Enzyme Aggregates Research Articles

Cross-linked β-Mannanase Aggregates: Preparation, Characterization, and Application for Producing Partially Hydrolyzed Guar Gum.

β-Mannanase capable of hydrolyzing β-1,4-linkages in guar gum was immobilized as cross-linked enzyme aggregates (M-CLEAs). The aggregation and cross-linking process was optimized by response surface methodology (RSM) for maximum activity. The resulting M-CLEAs were characterized by FTIR, DSC, SEM, and SDS-PAGE. The M-CLEAs showed higher pH stability, improved thermal and storage stability, and reusability than free β-mannanase. For example, M-CLEAs were stable over broader pH range (5.5-8.5) with highest relative of activity of 98.17% at pH 6.5 and retained almost double activity than free mannanase at 50°C after 4h. Moreover, Km and Vmax of M-CLEAs were altered significantly, with a 1.5-fold increase and 0.98-fold decrease, respectively, than free β-mannanase. The prepared M-CLEAs could hydrolyze native guar gum (MW = 588,147Da) to yield partially hydrolyzed guar gum (PHGG) (MW = 8023Da).

An effective immobilization of β-glucosidases by partly cross-linking enzyme aggregates

Enzyme immobilization provides ideal operating conditions for enzymes stabilization and sustainable recycling. In this work, as a kind of clay material, montmorillonite (MTL) was chosen for immobilizing the β-glucosidase extracted from Agrocybe aegirit. The immobilized β-glucosidase via partly cross-linking enzyme aggregates (pCLEAs) formed by self-catalysis provided biocatalysts with satisfactory thermal and pH stability. Compared to the glutaraldehyde cross-linked, the immobilized β-glucosidase (β-G-pCLEAs@MTL) exhibited significantly higher immobilization efficiency (IE) and immobilization yield (IY), which were 80.6% and 76.9%, respectively. The β-G-pCLEAs@MTL also showed better stability and preferable reusability. And the activity of the β-G-pCLEAs@MTL remained 85.0% after 5 cycles and 74.7% after 10 cycles. Therefore, the method based on the pre- crosslinking to form pCLEAs and after-immobilization can effectively improve IY and IE. In addition, MTL seems to be a good alternative carrier to immobilize other enzymes for industrial application.

Determination of effective assay parameters on the activity of magnetite cross-linked invertase aggregates by personal glucose meter

The invertase enzyme has been immobilized onto various support materials to enhance operational stability, shelf life, and reusability compared to free ones. Among the immobilization methods, carrier-free immobilization, such as cross-linked enzyme aggregates (CLEAs), attracts attention. However, recovery of the CLEAs from the reaction environment by centrifugation or filtration is a great challenge. The use of magnetic nanoparticles (Fe3O4) can overcome these separation limitations of the aggregates. For this aim, in this study, the surface of magnetite nanoparticles was functionalized with a significant number of free amino groups through silanization reaction. After precipitation of these aggregates and then cross-linking to the functionalized structure, the resulting magnetite cross-linked invertase aggregates (MCLIAs) were obtained, having different enzyme and glutaraldehyde concentrations. Magnetic nanoparticles were characterized by SEM, VSM, and FTIR analysis. The assay parameters, both constructional and experimental ones on the activity of MCLIAs, were practically determined by a personal glucose meter (PGM). The results indicate that magnetite nanoparticles with superparamagnetic behaviour were successfully functionalized with amino groups. The activity results demonstrated that experimental parameters were more effective than constructional parameters. MCLIAs also exhibited maximum activity at pH 5. Furthermore, 30-min incubation time and 35 °C were the best activity assay conditions.

Co-Immobilizing Two Glycosidases by Combined Cross-Linked Enzyme Aggregates with Enhanced Enzymatic Properties Achieves High Titer Icaritin Biosynthesis

Co-Immobilizing Two Glycosidases by Combined Cross-Linked Enzyme Aggregates with Enhanced Enzymatic Properties Achieves High Titer Icaritin Biosynthesis

Magnetic cross-linked enzyme aggregate based on ionic liquid modification as a novel immobilized biocatalyst for phytosterol esterification

Novel immobilized enzyme CRL-FIL-CLEAs@Fe3O4 with enhanced activities and stabilities was successfully prepared by a cross-linked lipase aggregate method for phytosterol esterification.

Regenerable temperature-responsive biocatalytic nanofiltration membrane for organic micropollutants removal

SummaryBiocatalytic nanofiltration membranes (BNMs) exhibit great potentials in organic micropollutants removal attributed to its synergistic effect between enzyme catalysis and membrane separation. However, the difficulties in regeneration of the BNMs halted their economic practicality. Inspired by cell membranes with stimuli-responsive channels, we have developed the temperature-responsive BNMs with nanogating function by poly(N-isopropyl acrylamide) (PNIPAM) modification. PNIPAM modification increases the geometric confinement of the support layer to enzymes, thus improving enzyme loading, inhibiting enzyme leakage, and preventing membrane permeability decline caused by enzyme excess migration and aggregation. By optimizing the concentration of reaction monomers, modification time, and strategies, the PNIPAM-based BNMs show high bisphenol A (BPA) removal efficiency and long-term stability. Furthermore, the PNIPAM-polyethyleneimine-based BNMs can be easily regenerated at 38°C, and the laccase activity and BPA removal efficiency are fully recovered. This work would promote the real application of BNMs in bioconversion, drug delivery, and biosensors.

Cross-Linked Enzyme-Adhered Nanoparticles (CLEANs) for Continuous-Flow Bioproduction.

Nanostructured but micro-sized biocatalysts were created by bottom-up technology using multi-functionalized silica nanoparticles (NPs) as nano-sized building blocks to form cross-linked enzyme-adhered nanoparticles (CLEANs) as robust micro-sized particles with beneficial internal structure and good mechanical properties. Systematic surface modification of NPs with a grafting mixture consisting of organosilanes with reactive (aminopropyl) and inert (e. g., vinyl, propyl, phenyl, or octyl) functions resulted in functional NPs enabling cross-linking agents, such as glutardialdehyde or bisepoxides (glycerol diglycidyl ether, neopentylglycol diglycidyl ether, and poly(propylene glycol) diglycidyl ether), to bind and cross-link enzymes covalently and to form macroporous microparticles. These CLEANs were able to diminish several weaknesses of traditional cross-linked enzyme aggregates as biocatalysts, such as poor mechanical resistance, difficult recovery, and storage, strengthening their use for packed-bed enzyme reactors. Lipase B from Candida antarctica (CaLB) was selected as model enzyme for development of robust CLEANs, which were successfully tested for various industrially relevant applications including a kinetic resolution of a racemic alcohol and the production of various natural fragrance compounds under continuous-flow conditions.

Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase.

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Cross-Linked Enzyme Aggregates and Their Application in Enzymatic Pretreatment of Microalgae: Comparison Between CLEAs and Combi-CLEAs.

Carrier-free immobilization is a key process to develop efficient biocatalysts able to catalyze the cell wall degradation in microalgae where the traditional solid supports cannot penetrate. Thus, the insolubilization of commercial Celluclast®, Alcalase®, and Viscozyme® enzymes by carrier-free immobilization and their application in microalgae pretreatment was investigated. In this study, different precipitants at different ratios (ethanol, acetone, and polyethylene glycol 4000) were tested in the first part of the method, to establish the precipitation conditions. The screening of the best precipitant is needed as it depends on the nature of the enzyme. The best results were studied in terms of immobilization yield, thermal stability, and residual activity and were analyzed using scanning electron microscopy. Moreover, a novel strategy was intended including the three enzymes (combi-CLEAs) to catalyze the enzymatic degradation of Nannochloropsis gaditana microalgal cell wall in one pot. The carrier-free immobilized derivatives were 10 times more stable compared to soluble enzymes under the same. At the best conditions showed its usefulness in the pretreatment of microalgae combined with ultrasounds, facilitating the cell disruption and lipid recovery. The results obtained suggested the powerful application of these robust biocatalysts with great catalytic properties on novel and sustainable biomass such as microalgae to achieve cost-effective and green process to extract valuable bioactive compounds.

Cascade Biocatalytic Processes through Combined Cross-linked Enzyme Aggregates

Cascade reactions catalyzed by immobilized multi enzymatic systems are emerging as a tool to address the increasing need for green and sustainable chemistry. In this regard, the carrier-free immobilization strategy of combined cross-linked enzymes aggregates (combi- CLEAs) is of great interest. In a combi-CLEAs two or more enzymes are co-immobilized into a single unit where they are accommodated in close proximity in order to minimize the intermediates diffusion in the reaction medium, at the same time allowing the access of substrates and coenzymes to the active sites. The use of CLEAs provides a simple and low cost methodology that enhances operational and storage enzyme stability and facilitates biocatalyst recovery and recycle. Some significant applications of cascade reactions catalyzed by combi- CLEAs are reported in this mini-review. These examples have been applied on a multigram scale and have furnished practical steps towards the implementation of enzymes on an industrial scale.

Enhancing stability and by-product tolerance of β-glucuronidase based on magnetic cross-linked enzyme aggregates

β-glucuronidase is an important catalyst which is highly specific for β-glucuronides. Here, we constructed magnetic cross-linking β-glucuronidase aggregates (MCLEAs) to for the production of glycyrrhetinic acid (GA). Before crosslinking via glutaraldehyde, we used carbodiimide to enhance the interaction between enzymes and carboxyl-functionalized Fe3O4, efficiently improving the activity recovery. Compared to free enzymes, both kcat and kcat/Km enhanced, indicating that crosslinking and aggregation brought higher catalytic efficiency to enzymes. MCLEAs enhanced pH and thermal stabilities and retained 63.3% of catalytic activity after 6 cycles. More importantly, it was first found that the glucuronic acid tolerance of β-glucuronidase after the formation of MCLEAs enhanced 221.5% in 10 mM of glucuronic acid. According to the Raman spectroscopy, the ordered structure of β-glucuronidase increased from 43.9% to 50.6% after immobilization, which explained the increased stability and tolerance. To sum up, MCLEAs provided an efficient strategy for immobilization of enzymes, which enhanced stability and glucuronic acid tolerance of enzymes. It might be an effective solution to the serious inhibition caused by by-products during the preparation of aglycone from natural glycosides, having a significant applied prospect in industry.

Preparation of combined cross-linked enzyme aggregates containing galactitol dehydrogenase and NADH oxidase for L-tagatose synthesis via in situ cofactor regeneration.

The combined cross-linked enzyme aggregates (combi-CLEAs) containing galactitol dehydrogenase (Gdh) and NADH oxidase (Nox) were prepared for L-tagatose synthesis. To prevent the excess consumption of cofactor, Nox in the combi-CLEAs was used to in situ regenerate NAD+. In the immobilization process, ammonia sulfate and glutaraldehyde were used as the precipitant and cross-linking reagent, respectively. The preparation conditions were optimized as follows: 60% ammonium sulfate, 1:1 (molar ratio) of Gdh to Nox, 20:1 (molar ratio) of protein to glutaraldehyde, and 6h of cross-linking time at 35°C. Under these conditions, the activity of the combi-CLEAs was 210Ug-1. The combi-CLEAs exhibited higher thermostability and preserved 51.5% of the original activity after eight cycles of reuses at 45°C. The combi-CLEAs were utilized for the preparation of L-tagatose without by-products. Therefore, the combi-CLEAs have the industrial potential for the bioconversion of galactitol to L-tagatose.

Preparation and characterization of a nontoxic cross‐linked lipase aggregate by using sodium tripolyphosphate and chitosan

AbstractCross‐linked enzyme aggregates (CLEAs) have the advantages of high‐enzyme activity and stability, and have an application potential in food and feed processing. In order to address the food and feed safety of CLEAs and have ideal enzymatic properties, a new design of CLEAs of lipase from Aspergillus niger is developed by using sodium tripolyphosphate (TPP) as a cross‐linking agent and chitosan as an auxiliary agent. The enzyme activity recovery of the developed CLEAs reaches 126%. The half‐life of the CLEAs is 11.4‐fold higher than that of free enzyme at the optimum reaction temperature of CLEAs. The surface structure of the CLEAs reveals cluster and porous structure. The variations of the secondary structures of the CLEAs indicate an increase of α‐helix and decrease of β‐sheet, β‐turn, and random coil. The adding of chitosan is beneficial to enzyme activity recovery and thermal stability of the CLEAs. TPP and chitosan are safe and nontoxic, and the CLEAs prepared with TPP and chitosan is suitable for the food and feed industries.

Production of combined-cross-linked hemicellulosic enzyme aggregates from paperboard residues

Production of combined-cross-linked hemicellulosic enzyme aggregates from paperboard residues

Immobilization and Biochemical Characterization of Keratinase 2S1 onto Magnetic Cross-Linked Enzyme Aggregates and its Application on the Hydrolysis of Keratin Waste

Immobilization and Biochemical Characterization of Keratinase 2S1 onto Magnetic Cross-Linked Enzyme Aggregates and its Application on the Hydrolysis of Keratin Waste

Improved catalytic performance of carrier-free immobilized lipase by advanced cross-linked enzyme aggregates technology.

The cross-linked enzyme aggregates (CLEAs) are one of the technologies that quickly immobilize the enzyme without a carrier. In this study, ionic liquid with amino group (1-aminopropyl-3-methylimidazole bromide, FIL) was used as the novel functional surface molecule to modify CRL (Candida rugosa lipase, CRL). The enzymatic properties of CRL-FIL-CLEAs were investigated. The activity of CRL-FIL-CLEAs (5.51 U/mg protein) was 1.9 times higher than that of CRL-CLEAs (2.86 U/mg protein) without surface modification. After incubating in a centrifuge tube for 50min at 60°C, CRL-FIL-CLEAs still maintained 61% of its initial activity, while the value for CRL-CLEAs was only 22%. After repeated use for five times, compared with the 22% residual activity of CRL-CLEAs, the value of CRL-FIL-CLEAs was 51%. Based on the above results, it was indicated that this method provided a new idea for the effective synthesis of immobilized enzyme.

Biocatalytic transesterification of algal oil employing a heterogenous methanol tolerant lipase enzyme aggregate from Bacillus mycoides strain CV18

Methanol induced lipase inactivation is the major hindrance in commercialization of enzyme catalyzed biodiesel production. Thus, methanol tolerance is highly needed for the lipase to be used in transesterification reaction. Hence, by virtue of such desirability, a methanol tolerant, extracellular lipase FDS8(L) was extracted from Bacillus mycoides strain CV18 and purified to homogeneity with 22.3 % yield. It was a non-position specific, alkaline lipase with 44 kDa of relative molecular mass and had no isoforms (confirmed by zymography). It showed optimal activity at pH 9.0 and 30 °C temperature, while stability in the pH range 7.0–10.0 and temperature upto 50 °C. Organic solvents (hexane, acetone and methanol) as well as metal ions (Mg2+, Mn2+ and Na+) had stimulatory effects on lipase activity. Lipase immobilization was done using glutaraldehyde to form cross linked lipase enzyme aggregate (CLEA) which further enhanced its stability in the presence of methanol. Owing to its methanol tolerance property, it was employed heterogeneously for catalyzing transesterification of algal lipids in methanol. The qualitative efficacy of the FDS8 lipase in converting algal lipids into biodiesel was confirmed after analyzing the hexane extracted transesterification products by TLC and FTIR spectroscopy.

Effect of the biological functionalization of nanoparticles on magnetic CLEA preparation

Lipase immobilization using adsorption on magnetic nanoparticles, cross-linked enzyme aggregates (CLEA), and a combination of both techniques was investigated.Experimental designs were used for the optimization of the immobilization observing that the pH and ionic strength play a principal role during the lipase immobilization and its activity. For adsorption on magnetic nanoparticles and CLEA synthesis the optimal condition was pH and 100 mM. Besides, during the CLEA synthesis, glutaraldehyde concentration showed to be a significant effect on the enzyme activity.A comparison between a magnetic CLEA prepared with (Lip@mCLEA) and without (mCLEA) biological functionalized magnetic nanoparticles was made observing that the use of functionalized support showed the best performance activity. All biocatalytic systems developed gives to the enzyme thermal stability between 45 and 70 °C, being Lip@mCLEA the more stable biocatalyst. Similar behavior was observed at different pH, where both Lip@mCLEA and mCLEA showed stability at a range of pH 5 to 8.The immobilized biocatalysts showed the same affinity of the subtract that the free enzyme suggested that the enzyme structure not modified the active site. The combination of both types of immobilization show evidenced the importance of the biological functionalization of the support when magnetic CLEA is produced.

Characterisation and optimisation of a novel laccase from Sulfitobacter indolifex for the decolourisation of organic dyes

Laccases are multi‑copper oxidases that possess the potential for industrial wastewater treatments. In this study, a putative laccase from Sulfitobacter indolifex was recombinantly produced and characterised. The enzyme was found to be stable and active at low to ambient temperature and across a range of pH conditions. The ability of the putative bacterial laccase to catalyse the decolourisation of seven common industrial dyes was also examined. Our results showed that the putative laccase could efficiently decolourise Indigo Carmine, Coomassie Brilliant Blue R-250, Congo Red, Malachite Green and Alizarin in the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a redox mediator. Furthermore, the use of enzyme immobilisation technology to improve the operational stability and reusability of the putative laccase was also investigated. We found that immobilising the enzyme through the cross-linked enzyme aggregate method significantly improved its tolerance towards extreme pH as well as the presence of organic solvents. This work expands the arsenal of bacterial laccases available for the bioremediation of dye-containing wastewater.

Encapsulation of Combi-CLEAs of Glycosidases in Alginate Beads and Polyvinyl Alcohol for Wine Aroma Enhancement

The aromatic expression of wines can be enhanced by the addition of specific glycosidases, although their poor stability remains a limitation. Coimmobilization of glycosidases as cross-linked enzyme aggregates (combi-CLEAs) offers a simple solution yielding highly stable biocatalysts. Nevertheless, the small particle size of combi-CLEAs hinders their recovery, preventing their industrial application. Encapsulation of combi-CLEAs of glycosidases in alginate beads and in polyvinyl alcohol is proposed as a solution. Combi-CLEAS of β-d-glucosidase and α-l-arabinofuranosidase were prepared and encapsulated. The effects of combi-CLEA loading and particle size on the expressed specific activity (IU/gbiocatalyst) of the biocatalysts were evaluated. Best results were obtained with 2.6 mm diameter polyvinyl alcohol particles at a loading of 60 mgcombi-CLEA/gpolyvinyl alcohol, exhibiting activities of 1.9 and 1.0 IU/gbiocatalyst for β-d-glucosidase and α-l-arabinofuranosidase, respectively. Afterwards, the stability of the biocatalysts was tested in white wine. All the encapsulated biocatalysts retained full activity after 140 incubation days, outperforming both free enzymes and nonencapsulated combi-CLEAs. Nevertheless, the alginate-encapsulated biocatalysts showed a brittle consistency, making recovery unfeasible. Conversely, the polyvinyl-encapsulated biocatalyst remained intact throughout the assay. The encapsulation of combi-CLEAs in polyvinyl alcohol proved to be a simple methodology that allows their recovery and reuse to harness their full catalytic potential.