lead to the generation of electromagnetic fields, which can affect enzymes. Herein, by means of atomic force microscopy (AFM) and spectrophotometry, a long-term effect of stopped flow of glycerol through a coiled heat exchanger on horseradish peroxidase (HRP) has been revealed. Samples of buffered HRP solution were incubated near either the inlet or the outlet sections of the heat exchanger after stopping the flow. It has been found that both the enzyme aggregation state and the number of mica-adsorbed HRP particles increase after such an incubation for 40 min. Moreover, the enzymatic activity of the enzyme incubated near the inlet section has been found to increase in comparison with that of the control sample, while the activity of the enzyme incubated near the outlet section remained unaffected. Our results can find application in the development of biosensors and bioreactors, in which flow-based heat exchangers are employed.

Crowding modulates the oxidant-induced cross-linking and aggregation of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase

Crowding modulates the oxidant-induced cross-linking and aggregation of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase from Bacillus lehensis G1 for maltooligosaccharides synthesis

Maltooligosaccharides (MOS) are functional oligosaccharides that can be synthesized through enzymatic cascade reaction between cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1. To address the problems of low operational stability and non-reusability of free enzymes, both enzymes were co-immobilized as combined cross-linked enzyme aggregates (Combi-CLEAs-CM) with incorporation of bovine serum albumin (BSA) and Tween 80 (Combi-CLEAs-CM-add). Combi-CLEAs-CM and Combi-CLEAs-CM-add showed activity recoveries of 54.12 % and 69.44 %, respectively after optimization. Combi-CLEAs-CM-add showed higher thermal stability at higher temperatures (40 °C) with longer half-life (46.20 min) as compared to those of free enzymes (36.67 min) and Combi-CLEAs-CM (41.51 min). Both combi-CLEAs also exhibited higher pH stability over pH 5 to pH 9, and displayed excellent reusability with >50 % of initial activity retained after four cycles. The reduction in Km value of about 22.80 % and 1.76-fold increase in starch hydrolysis in comparison to Combi-CLEAs-CM attested the improvement of enzyme-substrate interaction by Tween 80 and pores formation by BSA in Combi-CLEAs-CM-add. The improved product specificity of Combi-CLEAs-CM-add also produced the highest yield of MOS (492 mg/g) after 3 h. Therefore, Combi-CLEAs-CM-add with ease of preparation, excellent reusability and high operational stability is believed to be highly efficacious biocatalyst for MOS production.

Synthesis of magnetically recyclable porous cross-linked aggregates of Tramates versicolor MTCC 138 laccase for the efficient removal of pentachlorophenol from aqueous solution

The primary objective of this study is to synthesize the magnetically separable highly active porous immobilized laccase for the removal of pentachlorophenol (PCP) in an aqueous solution. Magnetic porous cross-linked enzyme aggregates (Mp-CLEAs) of laccase were synthesized using 1% starch solution with 5 mM glutaraldehyde followed by 10 h of cross-linking time with an activity recovery of 90.85 ± 0.2%. The biocatalytic efficiency of magnetic porous CLEAs (Mp-CLEAs) was 2-fold higher than that of magnetic CLEAs. The synthesized Mp-CLEAs were mechanically stable with enhanced catalytic efficiency, and reusability thus overcoming the mass transfer limitations and enzyme loss. At 40 °C, the thermal stability of the magnetic porous immobilized laccase was improved, with a 602 min half-life compared to 207 min half-life for the free enzyme. Using 40 U/mL of laccase for the removal of 100 ppm of PCP, M-CLEAs, and Mp-CLEAs removed 60.44% and 65.53% of PCP, respectively. Furthermore, to enhance PCP removal, a laccase-aided system was harnessed by optimizing various surfactants and mediators. Of these, 0.1 mM of rhamnolipid and 2,3 dimethoxy phenol had the highest PCP removal rates of 95.12% and 99.41%, respectively, for Mp-CLEAs. This study demonstrates the efficacy of the laccase-surfactant-mediator system for the removal of PCP from the aqueous solution, which can also be proposed for real-time application.

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.

Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media.

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5-5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.

Food processing applications of protease activity identified in the methotrexate degrading enzyme of Variovorax paradoxus

A 46 kDa dimeric methotrexate (MTX) degrading enzyme was reported from Variovorax paradoxus. This enzyme also showed caseinolytic protease activity. The optimum pH (5.5), and temperature (35 ℃) of protease activity were different from those of MTX degrading activity of the enzyme. Cross linked enzyme aggregates (CLEAs) of the purified soluble protease showed higher activity, higher thermostability, and lower Michaelis–Menten constant (Km) value on casein compared to the soluble enzyme. The soluble enzyme showed casein hydrolysis capacity in the zymogram reactions. The CLEAs could lead to faster milk clotting, lower bread hardness, shorten dough forming time, and increased dough and bread volumes compared to the soluble enzyme, which could be due to the higher affinity of CLEAs to the proteins over soluble enzyme. Majority of the panellists given ‘excellent’ in the five-point scale starting from ‘not satisfactory’ to ‘excellent’ to the CLEAs treated cheese, and bread samples compared to the soluble enzyme treated samples with respect to flavor, texture, and taste. These results suggest that CLEAs are advantageous over soluble enzyme during food processing applications.

Unspecific peroxygenases immobilized on Pd-loaded three-dimensional ordered macroporous (3DOM) titania photocatalyst for photo-enzyme integrated catalysis

The selective oxyfunctionalization of C-H and CC bonds is highly important and catalyzed by unspecific peroxygenases (UPOs) which suffer from inactivation by hydrogen peroxide (H2O2). Herein, photo-enzyme integrated catalysts were constructed by immobilizing AaeUPO as cross-linked enzyme aggregates (CLEAs) in Pd-loaded three-dimensional ordered macroporous titania (3DOM TiO2) with in situ photocatalytic H2O2 production. The prepared AaeUPO@3DOM TiO2-Pd obtained higher catalytic efficiency compared with CLEAs-AaeUPO. The conversion of ethylbenzene to (R)-1-phenethyl alcohol was significantly improved, reaching 99 % compared with free enzyme (38 %) and CLEAs-AaeUPO (49 %), with ee value of > 99 %. AaeUPO@3DOM TiO2-Pd exhibited good stability and reusability attributed to the protection of macropores and in situ H2O2 production. By using the designed 3DOM platform for photocatalysis and enzyme immobilization, the in situ photocatalytic generation of H2O2 was highly efficient, and the monodisperse CLEAs-AaeUPO in the macropores facilitated the contact of substrate and enzyme, thus obtaining excellent photo-enzyme coupled catalysis.

Tyrosinase Immobilization Strategies for the Development of Electrochemical Biosensors-A Review.

The development of enzyme biosensors has successfully overcome various challenges such as enzyme instability, loss of enzyme activity or long response time. In the electroanalytical field, tyrosinase is used to develop biosensors that exploit its ability to catalyze the oxidation of numerous types of phenolic compounds with antioxidant and neurotransmitter roles. This review critically examines the main tyrosinase immobilization techniques for the development of sensitive electrochemical biosensors. Immobilization strategies are mainly classified according to the degree of reversibility/irreversibility of enzyme binding to the support material. Each tyrosinase immobilization method has advantages and limitations, and its selection depends mainly on the type of support electrode, electrode-modifying nanomaterials, cross-linking agent or surfactants used. Tyrosinase immobilization by cross-linking is characterized by very frequent use with outstanding performance of the developed biosensors. Additionally, research in recent years has focused on new immobilization strategies involving cross-linking, such as cross-linked enzyme aggregates (CLEAs) and magnetic cross-linked enzyme aggregates (mCLEAs). Therefore, it can be considered that cross-linking immobilization is the most feasible and economical approach, also providing the possibility of selecting the reagents used and the order of the immobilization steps, which favor the enhancement of biosensor performance characteristics.

Recent Research on Lipase Immobilization with Multipoint Covalent Treatment by Glutaraldehyde

Abstract: Immobilized lipase has played an essential role in the chemical and biological sciences as a viable alternative to standard chemical catalysts. Glutaraldehyde is a low-cost crosslinking agent at risk of being superseded by developing crosslinking compounds with biocompatible, biodegradable, and non-toxic characteristics. The multipoint covalent treatment method using glutaraldehyde has both advantages and disadvantages. Immobilization techniques can be improved to improve the overall performance of immobilized lipase. The most recent update on lipase immobilization with multipoint covalent treatment by glutaraldehyde was summarized in this review. Covalent binding lipase on pre-activated support and aggregation-crosslinking lipase into crosslinked enzyme aggregates (CLEAs) or adsorptioncrosslinking lipase on support are the most common immobilization techniques. Based on the above technologies, the advancement trends in important domains, such as the advancement of supports, additives, reactors, and cross-linking agents, are summarized. In addition, the application of the improved immobilized lipase by glutaraldehyde in the production of fatty acids, glycerides, biodiesel, and drug precursors was reviewed. In view of this, we put forward further studies on multipoint covalent treatment in lipase immobilization with glutaraldehyde. Various analytical methods are required to provide additional information about the structure of glutaraldehyde and its crosslinked products for assisting the proper immobilization conditions. Applying the composite strategy can also bring new opportunities for improving the efficiency of biological catalysts.

(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.

Magnetic CLEAs of β-Galactosidase from Aspergillus oryzae as a Potential Biocatalyst to Produce Tagatose from Lactose

β-galactosidase is an enzyme capable of hydrolysing lactose, used in various branches of industry, mainly the food industry. As the efficient industrial use of enzymes depends on their reuse, it is necessary to find an effective method for immobilisation, maintaining high activity and stability. The present work proposes cross-linked magnetic cross-linked enzyme aggregates (mCLEAs) to prepare heterogeneous biocatalysts of β-galactosidase. Different concentrations of glutaraldehyde (0.6%, 1.0%, 1.5%), used as a cross-linking agent, were studied. The use of dextran-aldehyde as an alternative cross-linking agent was also evaluated. The mCLEAs presented increased recovered activity directly related to the concentration of glutaraldehyde. Modifications to the protocol to prepare mCLEAs with glutaraldehyde, adding a competitive inhibitor or polymer coating, have not been effective in increasing the recovered activity of the heterogeneous biocatalysts or its thermal stability. The biocatalyst prepared using dextran-aldehyde presented 73.6% recovered activity, aside from substrate affinity equivalent to the free enzyme. The thermal stability at 60 °C was higher for the biocatalyst prepared with glutaraldehyde (mCLEA-GLU-1.5) than the one produced with dextran-aldehyde (mCLEA-DEX), and the opposite happened at 50 °C. Results obtained for lactose hydrolysis, the use of its product to produce a rare sugar (D-tagatose) and operational and storage stability indicate that heterogeneous biocatalysts have adequate characteristics for industrial use.

Comparison of Four Immobilization Methods for Different Transaminases

Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HAGA-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from Luminiphilus syltensis. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChemTM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) invitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Design and Optimization of Laccase Immobilization in Cellulose Acetate Microfiltration Membrane for Micropollutant Remediation

The industrial and environmental applications of laccase, especially in wastewater treatment, have gained focus in recent years. Therefore, developing the proper laccase immobilization techniques, which could improve the stability of the enzymes and simplify the required downstream processes, is needed. A novel two-step immobilization process was developed, resulting in cross-linked enzyme aggregates (CLEA) in the pores of the membrane. Laccase adsorption on a biodegradable cellulose acetate microfiltration membrane along with cross-linking was investigated to maximize the enzyme load and immobilization efficiency. The optimization was done regarding the: pH, temperature, enzyme concentration, adsorption time, cross-linker concentration, and temperature. It was concluded that the highest immobilization efficiency (76%) could be achieved in acidic buffers at 29 °C with high surface activity (1174 U·m−2) at the cost of partial denaturation and membrane fouling. The membrane was successfully utilized for the enzymatic treatment of diclofenac, and 58% removal efficiency was achieved. The results indicated that cellulose acetate is a suitable carrier for adsorption-based immobilization of laccase for the potential for environmental utilisation.

Proteases: An overview on its recent industrial developments and current scenario in the revolution of biocatalysis

In recent years there is tremendous growth in the utilization of alkaline protease as an industrial catalyst. The extensive usage of chemicals for various applications in industries is usually associated with severe health effects and environmental problems. In this context, the scientific world mainly aims to substitute these toxic compounds with healthy environmental benign products for a good life. So, the chemicals are replaced by various enzymes among which microbial enzymes especially proteases are mostly employed in the modern corporate sectors in the manufacture of detergents, leather, food products and also in waste management techniques. The present review summarizes the various industrial applications of alkaline protease, different factors influencing its production like isolation source, optimum pH, temperature, the effect of inhibitor and metal ions on alkaline protease producing microorganisms. Furthermore the biocatalytic activity of alkaline protease in various organic transformations namely Knoevenagel condensation, Domino reaction, Aldol reaction, transesterification reaction, resolution of amino acids in organic solvents, kinetic resolution of carboxylic acids, strained amides, peptide synthesis by alcalase cross-linked enzyme aggregates (CLEA), enantioselective hydrolysis of prochiral dinitril, enzymatic desymmetrization of Ganciclovir, enantioselective aminoacid ester hydrolysis and fragmentation of p-amido benzyl ethers have been compiled in the current review.

Optimized Conditions for Preparing a Heterogeneous Biocatalyst via Cross-Linked Enzyme Aggregates (CLEAs) of β-Glucosidase from Aspergillus niger

This study mainly aims to find the optimal conditions for immobilizing a non-commercial β-glucosidase from Aspergillus niger via cross-linked enzyme aggregates (CLEAs) by investigating the effect of cross-linking agent (glutaraldehyde) concentration and soy protein isolate/enzyme ratio (or spacer/enzyme ratio) on the catalytic performance of β-glucosidase through the central composite rotatable design (CCRD). The influence of certain parameters such as pH and temperature on the hydrolytic activity of the resulting heterogeneous biocatalyst was assessed and compared with those of a soluble enzyme. The catalytic performance of both the soluble and immobilized enzyme was assessed by hydrolyzing ρ-nitrophenyl-β-D-glucopyranoside (ρ-NPG) at pH 4.5 and 50 °C. It was found that there was a maximum recovered activity of around 33% (corresponding to hydrolytic activity of 0.48 U/mL) in a spacer/enzyme ratio of 4.69 (mg/mg) using 25.5 mM glutaraldehyde. The optimal temperature and pH conditions for the soluble enzyme were 60 °C and 4.5, respectively, while those for CLEAs of β-glucosidase were between 50 and 65 °C and pH 3.5 and 4.0. These results reveal that the immobilized enzyme is more stable in a wider pH and temperature range than its soluble form. Furthermore, an improvement was observed in thermal stability after immobilization. After 150 days at 4 °C, the heterogeneous biocatalyst retained 80% of its original activity, while the soluble enzyme retained only 10%. The heterogeneous biocatalyst preparation was also characterized by TG/DTG and FT-IR analyses that confirmed the introduction of carbon chains via cross-linking. Therefore, the immobilized biocatalyst prepared in this study has improved enzyme stabilization, and it is an interesting approach to preparing heterogeneous biocatalysts for industrial applications.

Cross-linked lipase particles with improved activity; application of a non-toxic linker for cross-linking

The preparation of cross-linked enzymes is often performed through enzyme aggregation or crystallization prior to cross-linking by glutaraldehyde as the most common linker. Isocyanide-based multi-component reactions (ICMRs) provide a one-step process to prepare cross-linked preparations of enzymes directly from their soluble forms. ICMRs accept a variety of functional groups to be involved in the coupling reaction. We here introduce cysteamine as a novel non-toxic alternative for glutaraldehyde to be used as a cross-linker in cross-linking reactions implemented by ICMRs. The cross-linked enzymes (CLEs) of Rhizomucor miehei lipase (RML) and Thermomyces lanuginosa lipase (TLL) were prepared with high immobilization yields (up to 97%). The results showed the maximum activities of 43.7 U/mg and 213.35 U/mg for the immobilized derivatives of RML and TLL, respectively, presenting an 11-fold and 10-fold improvement compared to the activity of the corresponding soluble lipases. The immobilized derivatives showed improved thermal and co-solvent stability. Additionally, the CLEs of TLL showed a selectivity of 19.9 in the selective hydrolysis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil. The used linker provides an alternative strategy for CLEs production, particularly where the traditional cross-linking method has the possible risk of reaction contamination by weakly bonded glutaraldehyde molecules.