Hydroxytyrosol (HT) is a novel functional pharmaceutical or food ingredient that can be prepared using tyrosinase (TYR) as the catalyst. In this study, cross-linked agarose resin (AG-NTA-Ni2+) was ...selected as the carrier for the one-step immobilization and purification of the enzyme via specific adsorption of the His-tag on the protein and metal ions on the carrier particles. The immobilized TYR exhibited excellent thermal and pH stabilities and retained 82.3% of its relative activity after seven cycles. Using different reduction methods, immobilized TYR was used as a catalyst to synthesize HT via the controlled oxidation of tyrosol. The enzymatic cascade reaction of co-immobilized TYR and glucose dehydrogenase (GDH) as a biocatalyst served as a bioreduction method with an HT yield of 88%. For the chemical reduction using ascorbic acid (AA) as the reducing agent, an 80% HT yield was achieved. These results indicate that the immobilization method, which relies on the specific coordination between transition metal ions and certain protein amino acids, is a potential strategy for the effective co-immobilization of TYR and GDH. The proposed affinity co-immobilization technique is among the potential strategies for the efficient production of HT.
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•Co-immobilization of tyrosinase and glucose dehydrogenase for efficient synthesis of hydroxytyrosol (HT).•Affinity adsorption endows immobilized bienzyme with satisfactory activity and stability.•Conversion of HT prepared by multi-enzyme cascade reduction outperforms chemical reduction process.
Artificial Compartmentalized Cascade Systems
In article number 2300185, Wangsuk Oh, Dawoon Jeong, and Ji‐Woong Park use a membrane with reticulated nanopores to confine one enzyme and create a ...compartment for another enzyme that allow for a cascade of biocatalytic reactions. This membrane acts as both a bioactive barrier and a means of molecular transport between the internal and external media, facilitating the reactions.
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•The physical attachment of Hyal on PAMAM surface could significantly improve HA degradation.•Immobilized Hyal showed high loading capacity, catalytic activity, storage stability and ...thermal stability.•The inhibitory effect of ascorbic acid on PAMAM conjugated Hyal and native Hyal was compared.•The combining Hyal and PAMAM has a wide prospect for its applications in biomedical and bio-industries.
In this work hyper-branched poly (amide amine) (PAMAM) nanoparticles were conjugated with hyaluronidase (Hyal) to produce a robust nano-biocatalyst for hyaluronic acid (HA) degradation. The success enzyme attachment process was confirmed by Fourier transform infrared (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV–Vis. The influence of pH, temperature, and inhibitor on the enzymatic activity of hyaluronidase was also investigated. The optimum pH, temperature and storage time of Hyal-PAMAM nanocomplex were higher than free enzyme. Also, ascorbic acid showed more inhibitory effect on free enzyme, the IC50 values were determined to be around 55 ± 0.7 and 70 ± 0.3 mM for free Hyal and Hyal-PAMAM nanocomplex, respectively. Based on the greater υmax and lower Km, the Hyal-PAMAM showed a better catalytic efficiency for HA degradation. Moreover, the in silico screening of PAMAM/Hyal interactions further confirmed the experimental results. The novel strategy for combining Hyal and PAMAM dendrimer can hold great promise for applications in biomedical, sensing, and industrial catalysis.
Aspergillus oryzae β-D-galactosidase (β-Gal) efficiently hydrolyzes sesaminol triglucoside into sesaminol, which has higher biological activity. However, β-Gal is difficult to be separate from the ...reaction mixture and limited by stability. To resolve these problems, β-Gal was immobilized on amino-functionalized magnetic nanoparticles mesoporous silica pre-activated with glutaraldehyde (Fe3O4@mSiO2-β-Gal), which was used for the first time to prepare sesaminol. Under the optimal conditions, the immobilization yield and recovered activity of β-Gal were 57.9 ± 0.3 % and 46.5 ± 0.9 %, and the enzymatic loading was 843 ± 21 Uenzyme/gsupport. The construction of Fe3O4@mSiO2-β-Gal was confirmed by various characterization methods, and the results indicated it was suitable for heterogeneous enzyme-catalyzed reactions. Fe3O4@mSiO2-β-Gal was readily separable under magnetic action and displayed improved activity in extreme pH and temperature conditions. After 45 days of storage at 4 °C, the activity of Fe3O4@mSiO2-β-Gal remained at 92.3 ± 2.8 %, which was 1.29 times than that of free enzyme, and its activity remained above 85 % after 10 cycles. Fe3O4@mSiO2-β-Gal displayed higher affinity and catalytic efficiency. The half-life was 1.41 longer than free enzymes at 55.0 °C. Fe3O4@mSiO2-β-Gal was employed as a catalyst to prepare sesaminol, achieving a 96.7 % conversion yield of sesaminol. The excellent stability and catalytic efficiency provide broad benefits and potential for biocatalytic industry applications.
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•The amino-functionalized magnetic mesoporous silica was pre-activated by glutaraldehyde.•Aspergillus oryzae β-D-galactosidase was successfully immobilized.•The immobilized enzyme displayed enhanced stability, substrate affinity, and recycling compared to the free enzyme.•The immobilized enzyme exhibited remarkable catalytic ability in the preparation of sesaminol.
An immobilized lipase with metal‐organic frameworks (MOFs) was first used for the kinetic resolution of mandelic acid enantiomers by transesterification reaction using vinyl acetate as acyl donor in ...organic medium. Notably, the biocompatibility of the carrier and catalytic performance of lipase could be significantly improved via modification of NH2‐Ni‐MOF with polyvinylpyrrolidone. After extensively screening the enzymatic process, the optimal enantioselective resolution conditions and excellent outcomes were obtained for the enzyme‐catalyzed transesterification reaction. In addition, the recovered immobilized lipase displayed outstanding reusability after immobilization.
Polyvinylpyrrolidone‐modified NH2‐Ni‐metal organic framework was used as carrier for immobilization of Pseudomonas fluorescens lipase. The immobilized lipase was applied to the resolution of mandelic acid enantiomers by kinetic transesterification with vinyl acetate in organic medium. The biocompatibility of the carrier, catalytic performance, and reusability of lipase could be significantly improved.
Biomineralization is a natural process of mineral formation mediated by biomacromolecules, allowing access to hierarchical structures integrating biological, chemical, and material properties. In ...this contribution, we comprehensively investigate the biomineralization of zeolite imidazole frameworks (ZIFs) for one-step synthesis of an enzyme-MOF biocomposite, in terms of differential crystallization behaviors, fine microstructure of resultant ZIF biominerals, the enzyme's conformation evolution, and protective effect of ZIF mineral. We discover that the biomineralization ability is ZIF organic linker dependent and the biocatalytic function is highly related to the ZIF mineral species and their distinguishable topologies and defect structures. Importantly, a side-by-side analysis suggests that the protective effect of ZIF mineral toward the hosted enzyme is highly associated with the synergistic effect of size dimension and chemical microenvironment of the ZIF pores. This work provides important insight into the ZIF-dependent biomineralization behaviors and highlights the important role of the ZIF microstructure in its biocatalytic activity and durability, which has been underestimated previously.
This report describes an efficient procedure for enzyme encapsulation and its application for the hydrolysis of lactose. The enzymatic material that has been developed consists of hydrogel particles ...(ca. 3–4 mm of diameter) composed of either alginate or an alginate-agarose combination, in which bacterial cells loaded with a thermostable β-galactosidase are embedded. The cells were rendered fully permeable to the substrate, either chromogenic p-nitrophenyl galactose or lactose, by thermal treatment at 75 °C. Hydrogel particles made of a mixture of alginate and agarose displayed high catalytic activity (i.e. 1 g of beads hydrolyze the lactose equivalent of 100 mL of milk in 15 min) and thermal stability: they could be reused at 75 °C for complete hydrolysis of 5% (w/v) lactose solution at least six consecutive times without significant loss of activity.
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•Agarose-alginate hydrogel beads with lactose hydrolysis activity were prepared.•Bacterial cells loaded with a thermostable β-galactosidase were the core material.•Zymoactive beads can be operated at high temperature (75 °C).•Hydrogel beads can be reused at least six times without significant loss of activity.•1 g of beads hydrolyze the lactose equivalent of 100 mL of milk in 15 min.
Protein bioinformatics has been applied to a myriad of opportunities in biocatalysis from enzyme engineering to enzyme discovery, but its application in enzyme immobilization is still very limited. ...Enzyme immobilization brings clear advantages in terms of sustainability and cost‐efficiency but is still limited in its implementation. This, because it is a technique that remains tied to a quasi‐blind protocol of trial and error, and therefore, is regarded as a time‐intensive and costly approach. Here, we present the use of a set of bioinformatic tools to rationalize the results of protein immobilization that have been previously described. The study of proteins with these new tools allows the discovery of key driving forces in the process of immobilization that explain the obtained results, moving us a step closer to the final goal: predictive enzyme immobilization protocols.
Deciphering enzyme immobilization: Using bioinformatic tools in a case‐by‐case study of immobilized enzymes, allows rational decision making to be applied to a process currently linked to a trial‐and‐error approach. Here, we apply these tools to rationalize and investigate the guiding forces that affect a set of enzymes during their immobilization.
Electrospun nanofibers have gained much attention for enzyme immobilization due to their high surface‐to‐volume ratio. In this study, urease was immobilized on chitosan/poly(vinyl alcohol) (PVA) ...nanofibers by both adsorption and crosslinking methods. In order to obtain nanofibers with more desirable properties, solutions with different ratios of chitosan and PVA were electrospun and crosslinked using glutaraldehyde. Comparing SEM images of the nanofibers, before and after immersing them in phosphate buffer, it was shown that higher chitosan content leads to more stable fibers. So, the solution with the chitosan to PVA ratio of 40:60 was used for enzyme immobilization. Then, the effects of initial protein concentration, temperature, incubation time, and method of immobilization were investigated to reach the highest enzyme activity. Under similar immobilization conditions, covalently immobilized urease showed higher activity, compared to uncrosslinked immobilized enzyme. Besides, it retained 30% of its initial activity after 10 times usage. So, this method was chosen for further investigation. Not only the activity of the immobilized enzyme was much higher than the free enzyme in a wide range of pH and temperature, but also stability of the immobilized enzyme was improved. Immobilized urease was then used to remove thiourea which is a toxic compound. Findings indicated 60% hydrolysis of initial thiourea in 12 h. In conclusion, the findings showed that chitosan/PVA nanofibers are suitable candidates for the immobilization of urease.
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•The GICA was immobilized onto amino supports with very high recovery yield.•The adsorbed GICA showed an improvement on its thermo-stability.•An excellent operational stability in ...batch reactor cycles was observed at 85 °C.•A continuous packed bed reactor system was established for HFS production at 70 °C.
The glucose isomerase GICA from Caldicoprobacter algeriensis was immobilized by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The optimum temperature and pH of immobilized GICA were 90 °C and 7.0, respectively, similar to the corresponding values of free enzyme. Nevertheless, the adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3 h reaction cycles at 85 °C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70 °C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48 h of operation.
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