Search

Laccases are versatile oxidases that are capable of decolorizing various synthetic dyes. Recombinant Bacillus amyloliquefaciens laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) for application in dye decolorization. Several parameters influencing the activity recovery were evaluated during the synthesis of M-CLEAs. With ammonium sulfate as precipitant, maximum activity was recovered by cross-linking with 0.16% glutaraldehyde for 1 h. The prepared M-CLEAs exhibited improved activity under alkaline conditions. It remained 74% activity after incubation at 60 °C for 5 h. Enhanced tolerance towards NaCl was also observed for the M-CLEAs, with 68% activity remaining in the presence of 1 M NaCl. The immobilized laccase could rapidly decolorize more than 93% of reactive black 5 and indigo carmine in 1 h, while its catalytic efficiency towards reactive blue 19 was relatively low. After four cycles of consecutive reuse, the M-CLEAs could decolorize 92% of indigo carmine. The easy recovery and reusability of M-CLEAs facilitate the potential application of bacterial laccase in dye decolorization. [ABSTRACT FROM AUTHOR]

ß-galactosidases from Lactobacillus leichmannii 313 (LL 313) were immobilized for the first time by cross-linked enzyme aggregates (CLEA) technology. By using three precipitants (saturated ammonium sulfate (ASF), acetone (ACT), and isopropanol (IPA) in CLEA preparation and assessing performance in comparison with crude enzyme, recovered activities of 23% (ASFCLEA), 18% (ACT-CLEA), 13.6% (IPA-CLEA) were obtained. Immobilization did not change the optimal pH (7.0) and temperature (55 °C), except for ACT-CLEA (60 °C). CLEA retained activity ( -- 90% for ASF-CLEA) after 2 weeks storage at 4 °C in buffer; and for ACT-CLEA and IPACLEA, -- 50% of initial activity was retained after 10 cycles of use. Plackett-Burman (PB) and Response Surface Methodology (RSM) optimization with ASF as precipitant gave 23.5 mM of glutaraldehyde, reaction pH 6.9 and reaction time of 3.4 h as the optimum conditions giving highest recovered activity of 37.7%. The kinetics (Vmax and Km) were 0.06 mmol g-1 min-1 and 4.95 mM respectively for ASF-CLEA, compared with 0.0/ mmol g'1 min 1 and 4.08 iriM for crude enzyme. All CLEA types exhibited the capability to hydrolyze lactose and generate GOS. This study shows that CLEA is a suitable technique to immobilize ß-galactosidase from LL313, and the resulting enzyme system has promising applications in the food industry. © 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. [ABSTRACT FROM AUTHOR]

In the present study, Candida antarctica lipase B was immobilized on amine‐functionalized silica microspheres as cross‐linked enzyme aggregates (CLEA) and utilized for the biomanufacturing of rhamnolipids (RL). Lipase CLEA synthesized under optimized conditions of 2.0:1.0 by volume of silica microsphere/enzyme concentration, a 1.0:2.5 (v/v) ratio of enzyme/2‐propanol, 7 mM glutaraldehyde concentration, when incubated at pH 9.0 and 40 °C, for a cross‐linking time of 30 min were observed to exhibit superior biocatalytic properties and a maximum enzyme load of 770 U g−1. Lipase CLEA exhibited enhanced pH stability in acidic and alkaline media and increased temperature resistance as compared to free lipase. Both free and CLEA lipases were used to synthesize RL in different solvent systems. After 12 h, from initiation of the esterification, the degree of esterification (molar conversion yield) reached 46% and 71% in the batch mode. 1H and 13C nuclear magnetic resonance (NMR) and high‐performance liquid chromatographic (HPLC) analysis confirm RL production by CLEA lipase. The CLEA showed greater confrontation to enzyme‐mediated bioprocess approach as compared to its soluble counterpart and exhibited excellent RL production and catalytic activity even after its tenth successive reuse. [ABSTRACT FROM AUTHOR]

The present study focusses on the enhancement of the catalytic activity and stability of an acetylesterase enzyme isolated from Staphylococcus spp. as Cross-Linked Enzyme Aggregates (CLEAs). The various parameters governing the activity of CLEAs were optimized. The magnetite and graphene oxide nanoparticles were successfully prepared via the chemical co-precipitation and Hummer's method, respectively. These nanoparticles supported the preparation as magnetite nanoparticle-supported cross-Linked Enzyme Aggregates (MGNP-CLEAs) and graphene oxide-supported Cross-Linked Enzyme Aggregates (GO-CLEAs). The activity and stability of these immobilized CLEAs were compared with the free enzyme at various temperature, pH, and organic solvents along with its storage stability and reusability. The immobilized preparations were analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR) techniques. Acetylesterase precipitated with 60% saturated ammonium sulfate salt (SAS) solution and cross-linked with 100 mM glutaraldehyde for 4 h at 30 °C was found to be optimal to produce CLEAs with highest activity recovery of 99.8%. The optimal pH at 8.0 and temperature at 30 °C remained the same for both the free and immobilized enzyme, respectively. Storage stability significantly improved for the immobilized enzyme as compared to free enzyme. SEM showed type-I aggregate and FT-IR revealed the successful immobilization of the enzyme. MGNP-CLEAs were found to have better activity and stability in comparison to other immobilized preparations. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Preparation and characterization of CLEA- Bm HNL reported first time. • Reusability of CLEA- Bm HNL in (S)-cyanohydrin synthesis up to eight cycles without loss of conversion. • CLEA has improved enantioselectivity of (S)-cyanohydrin synthesis compared to the use of purified enzyme. • Among the 11 (S)-cyanohydrins synthesized here, 8 were not synthesized by any CLEA-HNL and 9 were not tested by BmHNL earlier. Abstract Hydroxynitrile lyase (HNL) catalyzed enantioselective C C bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity. We optimized the preparation of immobilization of Baliospermium montanum HNL (Bm HNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA- Bm HNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA- Bm HNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ∼99% ee and ∼60% conversion. Addition of organic solvent in CLEA- Bm HNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA- Bm HNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA- Bm HNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified Bm HNL. Nine aldehydes not tested earlier with Bm HNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA- Bm HNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA- Bm HNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins. [ABSTRACT FROM AUTHOR]

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%.

Ligninolytic enzymes from white-rot fungi are widely used in biotechnological processes. However, the application of these enzymes as free enzymes is limited due to their instability and lack of reusability. Enzyme stabilization is therefore a major challenge in biocatalytic process research, and immobilization methods are desirable. Using cross-linked enzyme aggregates (CLEAs) such as magnetic CLEAs, porous-CLEAs and combi-CLEAs is a promising technique for overcoming these issues. Cross-linking methods can stabilize and immobilize enzymes by interconnecting enzyme molecules via multiple bonds using cross-linking agents such as glutaraldehyde. The high catalyst density and microporous assembly of CLEAs guarantee high catalyst activity, which, together with their long shelf life, operational stability, and reusability, provide a cost-efficient alternative to matrix-assisted immobilization approaches. Here, we review current progress in ligninolytic enzyme immobilization and provide a comprehensive review of CLEAs. Moreover, we summarize the use of these CLEAs for biocatalysis processes, bioremediation such as dye decolourization, wastewater treatment or pharmaceutically active compound elimination. [ABSTRACT FROM AUTHOR]

An alkaline protease was produced by B. licheniformis with 132.43 ± 3.4 U/mL activity in LSF which was further enhanced by optimizing culture conditions. The optimum enzyme activity (148.9 ± 4.1 U/mL) was harvested at pH 7.5; temperature, 40 °C and inoculum, 1.5 mL after 48 h incubation. Alkaline protease was immobilization by forming cross linked enzyme aggregates (CLEAs) and the processes of CLEAs formation was also optimized. The protease CLEAs developed using 80% ammonium sulfate, 65 mM glutaraldehyde and 0.11 mM BSA showed best activity recovery (39.76%). Free protease and CLEAs were characterized and compared. It was observed that CLEAs of protease exhibited broad pH range with best activity at pH 10. The immobilized protease was also thermo-tolerant with optimum activity at 65 °C temperature. The V max and K m of protease-CLEAs were 125.5 U/mL and 18.97 μM, respectively as compared to 104.9 U/mL and 29 μM, respectively for free protease. It was concluded that immobilized enzyme in the form of CLEAs is a valuable catalyst for potential industrial applications. [ABSTRACT FROM AUTHOR]

Numerous state-of-the-art technologies and new types of carriers have been developed recently to improve enzyme immobilization. Cross-linked enzyme aggregate (CLEA) technology is a lucrative prospect, as several robust biocatalysts have been generated using this simple method of carrier-free immobilization with the possibility of using semipurified enzyme. However, the low lysine content in the enzyme remains a challenge for effective crosslinking. In this work, maghemite (γ- Fe 2 O 3 ), a recently sought after nanoparticle, was silanized with (3-aminopropyl) triethoxysilane (APTES) for use in the preparation of cross-linked enzyme aggregates of recombinant xylanase from Trichoderma reesei (Xyl-CLEA-silanized maghemite). Prior screening revealed ethanol to be the best precipitant, and a 0.2: 1 (v:v) glutaraldehyde to enzyme ratio was essential to form active CLEAs. The Xyl-CLEA-silanized maghemite succeeded in increasing the activity recovery 1.66- and 1.5-fold compared to conventional Xyl-CLEAs and Xyl-CLEA-BSA, respectively. The silanization of maghemite with APTES was proven feasible when a 0.0075:1 (v:v) maghemite to enzyme ratio was able to achieve a 78% activity recovery of the xylanase aggregates, whereas the non-silanized maghemite only achieved a 47% activity recovery. At an elevated temperature of 60 °C, Xyl-CLEA-silanized maghemite retained approximately 50% of its initial activity, compared to the free enzyme, for which the activity recovery had plummeted to 20%. Conversely, Xyl-CLEA suffered a total loss of activity at this temperature, whereas Xyl-CLEA-BSA retained only a 6% activity. Xyl-CLEA-silanized maghemite also successfully retained more than 50% of its activity after up to 6 cycles, whereas Xyl-CLEA retained approximately 10% of its initial activity after only 5 cycles. The surface morphology, particle size and loading of silanized maghemite were confirmed by field emission scanning electron microscopy (FESEM) and Fourier transform infrared (FTIR) spectroscopy. The combination of the effective surface modification, high enzyme activity recovery, improved stability and enhanced reusability of Xyl-CLEA-silanized maghemite presents an attractive process for xylanase immobilization and provides a promising catalyst for the biomass industry. [ABSTRACT FROM AUTHOR]

Interfacial activation via surfactant (Tween 80, Triton X-100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross-linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non-pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899-904, 2016 [ABSTRACT FROM AUTHOR]

Cross-linked enzyme aggregates (CLEAs) were prepared from several precipitant agents using glutaraldehyde as a cross-linking agent with and without BSA, finally choosing a 40% saturation of ammonium sulfate and 25 mM of glutaraldehyde. The CLEAs obtained under optimum conditions were biochemically characterized. The immobilized enzyme showed higher thermal activity and a broader range of pH and organic solvent tolerance than the free enzyme. Arylesterase from Gluconobacter oxydans showed activity toward cephalosporin C and 7-aminocephalosporanic acid. The CLEAs had a Kcat/KM of 0.9 M−1/S−1 for 7-ACA (7-aminocephalosporanic acid) and 0.1 M−1/S−1 for CPC (cephalosporin c), whereas free enzyme did not show a typical Michaelis-Menten kinetics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:36-42, 2016 [ABSTRACT FROM AUTHOR]

Glycosidases are frequently used in winemaking to liberate glycosidically bound aroma compounds. Since most of the glycosidases used for diglycoside hydrolysis act sequentially, their co-immobilization is an attractive alternative from a technical and economical perspective. The enzymes α-L-arabinosidase (ARA) and β-D-glucosidase (βG) from the preparation Rapidase® AR2000 were co-immobilized in CLEAs (combi-CLEAs), evaluating the effect of bovine serum albumin (BSA) addition and the concentration of glutaraldehyde (Glu) on enzyme immobilization yield and expressed activity. Combi-CLEAs prepared with a Glu to Rapidase protein mass ratio of 0.053 and BSA to Rapidase protein mass ratios of 1, 0.33, and 0.2 were selected, evaluating their stability at simulated winemaking conditions: 25 °C, pH 3.5, and 10% (v/v) of ethanol. All combi-CLEAs were more stable than the soluble enzymes, the best result being obtained at a BSA to Rapidase protein mass ratio of 0.33. Half-lives of (βG and ARA in combi-CLEAs were 43.9 and 54.9 days, respectively, whereas in the case of the soluble enzymes were only 1.3 and 6.2 days, respectively. Immobilization yields were 79.1 and 47.1% in terms of βG and ARA activity, respectively. Combi-CLEAs of glycosidases are technologically relevant robust biocatalysts for their application as aroma enhancers in winemaking. [ABSTRACT FROM AUTHOR]

Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve ( R, S)-2-octanol, and retained 100% activity and 100% after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates. Chirality 27:199-204, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The preparation of cross-linked enzyme aggregates (CLEAs) with albumin as an additive is demonstrated successfully with lipase Thermomyces lanuginosus (TL) and lipase B of Candida antarctica (CalB). Investigations regarding the albumin admixture and the temperature optimum are employed. Compared to non-cross-linked lipase, CalB delivers the best results at a mass ratio of 1:16 lipase/albumin, which leads to a 24-fold increase of activity; TL at a mass ratio of 1:12, which leads to a eightfold increased activity. A transesterification yield of 61% m/m ethyl ester can be reached with 2.5% m/m CLEAs of lipase TL applied on rapeseed oil within 1.5 h reaction time. A catalyst mass of 10% m/m yields 94% m/m ethyl ester in the same time. Within six cycles of reuse, the catalyst remains stable. CLEAs of CalB and Novozym 435 (a commercial immobilized lipase B of C. antarctica) show comparable rates in ethanolysis and glycerolysis reaction. Practical applications: CLEAs have become of increasing interest in terms of synthesis of organic compounds via bio-catalytic (green) approaches. The findings presented here are targeting at transesterification of fats and oils into corresponding alkyl esters applicable, e.g., as alternative fuel substitutes. High conversion rates coupled with hyperactivation effects as well as highly appealing reusability behavior indicate the practical benefits even for technical applications. Conversion of lipids into derivatives by CLEAs as demonstrated here, might extend utilization and conversion possibilities of biomass. [ABSTRACT FROM AUTHOR]

Abstract: The precipitation of enzyme causes the major activity loss in the conventional protocol for CLEAs preparation. Herein, a sugar-assisted strategy was developed to minimize the activity loss in the step of enzyme precipitation by adding sugar as the stabilizer, which contributed to improve the activity yield of resulting CLEAs. Penicillin G acylase (PGA) was employed as a model enzyme. The effects of glucose, sucrose and trehalose on the activity yields of CLEAs were investigated. The highest activity was obtained in the case of adding trehalose. Confocal laser scanning microscopy and Fourier transform infrared spectroscopy showed that the polar microenvironment and the secondary structure of native enzyme were preserved to some extent when PGA was prepared as sugar-assisted CLEAs, resulting in PGA''s higher activity than sugar-free CLEAs. Scanning electron microscope revealed the different inner morphologies, and the kinetic studies showed the higher affinity and resist-inhibition capacity of sugar-assisted CLEAs. Furthermore, stability experiments demonstrated that CLEAs prepared in sugar-assisted strategy remained higher thermal stability when it was incubated at high temperature. [Copyright &y& Elsevier]

Abstract: Cross-linked enzyme aggregates (CLEA®s) were prepared from Candida rugosa lipase (CrL) using glutaraldehyde as the cross-linker. The optimum conditions of the immobilization process were determined (precipitant: ethanol, crosslinker concentration: 25mM, enzyme concentration: 50mg/ml, crosslinking time: 45min.). CLEAs were shown to have several advantages compared to the free enzyme. They were more stable at 50°C and 60°C and had good reusability; retaining 40% of their initial activity after 15 recycles in aqueous media and remaining constant at that level thereafter, suggesting some initial leaching in water. The CLEAs catalyzed esterification reactions in cyclohexane, affording higher conversions than with the free enzyme, especially when longer fatty acids and alcohols were used as substrates. [Copyright &y& Elsevier]

Abstract: A recombinant esterase (EC 3.1.1.1) cloned from Bacillus subtilis 0554 (BSE) was carrier-freely immobilized with the method of cross-linked enzyme aggregates. The conditions for preparing the cross-linked aggregates of BSE (CLA-BSE) were optimized, including the type and concentration of precipitants, and the concentration of cross-linker, and a simple and efficient procedure for preparing CLA-BSE was developed, consisting of a precipitation step with 0.5gmL−1 (NH4)2SO4 and a cross-linking step with 60mM glutaraldehyde for a period of 3h as the cross-linking time. As a result, about 70% of the initial free BSE activity was incorporated into the CLA-BSE. The thermal stabilities of the immobilized enzyme at 30°C and 50°C were >360 and 14 times those of free BSE, respectively. More importantly, the operational stability of CLA-BSE was also considerably improved. In the kinetic resolution of dl-menthyl acetate to produce l-menthol with CLA-BSE gave ee p >94% at conversion of >40% and the CLA-BSE could be reused for 10 times with only about 8% reduction in activity. Therefore, the new biocatalyst immobilized through the methodology of CLEAs could significantly decrease the manufacturing cost of l-menthol and would be more beneficial for its practical applications. [Copyright &y& Elsevier]

The article presents a study on the immobilization of candida rugosa lipase on amino-functionalized magnetic beads using cross-linked enzyme aggregates (CLEA). It states that batch operation mode was used to examine immobilized lipase usability while the immobilized and free lipases were stored in a solution of sodium phosphate buffer. Results show that 80% of the immobilized lipase's initial activity was maintained.

Abstract: The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1unit (pH 8.0) and 5°C higher (45°C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30min of each reaction time, indicating stable reusability under aqueous medium. [Copyright &y& Elsevier]

Abstract: The microstructure and the catalytic properties of cross-linked enzyme aggregates (CLEA) of penicillin acylase (PA) obtained under different conditions were investigated. The period of time left between the enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation. Confocal fluorescent microscopy of the so-called “fresh” and “mature” CLEAs PA allowed to estimate the “characteristic” diameter of CLEA PA particles, which appeared to be about 1.6μm, and revealed that the “mature” type was composed of relatively big particles as compared to the “fresh” type. Complementary kinetic studies showed that the “mature” CLEA PA were more effective in both hydrolytic and synthetic reactions. It was suggested that the aggregate size might regulate the extent of covalent modification of PA and thereby influence the catalytic properties of CLEA. [Copyright &y& Elsevier]

Abstract: Tyrosinase from mushroom was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized tyrosinase was characterized. Sixty percent ammonium sulfate saturation and 2% glutaraldehyde were used; a 3-h cross-linking reaction at room temperature, at pH 7.0 was performed; particle sizes of the aggregates were reduced; consequently, 100% activity recovery was achieved in CLEAs with enhanced thermal and storage stabilities. Slight changes in optimum pH and temperature values of the enzyme were recorded after immobilization. Although immobilization did not affect V max, substrate affinity of the enzyme increased. Highly stable CLEAs were also prepared from crude mushroom tyrosinase with 100% activity recovery. [Copyright &y& Elsevier]

Abstract: Aspergillus oryzae β-galactosidase was immobilized by three different techniques, namely adsorption on celite, covalent coupling to chitosan and aggregation by cross-linking. These techniques were compared in terms of the yield of immobilized preparation, enzymatic characteristics, stability and efficiency in oligosaccharide synthesis. Immobilization led to increase in K m in each case. Immobilization on chitosan gave maximum enzyme yield and oligosaccharide synthesis. At 60°C, the chitosan-immobilized enzyme was stabilized (by 1.6-fold) due to protection effect of the matrix. However, at 65°C, the t 1/2 of cross-linked enzyme aggregates (CLEA) of β-galactosidase was 1.07h as compared to 0.79h in the case of free enzyme. Both chitosan-immobilized enzyme and CLEA were used for oligosaccharide synthesis. Using 20% (w/v) lactose, the chitosan-immobilized enzyme gave maximum oligosaccharide yield (17.3% of the total sugar) as compared to free enzyme (10.0%) in 2h at 40°C. CLEA were instead found effective in lactose hydrolysis yielding 78% monosaccharide in 12h. [Copyright &y& Elsevier]

[Display omitted] • Magnetite cross-linked inulinase aggregates were prepared for inulin hydrolysis. • The immobilization process was optimized using D-optimal Design. • 18.1 g/L fructose from inulin was obtained at 60℃ in 4 h. • The immobilized magnetic inulinase was recycled for 25 cycles. The utilization of advanced nanoengineered technologies opens up attractive horizons towards sustainable treatment of waste biomass to produce fine chemicals. Production of fructose syrup (FS) in a single step using inulinase is a major achievements of enzyme technology as it eliminates the need for a multi-enzyme system. Functionalization of particles was done by coating chitosan on the surface of the magnetite and further, the chitosan coated magnetic nanoparticle (cMNP) was added to the mixture of cross-linked inulinase aggregates, and the physio-chemical properties of the aggregates were characterized. The immobilized biocatalyst was further utilized for the production of FS. Upon inulin hydrolysis for 4 h at 60 °C, 18.1 g/L of fructose was obtained with immobilized system. The immobilized inulinase was recycled up to 25 cycles and could be separated from the solution in 20 s by an external magnetic field. [ABSTRACT FROM AUTHOR]

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]