Search Results (13)

An easy and versatile method was designed and applied successfully to obtain access to lipase-based cross-linked-enzyme aggregate-like copolymers (CLEA-LCs) using one-pot, consecutive cross-linking steps using two types of homobifunctional cross-linkers (glutaraldehyde and putrescine), mediated with amine activation through pH alteration (pH jump) as a key step in the process. Six lipases were utilised in order to assess the effectiveness of the technique, in terms of immobilization yields, hydrolytic activities, thermal stability and application in kinetic resolution. A good retention of catalytic properties was found for all cases, together with an important thermal and storage stability improvement. Particularly, the CLEA-LCs derived from Candida rugosa lipase showed an outstanding behaviour in terms of thermostability and capability for catalysing the enantioselective hydrolysis of racemic ibuprofen ethyl ester, furnishing the eutomer (S)-ibuprofen with very high conversion and enantioselectivity. Full article

Enzymes are used extensively as industrial bio-catalysts in various manufacturing and processing sectors. However, commercial enzymes are expensive in part due to the high cost of the nutrient medium for the biomass culture. Activated sludge (AS) is a waste product of biological wastewater treatment and consists of microbial biomass that degrades organic matter by producing substantial quantities of hydrolytic enzymes. Recovering enzymes from AS therefore offers a potential alternative to conventional production techniques. A carrier-free, cross-linked enzyme aggregate (CLEA) was produced from crude AS enzyme extract for the first time. A major advantage of the CLEA is the combined immobilization, purification, and stabilization of the crude enzymes into a single step, thereby avoiding large amounts of inert carriers in the final enzyme product. The AS CLEA contained a variety of hydrolytic enzymes and demonstrated high potential for the bio-conversion of complex organic substrates. Full article

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. Full article

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 HA_GA-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 SpinChem^TM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability. Full article

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. Full article

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 $\rho$-nitrophenyl-β-D-glucopyranoside ($\rho$-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. Full article

Eversa^® Transform 2.0 has been launched to be used in free form, but its immobilization may improve its performance. This work aimed to optimize the immobilization of Eversa^® Transform 2.0 by the crosslinked enzyme aggregates (CLEAs) technique, using almost all the available tools to improve its performance. Several variables in the CLEA preparation were optimized to improve the recovered activity, such as precipitant nature and crosslinker concentration. Moreover, some feeders were co-precipitated to improve the crosslinking step, such as bovine serum albumin, soy protein, or polyethyleneimine. Starch (later enzymatically degraded) was utilized as a porogenic agent to decrease the substrate diffusion limitations. Silica magnetic nanoparticles were also utilized to simplify the CLEA handling, but it was found that a large percentage of the Eversa activity could be immobilized on these nanoparticles before aggregation. The best CLEA protocol gave a 98.9% immobilization yield and 30.1% recovered activity, exhibited a porous structure, and an excellent performance in the transesterification of soybean oil with ethanol: 89.8 wt% of fatty acid ethyl esters (FAEEs) yield after 12 h of reaction, while the free enzyme required a 48 h reaction to give the same yield. A caustic polishing step of the product yielded a biodiesel containing 98.9 wt% of FAEEs and a free fatty acids content lower than 0.25%, thus the final product met the international standards for biodiesel. The immobilized biocatalyst could be reused for at least five 12 h-batches maintaining 89.6% of the first-batch yield, showing the efficient catalyst recovery by applying an external magnetic field. Full article

Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing an organic solvent tolerant protease using a CLEA is disclosed and optimized for better biochemical properties, followed by an analysis of the structure of this CLEA-treated protease. The said organic solvent tolerant protease is a metalloprotease known as elastase strain K in which activity of the metalloprotease is measured by a biochemical interaction with azocasein. Results showed that when a glutaraldehyde of 0.02% (v/v) was used under a 2 h treatment, the amount of recovered activity in CLEA-elastase was highest. The recovered activity of CLEA-elastase and CLEA-elastase-SB (which was a CLEA co-aggregated with starch and bovine serum albumin (BSA)) were at an approximate 60% and 80%, respectively. The CLEA immobilization of elastase strain K allowed the stability of the enzyme to be enhanced at high temperature and at a broader pH. Both CLEA-elastase and CLEA-elastase-SB end-products were able to maintain up to 67% enzyme activity at 60 °C and exhibiting an enhanced stability within pH 5–9 with up to 90% recovering activity. By implementing a CLEA on the organic solvent tolerant protease, the characteristics of the organic solvent tolerant were preserved and enhanced with the presence of 25% (v/v) acetonitrile, ethanol, and benzene at 165%, 173%, and 153% relative activity. Structural analysis through SEM and dynamic light scattering (DLS) showed that CLEA-elastase had a random aggregate morphology with an average diameter of 1497 nm. Full article

Due to the heterogeneity of oils, the use of mixtures of lipases with different activity for a large number of glycerol-linked carboxylic acids that compose the substrate has been proposed as a better alternative than the use of one specific lipase preparation in the enzymatic synthesis of biodiesel. In this work, mixtures of lipases from different sources were evaluated in their soluble form in the ethanolysis of soybean oil. A mixture of lipases (50% of each lipase, in activity basis) from porcine pancreas (PPL) and Thermomyces lanuginosus lipase (TLL) gave the highest fatty acid ethyl ester (FAEE) yield (around 20 wt.%), while the individual lipases gave FAEE yields 100 and 5 times lower, respectively. These lipases were immobilized individually by the cross-linked enzyme aggregates (CLEAs) technique, yielding biocatalysts with 89 and 119% of expressed activity, respectively. A mixture of these CLEAs (also 50% of each lipase, in activity basis) gave 90.4 wt.% FAEE yield, while using separately CLEAs of PPL and TLL, the FAEE yields were 84.7 and 75.6 wt.%, respectively, under the same reaction conditions. The mixture of CLEAs could be reused (five cycles of 6 h) in the ethanolysis of soybean oil in a vortex flow-type reactor yielding an FAEE yield higher than 80% of that of the first batch. Full article

The preparation of Cross-Linked Enzyme Aggregates (CLEAs) is a simple and cost-effective technique capable of generating insoluble biocatalysts with high volumetric activity and improved stability. The standard CLEA preparation consists of the aggregation of the enzyme and its further crosslinking, usually with glutaraldehyde. However, some enzymes have too low a content of surface lysine groups to permit effective crosslinking with glutaraldehyde, requiring co-aggregation with feeders rich in amino groups to aid the formation of CLEAs. The co-aggregation with magnetic particles makes their handling easier. In this work, CLEAs of a commercial amyloglucosidase (AMG) produced by Aspergillus niger were prepared by co-aggregation in the presence of polyethyleneimine (PEI) or starch with aminated magnetic nanoparticles (MNPs) or bovine serum albumin (BSA). First, CLEAs were prepared only with MNPs at different glutaraldehyde concentrations, yielding a recovered activity of around 20%. The addition of starch during the precipitation and crosslinking steps nearly doubled the recovered activity. Similar recovered activity (around 40%) was achieved when changing starch by PEI. Moreover, under the same conditions, AMG co-aggregated with BSA was also synthesized, yielding CLEAs with very similar recovered activity. Both CLEAs (co-aggregated with MNPs or BSA) were four times more stable than the soluble enzyme. These CLEAs were evaluated in the hydrolysis of starch at typical industrial conditions, achieving more than 95% starch-to-glucose conversion, measured as Dextrose Equivalent (DE). Moreover, both CLEAS could be reused for five cycles, maintaining a DE of around 90%. Although both CLEAs had good properties, magnetic CLEAs could be more attractive for industrial purposes because of their easy separation by an external magnetic field, avoiding the formation of clusters during the filtration or centrifugation recovery methods usually used. Full article

Enzymes are biocatalysts. They are useful in environmentally friendly production processes and have high potential for industrial applications. However, because of problems with operational stability, cost, and catalytic efficiency, many enzymatic processes have limited applications. The use of cross-linked enzyme aggregates (CLEAs) has been introduced as an effective carrier-free immobilization method. This immobilization method is attractive because it is simple and robust, and unpurified enzymes can be used. Coimmobilization of different enzymes can be achieved. CLEAs generally show high catalytic activities, good storage and operational stabilities, and good reusability. In this review, we summarize techniques for the preparation of CLEAs for use as biocatalysts. Some important applications of these techniques in chemical synthesis and environmental applications are also included. CLEAs provide feasible and efficient techniques for improving the properties of immobilized enzymes for use in industrial applications. Full article

Barley β-amylase was immobilized using different techniques. The highest global yield was obtained using the cross-linked enzyme aggregates (CLEA) technique, employing bovine serum albumin (BSA) or soy protein isolate (SPI) as feeder proteins to reduce diffusion problems. The CLEAs produced using BSA or SPI showed 82.7 ± 5.8 and 53.3 ± 2.4% global yield, respectively, and a stabilization effect was observed upon immobilization at neutral pH value, e.g., after 12 h at 55 °C, the free β-amylase is fully inactivated, while CLEAs retained 25 and 15% of activity (using BSA and SPI, respectively). CLEA using SPI was selected because of its easier recovery, being chosen to convert the residual starch contained in cassava bagasse into maltose. This biocatalyst permitted to reach almost 70% of maltose conversion in 4 h using 30.0 g/L bagasse starch solution (Dextrose Equivalent of 15.88) and 1.2 U of biocatalyst per gram of starch at pH 7.0 and 40 °C. After 4 reuses (batches of 12 h) the CLEA using SPI maintained 25.50 ± 0.01% of conversion due to the difficulty of recovering. Full article

Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed. Full article