Potential and Scale-Up of Pore-Through-Flow Membrane Reactors for the Production of Prebiotic Galacto-Oligosaccharides with Immobilized β-Galactosidase

The production of prebiotics like galacto-oligosaccharides (GOS) on industrial scale is becoming more important due to increased demand. GOS are synthesized in batch reactors from bovine lactose using the cost intensive enzyme β-galactosidase (β-gal). Thus, the development of sustainable and more efficient production strategies, like enzyme immobilization in membrane reactors are a promising option. Activated methacrylatic monoliths were characterized as support for covalent immobilized β-gal to produce GOS. The macroporous monoliths act as immobilized pore-through-flow membrane reactors (PTFR) and reduce the influence of mass-transfer limitations by a dominating convective pore flow. Monolithic designs in the form of disks (0.34 mL) and for scale-up cylindric columns (1, 8 and 80 mL) in three different reactor operation configurations (semi-continuous, continuous and continuous with recirculation) were studied experimentally and compared to the free enzyme system. Kinetic data, immobilization efficiency, space-time-yield and long-term stability were determined for the immobilized enzyme. Furthermore, simulation studies were conducted to identify optimal operation conditions for further scale-up. Thus, the GOS yield could be increased by up to 60% in the immobilized PTFRs in semi-continuous operation compared to the free enzyme system. The enzyme activity and long-time stability was studied for more than nine months of intensive use.

Tailoring surface properties of functionalized graphene papers aiming to enzyme immobilization

The use of enzymes as catalysts requires recovery and reuse to make the process viable. Enzymatic immobilization changes enzyme stability, activity, and specificity. It is very important to explore new substrates for immobilization with appropriate composition and structure to improve the efficiency of the immobilized enzymes. This work explores the use of two different graphene oxide papers, one produced by oxidation route (GO) and the other by electrochemical synthesis (EG), aiming for β-galactosidase immobilization. The chemical and structural properties of these two papers were characterized by Raman spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction. Atomic force microscopy images showed that EG paper ensured more efficient immobilization of the enzymes on the surface of the paper. Cyclic voltammetry was used to monitor the reaction of conversion of lactose to glucose in the free enzyme solution and graphene paper immobilized enzyme solutions. The cyclic voltammetry analysis showed that immobilized enzymes on GO paper showed an improvement in the activity of β-galactose when compared to free enzyme solution, as well as enzyme immobilized on a glassy carbon electrode.

Immobilization of Enzymes by Polymeric Materials

Enzymes are the highly efficient biocatalyst in modern biotechnological industries. Due to the fragile property exposed to the external stimulus, the application of enzymes is highly limited. The immobilized enzyme by polymer has become a research hotspot to empower enzymes with more extraordinary properties and broader usage. Compared with free enzyme, polymer immobilized enzymes improve thermal and operational stability in harsh environments, such as extreme pH, temperature and concentration. Furthermore, good reusability is also highly expected. The first part of this study reviews the three primary immobilization methods: physical adsorption, covalent binding and entrapment, with their advantages and drawbacks. The second part of this paper includes some polymer applications and their derivatives in the immobilization of enzymes.

Metal ion effects on Polyphenol Oxidase Covalently immobilized on a Bio-Composite

Biosensors can be developed using different immobilization methods. Interest in immobilization methods have increased because biosensors have been important for science. Polyphenol oxidase (PPO) was used generally in biosensor applications. For this purpose, Polyphenol oxidase from banana was purified and covalently immobilized on chitosan-gelatin bio-composite. The properties of immobilized enzyme were investigated and compared to free enzyme. Various parameters were studied such as pH, temperature and storage stability on immobilized and free enzyme. Kinetic parameters were also evaluated by different substrates on immobilized and free enzyme. Catechol was determined the best substrate for immobilized enzyme with optimum condition. In vitro effects of metal ions were studied on immobilized enzyme. Concentration range of metal ions is 1.0-10.0 x10-6 mol/L. The activity of immobilized PPO was increased by Fe+2 and Ag+1 ion. Co+3 and Cu+1 had very strong inhibitory effects with IC50 values of 19.69x10-3 mol/L and 23.49 x10-3 mol/L, respectively. Inhibition constants (Ki) and inhibition types of metal ions were determined with immobilized enzyme. Zn+2 and Cr+3 ions were showed competitive inhibition and Pb+2 ions were determined non-competitive inhibition with immobilized enzyme. Mixed type inhibition was obtained with Co+3 ion using catechol as substrate with 3.33x10-5 mol/L Ki value on immobilized PPO. Immobilized PPO can be evaluated for biosensor for the purpose of measurements of metal ions.

Immobilization of Fusarium solani Cutinase onto Magnetic Genipin-Crosslinked Chitosan Beads

Genipin was used as a crosslinking agent to prepare magnetic genipin-crosslinked chitosan beads, which were then used as a carrier for immobilizing recombinant cutinase from Fusarium solani (FSC) to obtain immobilized FSC. The optimal temperature for the immobilized FSC was 55 °C, which was 5 °C higher than that of the free enzyme, whereas its optimal pH was increased from 8.0 to 9.0; this indicates that the immobilized FSC had improved pH and thermal stability. After repeated use for 10 cycles, the activity of the immobilized FSC remained at more than 50%; after being stored at 4 °C for 30 days, its activity was still approximately 88%. We also found that the Km of the immobilized FSC was higher than that of the free enzyme. These results indicate that the performance of FSC was improved after immobilization, which is an important basis for the subsequent application of FSC in industrial production.

Immobilization of Phospholipase A1 Using a Protein-Inorganic Hybrid System

In this study, four kinds of phospholipase A1-metal (Al/Co/Cu/Mn) hybrid nanostructures were prepared for enhancing the stability of the free PLA1. The formed hybrid complexes were characterized by scanning electron microscope (SEM), Fourier infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The stability and substrate specificity of immobilized enzymes were subsequently determined. After immobilization, the temperature tolerance of PLA1–metal hybrid nanostructures was enhanced. The relative activity of PLA1–Al/Co/Cu hybrid nanostructures remained above 60% at 50 °C, while that of free enzyme was below 5%. The thermal transition temperature measured by differential scanning calorimetry (DSC) was found to increase from 65.59 °C (free enzyme) to 173.14 °C, 123.67 °C, 96.31 °C, and 114.79 °C, referring to PLA1–Cu/Co/Al/Mn hybrid nanostructures, respectively. Additionally, after a storage for fourteen days at 4 °C, the immobilized enzymes could exhibit approximately 60% of the initial activity, while the free PLA1 was inactivated after four days of storage. In brief, using Co2+, Cu2+, Al3+, and Mn2+ as the hybridization materials for immobilization could improve the catalytic properties and stability of the free PLA1, suggesting a promising method for a wider application of PLA1 in many fields such as food, cosmetics, and the pharmaceutical industry.

Immobilization of Mutant Phosphotriesterase on Fuller’s Earth Enhanced the Stability of the Enzyme

Immobilization is a method for making an enzyme more robust in the environment, especially in terms of its stability and reusability. A mutant phosphotriesterase (YT PTE) isolated from Pseudomonas dimunita has been reported to have high proficiency in hydrolyzing the Sp and Rp-enantiomers of organophosphate chromophoric analogs and therefore has great potential as a decontamination agent and biosensor. This work aims to investigate the feasibility of using Fuller’s earth (FE) as a YT PTE immobilization support and characterize its biochemical features after immobilization. The immobilized YT PTE was found to show improvement in thermal stability with a half-life of 24 h compared to that of the free enzyme, which was only 8 h. The stability of the immobilized YT PTE allowed storage for up to 4 months and reuse for up to 6 times. The immobilized YT PTE showed high tolerance against all tested metal ions, Tween 40 and 80 surfactants and inorganic solvents. These findings showed that the immobilized YT PTE became more robust for use especially with regards to its stability and reusability. These features would enhance the future applicability of this enzyme as a decontamination agent and its use in other suitable industrial applications.

Fungal α-amylase entrapped in agar-agar organic matrix “beads” enhances fabric starch-desizing potentials and α-amylase-detergent compatibility

Aureobasidium pullulans α-amylase (ApAmy) mixed with melted agar-agar solution and drop-wisely added to a mixture of organic solvent solution allowed for the entrapment of the α-amylase in the agar-agar organic matrix as beads. The immobilized ApAmy’s characteristics and wash performance were elucidated in comparison with the soluble ApAmy. Agar-agar at 2.0 % (w/v) and toluene: chloroform at 3:1 resulted in the highest immobilization yield retaining about 98% residual activity after ten catalytic cycles. The optimum temperature and pH for the immobilized enzyme were 60ºC and 6.5 respectively. The immobilized ApAmy hydrolysed branched and linear substrates thus establishing its broad substrate specificity. Relatively, the immobilized ApAmy (iApAmy) was more tolerant to organic solvents than the free enzyme. The iApAmy was mildly inhibited by cobalt but metals such as zinc, manganese, calcium and sodium enhanced the free and immobilized ApAmy activity. The iApAmy had a higher washing efficiency (77%) in the presence of detergents than the free enzyme (68%) and control (36%). The iApAmy showed good potentials as a detergent additive and from its characteristics, it could be useful in other industrial applications.

Immobilization of Catalase on Chitosan/ZnO and Chitosan/ZnO/Fe2O3 Nanocomposites: A Comparative Study

The strong catalytic performance, eco-friendly reaction systems, and selectivity of enzyme-based biocatalysts are extremely interesting. Immobilization has been shown to be a good way to improve enzyme stability and recyclability. Chitosan-incorporated metal oxides, among other support matrices, are an intriguing class of support matrices for the immobilization of various enzymes. Herein, the cross-linked chitosan/zinc oxide nanocomposite (CS/ZnO) was synthesized and further improved by adding iron oxide (Fe2O3) nanoparticles. The final cross-linked CS/ZnO/Fe2O3 nanocomposite was used as an immobilized support for catalase and is characterized by SEM, EDS, and FTIR. The nanocomposite CS/ZnO/Fe2O3 enhanced the biocompatibility and immobilized system properties. CS/ZnO/Fe2O3 achieved a higher immobilization yield (84.32%) than CS/ZnO (37%). After 10 repeated cycles, the remaining immobilized catalase activity of CS/ZnO and CS/ZnO/Fe2O3 was 14% and 45%, respectively. After 60 days of storage at 4 °C, the remaining activity of immobilized enzyme onto CS/ZnO and CS/ZnO/Fe2O3 was found to be 32% and 47% of its initial activity. The optimum temperature was noticed to be broad at 25–30 °C for the immobilized enzyme and 25 °C for the free enzyme. Compared with the free enzyme optimum pH (7.0), the optimum pH for the immobilized enzyme was 7.5. The Km and Vmax values for the free and immobilized enzyme on CS/ZnO, and the immobilized enzyme on CS/ZnO/Fe2O3, were found to be 91.28, 225.17, and 221.59 mM, and 10.45, 15.87, and 19.92 µmole ml−1, respectively. Catalase immobilization on CS/ZnO and CS/ZnO/Fe2O3 offers better stability than free catalase due to the enzyme’s half-life. The half-life of immobilized catalase on CS/ZnO/Fe2O3 was between 31.5 and 693.2 min.