Dextran Polyaldehyde Research Articles

Immobilization of BoPAL3 Phenylalanine Ammonia-Lyase on Electrospun Nanofibrous Membranes of Polyvinyl Alcohol/Nylon 6/Chitosan Crosslinked with Dextran Polyaldehyde.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is common in plants and catalyzes the formation of trans-cinnamic acid and ammonia via phenylalanine deamination. Recombinant Bambusa oldhamii BoPAL3 protein expressed in Escherichia coli was immobilized on an electrospun nanofibrous membrane using dextran polyaldehyde as a crosslinker. The immobilized BoPAL3 protein exhibited comparable kinetic properties with the free BoPAL3 protein and could be recycled for six consecutive cycles compared with the free BoPAL3 protein. The residual activity of the immobilized BoPAL3 protein was 84% after 30 days of storage at 4 °C, whereas the free BoPAL3 protein retained 56% residual activity in the same storage conditions. Furthermore, the resistance of the immobilized BoPAL3 protein to chemical denaturants was greatly increased. Therefore, the BoPAL3 protein can be immobilized using the natural dextran polyaldehyde crosslinker in place of the conventional chemical crosslinker. Nanofibrous membranes made from polyvinyl alcohol (PVA), nylon 6, and chitosan (CS) are incredibly stable and useful for future industrial applications.

Preparation of cross-linked cell aggregates (CLCAs) of recombinant E. coli harboring glutamate dehydrogenase and glucose dehydrogenase for efficient asymmetric synthesis of l-phosphinothricin

The enzymatic asymmetric synthesis of L-phosphinothricin (L-PPT) from 2-carboxyl-4-(hydroxymethylphosporyl) butyric acid (PPO) is a green and efficient method. In our previous works, we constructed a glutamate dehydrogenase (GluDH) and glucose dehydrogenase (GDH) co-expression strain, Escherichia coli BL21 (DE3)/pETDuet-gludh-gdh, which can asymmetrically reduce PPO to L-PPT. To improve the stability and recyclability of the biocatalyst, we developed a novel method for whole-cell immobilization with cross-linked cell aggregates (CLCAs) and employed immobilized cells in L-PPT production. Montmorillonite was used as a carrier, and dextran polyaldehyde (DPA) was developed as a new cross-linker for cell immobilization. The highest activity recovery (79.2%) was obtained under the following conditions: 20 g/L cell concentration, 0.9% montmorillonite concentration, 1.2% polyethyleneimine (PEI) concentration, and 1.2% DPA concentration. The half-lives of CLCAs were 169.10 h (35 °C), 17.39 h (45 °C), and 10.65 h (55 °C), which were 1.87-, 3.70-, and 10.78-fold than that of free cells, respectively. The CLCAs showed good reusability, retaining over 85% of their initial activities after 10 cycles of utilization. The total yield of L-PPT reached 667 mmol/g cells, and the enantiomeric excess value was more than 99%. The immobilized whole-cell in the form of CLCAs is a valuable biocatalyst for industrial applications.

Cross-Linked Gelatine by Modified Dextran as a Potential Bioink Prepared by a Simple and Non-Toxic Process.

Essential features of well-designed materials intended for 3D bioprinting via microextrusion are the appropriate rheological behavior and cell-friendly environment. Despite the rapid development, few materials are utilizable as bioinks. The aim of our work was to design a novel cytocompatible material facilitating extrusion-based 3D printing while maintaining a relatively simple and straightforward preparation process without the need for harsh chemicals or radiation. Specifically, hydrogels were prepared from gelatines coming from three sources—bovine, rabbit, and chicken—cross-linked by dextran polyaldehyde. The influence of dextran concentration on the properties of hydrogels was studied. Rheological measurements not only confirmed the strong shear-thinning behavior of prepared inks but were also used for capturing cross-linking reaction kinetics and demonstrated quick achievement of gelation point (in most cases < 3 min). Their viscoelastic properties allowed satisfactory extrusion, forming a self-supported multi-layered uniformly porous structure. All gelatin-based hydrogels were non-cytototoxic. Homogeneous cells distribution within the printed scaffold was confirmed by fluorescence confocal microscopy. In addition, no disruption of cells structure was observed. The results demonstrate the great potential of the presented hydrogels for applications related to 3D bioprinting.

Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers

Phenylalanine ammonia-lyase (PAL) catalyzes the nonoxidative deamination of phenylalanine to yield trans-cinnamic acid and ammonia. Recombinant Bambusa oldhamii BoPAL1/2 proteins were immobilized onto electrospun nanofibers by dextran polyaldehyde as a cross-linking agent. A central composite design (CCD)-response surface methodology (RSM) was utilized to optimize the electrospinning parameters. Escherichia coli expressed eBoPAL2 exhibited the highest catalytic efficiency among four enzymes. The optimum conditions for fabricating nanofibers were determined as follows: flow rate of 0.10 mL/h, voltage of 13.8 kV, and distance of 13 cm. The response surface models were used to obtain the smaller the fiber diameters as well as the highest PAL activity in the enzyme immobilization. Compared with free BoPALs, immobilized BoPALs can be reused for at least 6 consecutive cycles. The remained activity of the immobilized BoPAL proteins after storage at 4 °C for 30 days were between 75 and 83%. In addition, the tolerance against denaturants of the immobilized BoPAL proteins were significantly enhanced. As a result, the dextran polyaldehyde natural cross-linking agent can effectively replace traditional chemical cross-linking agents for the immobilization of the BoPAL enzymes. The PAL/nylon 6/polyvinyl alcohol (PVA)/chitosan (CS) nanofibers made are extremely stable and are practical for industrial applications in the future.

Immobilization of laccase via cross-linked enzyme aggregates prepared using genipin as a natural cross-linker

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs.

A Millifluidic Device with Embedded Cross-Linked Enzyme Aggregates for Degradation of H2O2.

Millifluidic devices decorated with enzymes have been used for enzymatic reactions in continuous processes, but low enzymatic activity and enzyme leaching remain as challenges. Herein, we develop a strategy to embed cross-linked enzyme aggregates (CLEAs) on the surfaces of millifluidic devices to achieve higher enzymatic activity and better stability. Catalase was chosen as a model enzyme to degrade H2O2 in wastewater samples. First, CLEA of catalase (153 ± 10 nm) was formed by simultaneous precipitation and cross-linking with 25.0 wt % acetonitrile containing 0.025 wt % glutaraldehyde in a millifluidic device. To immobilize CLEA, we first swell a piece of plastic tubing by using 5.0 wt % acetonitrile and then immerse it in an aqueous solution with 5.0 wt % (3-aminopropyl)triethoxysilane (APTES) and 5.0 wt % dextran polyaldehyde (DPA) subsequently. After CLEA is absorbed inside the expanded polymer network of the tubing, the tubing is tightened by using a vacuum to secure the immobilized CLEA. The millifluidic device decorated with CLEA of catalase has total activity of 660 U for degradation of H2O2, and it shows good stability under a flow rate of 200 μL/min. The tubing can be used to degrade 0.1 wt % H2O2 solution continuously for 3 h or remove 2 wt % residual H2O2 in wastewater for 2 h. The technique is general enough and can be applied to other types of enzymes for continuous enzymatic reactions.

Cross-linked enzyme lyophilisates (CLELs) of urease: A new method to immobilize ureases

In this study, we presented a new approach for immobilizing JBU (Jack bean urease), by producing urease cross-linked enzyme lyophilisates (CLELs). Through the use of bovine serum albumin (BSA), lyophilisation, cross-linking with dextran polyaldehyde (DPA), and optimizing cross-linker pH, the urease-CLELs produced show an increase in relative catalytic activity that is 1.47 times higher than that of free urease, while remaining stable up to temperatures of 85 °C. Urease-CLEL activity increases in direct proportion with the increasing BSA content due to the offered additional lysine (Lys) groups which are potential cross-linking points providing better immobilization and retention of JBU, while lyophilisation also enables stabilization by eliminating solvating water molecules and intra-molecular reactions that may block the cross-linking residues. Two most commonly used cross-linkers that are reacting with the available Lys groups, i.e.glutaraldehyde (GA) and bulkier alternative DPA, have been selected for the immobilization of urease. The catalytic activity increase with DPA suggests an improved access to the active site through hindering blockage, while the increase with alkaline pH of the cross-linkers indicates decreased buffer inhibition. The long lifetime (113% residual activity after 4 weeks), recyclability (132% residual activity after 10 cycles) and thermal stability (276% relative activity at 85 °C) of these urease-CLELs demonstrate that they are technologically attractive as green biocatalysts, while our immobilization approach offers an alternative to conventional methods for proteins that are difficult to immobilise.

Fine-tuned preparation of cross-linked laccase nanoaggregates

This study focuses on well-known but commonly overlooked or unreported factors in the preparation of cross-linked enzyme nano-aggregates (nano-CLEAs). The parameters including the ionic strength of the protein solution, protein, precipitant and cross-linker concentrations, pH and addition order of the reagents were fine-tuned for nanoaggregate preparation without the need of non-protein support material, special equipment or sophisticated procedures. For this purpose, precipitation as nano-aggregates and then cross-linking while maintaining submicron size distribution were studied independently. Moreover, nano-aggregate formation from reverse micellar solutions was also investigated to improve the scope of the method to membrane-bound enzymes. Five different precipitation agents together with three different cross-linkers were investigated for immobilization of the Trametes versicolor laccase as cross-linked nano-aggregates. Although complete activity recovery was possible for micro-aggregates, the best activity results for nano-aggregates were 40.5%±5.0. The Km values of the immobilized enzymes were slightly lower than the Km values of the free counterparts which indicate little or no mass transfer limitation due to the nano-immobilization process. However, Vmax values were also lower. The activity loss and Vmax reduction upon immobilization were found to mainly result from the modification of the amine groups instead of excess crosslinking. The thermal stabilities of the crosslinked laccase nano-aggregates were significantly higher (∼10–30 fold at 60 °C) compared to free laccase and the nano-CLEAs retained up to 30% of their initial activities upon 7 consequent usages. The sizes of the obtained immobilized enzyme products were found to be greatly variable depending on the cross-linker type. The smaller particles (∼200 nm radius) were obtained when EDAC was used as the cross-linker. The larger products (∼600 nm radius) were prepared when the cross-linker was dextran poly-aldehyde. The addition order of the reagents was found to be effective on particle size and thermal stability values.

Immobilization of Enzymes on Flexible Tubing Surfaces for Continuous Bioassays.

Immobilized enzymes can be used to catalyze biochemical reactions in a batch process, however, it is more difficult to use them in a continuous process. Herein, we develop an enzyme immobilization technique for flexible tubing surfaces, which can be used to catalyze biochemical reactions in a continuous process. In this technique, the tubing is first treated with (3-aminopropyl)triethoxysilane at 50 °C and baked at 100 °C in vacuum to form a network of reactive amine functional group on the inner tubing surface. Subsequently, dextran polyaldehyde, a polymeric cross-linker, is used to immobilize crude protease extract and catalase for hydrolyzing casein and degrading H2O2, respectively, in a continuous process. The immobilized proteases are highly stable even after a long-term storage at 4 °C. After 12 weeks of storage, 90% of the original protease activity can be preserved. Meanwhile, the immobilized catalase is able to degrade 0.1% H2O2 solution flowing at 5 μL/min. The immobilization technique is potentially useful for bioassays and industrial wastewater treatments when continuous processes are preferred.

Chitosan-immobilized pectinolytics with novel catalytic features and fruit juice clarification potentialities

Biological macromolecules are primarily composed of complex polysaccharides that strengthen microbial growth for the production of industrially relevant enzymes. The presence of polysaccharides in the form of the disrupted cell wall and cell materials are among major challenges in the fruit juice industry. The breakdown of such biological macromolecules including cellulose and pectin is vital for the juices processing. In this background, pectinolytic enzymes including polygalacturonase (PG), pectin lyase (PL), and pectin methylesterase (PME) were isolated from Aspergillus ornatus, statistically optimized and purified via ammonium sulfate fractionation (ASF), dialysis, and Sephadex G-100 gel permeation chromatography. After passing through Sephadex G-100 column, PG, PL, and PME were 2.60-fold, 3.30-fold, and 4.52-fold purified with specific activities of 475.2U/mg, 557.1U/mg, and 205.7U/mg. The active PG, PL, and PME, each separately, were surface immobilized using various concentrations of chitosan and dextran polyaldehyde as a macromolecular crosslinking agent. Prior to exploit for juice clarification purposes, various parameters including pH, thermal and Michaelis-Menten kinetic constants of purified and chitosan-immobilized fractions were investigated. A considerable improvement in the pH and thermal profiles was recorded after immobilization. However, the negligible difference between the Km and Vmax values of purified free and chitosan-immobilized fractions revealed that the conformational flexibility of pectinolytics was retained as such. A significant color and turbidity reductions were recorded after 60min treatment with CTS-PG, followed by CTS-PME, and CTS-PL. It can be concluded that the clarification of apples, mango, peach, and apricot juices was greatly affected by CTS-PG, CTS-PME, and CTS-PL treatments rendering them as potential candidatures for food industry applications.

Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker

Pectinase was immobilized onto chitosan magnetic nanoparticles (CMNPs) by dextran polyaldehyde as a macromolecular cross-linking agent. The parameters like cross-linking concentration, time and CMNPs to enzyme ratio were optimized. Further, prepared magnetic pectinase nanobiocatalyst was characterized by FT-IR and XRD. The thermal kinetic studies for immobilized pectinase showed two folds improved thermal stability in the range of 55–75°C as compared to free form. The Vmax and Km values of immobilized pectinase were found to be nearly equal to native form which indicated that conformational flexibility of pectinase was retained even after immobilization. The residual activity of immobilized pectinase was 85% after seven successive cycles of reuse, while it retained upto 89% residual activity on storage of fifteen days which exhibited excellent stability and durability. The conformational changes in pectinase after immobilization were evaluated by FT-IR spectroscopy data analysis tools. Finally, magnetic pectinase nanobiocatalyst was employed for apple juice clarification which showed turbidity reduction upto 74% after 150min treatment.

Preparation of cross-linked enzyme aggregates of nitrile hydratase ES-NHT-118 from E. coli by macromolecular cross-linking agent

Cross-linked enzyme aggregates (CLEAs) of nitrile hydratase (NHase) ES-NHT-118 from Escherichia coli were prepared by using ammonium sulfate as precipitating agent followed by cross-linking with dextran polyaldehyde for the first time. In this process, egg white was added as protein feeder for facilitating the formation of CLEAs. The optimal conditions of the immobilization process were determined. Michaelis constants (Km) of free NHase and NHase CLEAs were also determined. The NHase CLEAs exhibited increased stability at varied pH and temperature conditions compared to its free counterpart. When exposed to high concentrations of acrylamide, NHase CLEAs also exhibited effective catalytic activity.

Исследование острой токсичности конъюгата полиальдегиддекстрана с гидразидом изоникотиновой кислоты

Проведено исследование острой токсичности противотуберкулезной фармацевтической субстанции – конъюгата полиальдегиддекстрана с гидразидом изоникотиновой кислоты (декстразид). Исследования проводились на животных и культурах клеток линий HepaRG и фибробластах кожи человека FRSN. В результате исследования были определенны среднепереносимые (ЛД10, ЛД16), среднесмертельные (ЛД50) и летальные (ЛД84) дозы декстразида при внутрибрюшинном введении мышам и крысам и при внутривенном введении кроликам.

Immobilisation of Aspergillus oryzae α-amylase and Aspergillus niger glucoamylase enzymes as cross-linked enzyme aggregates

AbstractCross-linked enzyme aggregates (CLEA) of Aspergillus oryzea α-amylase (AoAA) and Aspergillus niger glucoamylase (AnGA) were prepared using glutaraldehyde and dextran polyaldehyde as crosslinkers. The maximum activity recoveries for glutaraldehyde cross-linking were 21.8 % and 41.2 %, respectively. The addition of a proteic feeder (bovine serum albumin) exhibited a negative effect on the activity recoveries for both enzymes. Dextran polyaldehyde was used as a cross-linking agent instead of glutaraldehyde to reduce the activity losses. As a result, an activity recovery of 60.0 % was obtained for Aspergillus oryzea α-amylase. On the other hand, no activity recovery was observed for Aspergillus niger glucoamylase due to the latter enzyme’s affinity for dextran.

Formation of Nitrile Hydratase Cross-Linked Enzyme Aggregates in Mesoporous Onion-like Silica: Preparation and Catalytic Properties

Nitrile hydratase cross-linked enzyme aggregates (CLEAs) were formed in mesoporous onion-like silica (NHase-CLEAs@MOS) by using macromolecular dextran polyaldehyde as a cross-linker through the carrier-bound CLEAs method. The effect of preparation parameters on the recovery of enzyme activity was investigated. The properties such as pH, thermal and storage stability, and kinetic parameters of NHase-CLEAs@MOS were also studied. The maximum amount of NHase absorbed in MOS was 535 mg/g. Under optimized conditions, the maximum activity recovery of NHase-CLEAs@MOS was 48.2%. The stabilities of NHase-CLEAs@MOS were improved significantly compared to the NHase@MOS prepared by physical adsorption and free NHase. This work demonstrated that the mesoporous onion-like silica can be efficiently employed as host materials for NHase immobilization, and the carrier-bound CLEAs method can lead to enhanced activity and stability of the immobilized enzymes.

Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates

We employed a cross-linked enzyme aggregate (CLEA) method to immobilize formate dehydrogenase (FDH) from Candida boidinii. The optimal conditions for the preparation of CLEAs were determined by examining effects of various parameters: the nature and amount of cross-linking reagent, additive concentration, cross-linking time, and pH during CLEA preparation. The recovered activities of CLEAs were significantly dependent on the concentration of glutaraldehyde; however, the recovered activity was not severely influenced by the content of dextran polyaldehyde as a mild cross-linker. Bovine serum albumin (BSA) was also used as a proteic feeder and enhanced the activity recovery by 130%. The highest recovered activity of CLEA was 18% for formate oxidation reaction and 25% for CO2 reduction reaction. The residual activity of CLEA prepared with dextran polyaldehyde (Dex-CLEA) was over 95% after 10 cycles of reuse. The thermal stability of Dex-CLEA was increased by a factor of 3.6 more than that of the free enzyme. CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO2 reduction.

Preparation of β-mannanase CLEAs using macromolecular cross-linkers

Cross-linked enzyme aggregates (CLEAs) of β-mannanase were prepared by precipitation and subsequent cross-linking. The macromolecular cross-linkers (dialdehyde starch and dextran polyaldehyde) with different molecular weight were developed instead of traditional glutaraldehyde so as to improve the activity of CLEAs toward macromolecular substrates. Scanning electron microscopy revealed that CLEAs prepared using linear dextran polyaldehyde (MW = 2000 kDa) presented a porous structure with low steric hindrance, and thus exhibited excellent activity toward macromolecular substrates, which was 16 times higher than that prepared using glutaraldehyde.

Method for determining the number of aldehydes in dextran polyaldehyde using 2,4-dinitrophenylhydrazine

A method for determining the number of aldehydes in dextran polyaldehyde (DPA) was proposed. The method was based on the production of the DPAconjugate with 2,4-dinitrophenylhydrazine, reaction of which with base formed a colored product. The developed method for quantitative determination of aldehydes was distinguished from others by its simplicity and high sensitivity. Results of physicochemical studies of DPA and their derivatives were presented in order to identify their structures and formation mechanism in the presence of KMnO4.

Cross-Linked Enzyme Aggregates as Industrial Biocatalysts

The immobilization of enzymes as cross-linked enzyme aggregates (CLEAs) and their applications in industrially relevant biotransformations are reviewed. The preparation of CLEAs involves precipitation from aqueous buffer followed by cross-linking with a bifunctional reagent, usually a dialdehyde such as glutaraldehyde or dextran polyaldehyde. The technique is exquisitely simple and broadly applicable. CLEAs have several benefits in the context of industrial biocatalysis. They have a significantly enhanced shelf life and operational stability, are easy to recover and reuse, and are completely stable towards leaching in aqueous media. They circumvent the use of, often expensive, carriers and have high volumetric and catalyst productivities as a result of the absence of noncatalytic ballast. The applications of CLEAs for a wide variety of enzymes, including various types of hydrolases, oxidoreductases, and lyases in industrially relevant biotransformations, are discussed. Co-aggregation and cross-linking of two or more enzymes affords combi-CLEAs that are ideally suited for catalyzing one-pot, enzymatic cascade processes, for example, the successful application of a triple-decker combi-CLEA comprising a hydroxynitrile lyase, a nitrilase, and an amidase in the one-pot conversion of benzaldehyde to S-mandelic acid in >99% ee at 96% conversion. Finally, reactor concepts are discussed in the context of industrial applications.

Dextran Coatings for Aggregation Control of Layer-by-Layer Assembled Polyelectrolyte Microcapsules

We propose dextran and dextran polyaldehyde (DPA) coatings for modification of layer-by-layer (LbL) assembled polyelectrolyte microcapsules which provide stability against aggregation in 0.75 M aqueous solutions of mono- and bivalent ions (Na(+), Cl(-), Ca(2+), HPO(4)(2-)). The microcapsules were prepared of three bilayers of poly(4-styrenesulfonate) (PSS) and poly(allylamine) (PAH). Dextran and its derivatives were attached to amino-terminated surface of the microcapsules via three types of chemical bonds of subsequently increasing strength: (1) hydrogen bonds, (2) hydrolyzable covalent cross-links resulting from aldehydes and primary amines coupling, and (3) nonhydrolyzable covalent C-N single bonds of secondary amines. Attachment of the DPA materials via the latter two types of bonds resulted in strengthening the capsules' walls which preserved a fraction of the microcapsules from disintegration upon electrostatic swelling in 0.1 M NaOH. The non-disintegrated fraction of the DPA-coated microcapsules restored their initial size after pH was decreased back to neutral. The microcapsules coated with the original dextran immobilized via hydrogen bonds and the bare microcapsules were fully dissolved under the alkaline conditions. The preserved fraction of the microcapsules was higher for the DPA materials with higher contents of the aldehyde groups and after conversion of the hydrolyzable covalent cross-links to the nonhydrolyzable secondary amines via reduction with NaBH(4). The higher contents of the aldehyde groups and the reduction led to the lower limiting swelling degree of the DPA-coated microcapsules at alkaline pH. The proposed coatings can be used for colloid stabilization of polyelectrolyte microcapsules in aqueous medium, encapsulation of pH-insensitive macromolecules at the postpreparation stage, and pH-triggered release of encapsulated material.