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1.
Small ; 18(10): e2105880, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34989480

RESUMO

Glyconanoparticles (GNPs) made by self-assembly of carbohydrate-based polystyrene-block-ß-cyclodextrin copolymer are used as a building block for the design of nanostructured biomaterials of electrode. The firm immobilization of GNPs is carried out on electrochemically generated polymer, poly(pyrrole-adamantane), and copolymer, poly(pyrrole-adamantane)/poly(pyrrole-lactobionamide) via host-guest interactions between adamantane and ß-cyclodextrin. The ability of GNPs for the specific anchoring of biological macromolecules is investigated using glucose oxidase enzyme modified by adamantane groups as a protein model (GOx-Ad). The immobilization of GOx-Ad is carried out by incubation of an aqueous enzyme solution on a coating of GNPs adsorbed on a platinum electrode. The presence of immobilized GOx-Ad is evaluated in aqueous glucose solution by potentiostating the underlying platinum electrode at 0.7 V/SCE for the electro-oxidation of H2 O2 generated by the enzyme. The analytical performance of the bioelectrodes for the detection of glucose is compared to control electrodes prepared without GNPs or without electropolymerized films. The better permeability of copolymer compared to polymer and the possibility to elaborate two alternating layers of GNPs and GOx-Ad are clearly observed. The best amperometric response is recorded with a multilayered bioelectrode displaying a wide linear range linear range of the calibration curve: 68 µmol L-1 to 0.1 mol L-1 .


Assuntos
Técnicas Biossensoriais , Nanopartículas , beta-Ciclodextrinas , Eletrodos , Enzimas Imobilizadas/química , Glucose/química , Glucose Oxidase/química , Nanopartículas/química , Pirróis/química , beta-Ciclodextrinas/química
2.
Anal Chem ; 94(24): 8580-8587, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35678765

RESUMO

Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 µm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (µIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the µIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed µIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.


Assuntos
Enzimas Imobilizadas , Espectrometria de Massas em Tandem , Cromatografia Líquida , Enzimas Imobilizadas/química , Mapeamento de Peptídeos/métodos , Impressão Tridimensional
3.
Int J Biol Macromol ; 213: 43-54, 2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35644313

RESUMO

Lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida rugosa (CRL), forms A and B of lipase from Candida antarctica (CALA and CALB) and Eversa Transform 2.0 have been immobilized on octyl-agarose beads at two different loads (1 mg/g and saturated support) and treated with phosphate and/or some metallic salts (Zn2+, Co2+, Cu2+). They have been also immobilized on the support modified by the metallic phosphate, usually driving to biocatalyst with lower stability or marginal improvements. The effects of the phosphate/metal modification on enzyme features depended on the loading of the support. Some enzymes (TLL, CRL or CALA), mainly using the highly loaded biocatalysts, showed very significant improvement on enzyme stability after the treatment with some of the metal phosphates (next to a 20-fold factor), improvements that were not justified by the presence of metallic or phosphate ions in solution, as they had negative effects on enzyme stabilities. In some other cases, a significant increase in enzyme activity was detected (e.g., CALB). This could be explained by the modification of the nucleation places of the enzymes by the metallic phosphate, and this could help to explain the good results obtained in the nanoflower immobilization of many enzymes.


Assuntos
Enzimas Imobilizadas , Sais , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Fosfatos
4.
Int J Biol Macromol ; 213: 70-82, 2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35644316

RESUMO

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.


Assuntos
Enzimas Imobilizadas , Engenharia de Proteínas , Aminoácidos , Reagentes para Ligações Cruzadas , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glicosídeo Hidrolases , Temperatura
5.
Methods Mol Biol ; 2487: 133-150, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687233

RESUMO

In recent years, three key techniques including random co-immobilization, positional co-immobilization, and compartmentalization for multi-enzyme immobilization were extensively considered. Herein, we investigate random co-immobilization and positional co-immobilization techniques for multi-enzyme systems in detail. We describe randomly co-immobilized glucose oxidase (GOx) and horseradish peroxidase (HRP) on reduced graphene oxide (rGO) as the most used methods. Materials and methods are presented in terms of preparation of GO and rGO as well as enzyme immobilization procedure. Moreover, the principles of positional co-immobilization have been reviewed, and the relevant methods based on microfluidic systems and DNA structure considering HRP and GOx enzymes have been individually studied. It is believed that the benefits of using the methods associated with random and specifically positional immobilized multi-enzyme systems include not only enhanced cascade enzymatic activity via manipulated surface such as microfluidic systems (including porous materials) and DNA structure but also improved enzyme stability and ease of recovery for recycle.


Assuntos
Enzimas Imobilizadas , Glucose Oxidase , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Microfluídica
6.
Methods Mol Biol ; 2487: 151-162, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687234

RESUMO

The methods of compartmentalized immobilization in multi-enzyme systems containing inorganic complexes and organic scaffolds (i.e. nucleic acid (RNA and DNA), protein and lipid) have been thoroughly investigated. Compartmentalization mostly focuses on dividing individual enzyme(s) into specific location or orientation of the enzymes cooperating in cascade reaction. Organic scaffolds are preferred because of their capability for simultaneous synthesis in biological systems. Besides, the most required methods of horseradish peroxidase (HRP) and glucose oxidase (GOD) enzymes including enzyme activity measurement, enzyme immobilization, removal, and re-hybridization, and enzyme attaching have been provided because they have been extensively applied in multi-enzyme systems. Organic scaffolds have a wide range and properties. Therefore, two methods including dockerin-cohesin linker and nucleotides interaction have been demonstrated for immobilization of enzyme on protein and DNA scaffold, respectively.


Assuntos
Enzimas Imobilizadas , Glucose Oxidase , DNA , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/metabolismo , Peroxidase do Rábano Silvestre/metabolismo
7.
Methods Mol Biol ; 2487: 163-175, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687235

RESUMO

3D printing is lately utilized in biological sciences under the scope to develop customized scaffolds that will host biomolecules, either whole cells or parts of them, like enzymes. In the present work, we present a protocol to modify the surface of 3D printed polylactic acid (PLA) well-plates with the aim to co-immobilize multiple enzymes that will perform cascade reactions. Detailed steps to design and print the final models are described. The developed protocol for surface modification is based on coating with chitosan biopolymer and covalent immobilization of the enzymes ß-glucosidase, glucose oxidase, and peroxidase via glutaraldehyde cross-linking. Enzymatic activity measurements indicative of the catalytic performance of the system are also presented.


Assuntos
Quitosana , Poliésteres , Enzimas Imobilizadas , Impressão Tridimensional
8.
Methods Mol Biol ; 2487: 189-195, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687237

RESUMO

As the vast majority of enzymes are glycosylated, lectins can serve as molecular glues to agglutinate multiple glycoenzymes for preparing multienzyme catalysts in an efficient and biocompatible way. Taking glucose oxidase and horseradish peroxidase as a model cascade, we describe in this protocol the coimmobilization of cascade glycoenzymes through lectin-mediated protein agglutination with and without magnetic nanoparticles as carriers.


Assuntos
Enzimas Imobilizadas , Lectinas , Concanavalina A , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/metabolismo , Peroxidase do Rábano Silvestre , Lectinas/metabolismo
9.
Methods Mol Biol ; 2487: 205-225, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687239

RESUMO

This chapter reports a single-step preparation of nanoarmored bi-enzyme systems assembled on 1-D and 2-D nanomaterials, with glucose oxidase and peroxidase enzymes as model systems for cascade bio-catalysis. This is a simple and facile method to both exfoliate the bulk 1D (carbon nanotubes, CNT) and 2D nanomaterials (α-Zirconium phosphate, α-ZrP) and bind the enzymes in a single step. Exfoliation of the bulk material enhances the accessible surface area of the materials for the enzyme binding, and it also boosts the diffusion of reagents from the bulk phase to the active sites of the bio-catalysts. For example, a mixture of horseradish peroxidase, glucose oxidase, and bovine serum albumin (BSA) were adsorbed on the surfaces of the α-ZrP nanoplates or carbon nanotubes (CNT) as the bulk materials are exfoliated simultaneously, in a one-step process. The resulting bio-catalysts were thoroughly characterized by powder X-ray diffraction, electron microscopy, biochemical and biophysical methods, while enzyme activity studies proved successful binding of enzymes with retention of activities or even enhancements in their specific activities. For example, GOx/HRP/BSA/CNT displayed 6 times the activity of a mixture of GOx/HRP/BSA, under otherwise identical conditions. Similarly, GOx/HRP/BSA/ZrP had 3.5 times the activity of the corresponding mixture of GOx/HRP/BSA, in the absence of the nanoplates. These robust nano-dispersions worked extraordinarily well as active bio-catalysts. These two kinds of fabricated biocatalyst dispersions are also highly stable.


Assuntos
Glucose Oxidase , Nanotubos de Carbono , Catálise , Enzimas Imobilizadas/química , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Soroalbumina Bovina/química
10.
Methods Mol Biol ; 2487: 263-278, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687241

RESUMO

Cascade reactions catalyzed by multi-enzymatic systems have attracted enormous scientific interest over the last decade. They are an emerging technology that significantly expands the applicability of biocatalysts in several biotechnological processes, such as the synthesis of high value-added products. Immobilization of enzymes on a solid carrier is a commonly used strategy to improve the stability and reuse of multiple enzyme systems. Magnetic nanoparticles have been applied as promising nanocarriers for either the immobilization of one enzyme or the co-immobilization of multiple enzymes. In this chapter, we describe the preparation of magnetic iron oxide nanoparticles γ-Fe2O3 modified with 3-(aminopropyl)-triethoxysilane (APTES), for the simultaneous covalent co-immobilization of oxidoreductases and hydrolytic enzymes, such as cellulase, ß-glucosidase (bgl), glucose oxidase (GOx), and horseradish peroxidase (HRP). Several spectroscopic techniques that are used to characterize the structure and the catalytic performance of such systems are also described.


Assuntos
Celulase , Enzimas Imobilizadas , Enzimas Imobilizadas/química , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Oxirredutases
11.
Methods Mol Biol ; 2487: 279-296, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687242

RESUMO

Multi-enzymatic assemblies offer the opportunity of bringing in proximity several enzymes that are enabled to work together for the catalysis of multi-step reactions. Especially, the development of robust nanobiocatalytic systems comprising of several enzymes has gained considerable attention over the last few years for the catalysis of complex reactions and the production of high added-value products. In the present chapter, we describe the methodology for the development of a bi-enzymatic nanobiocatalyst consisting of the enzymes ß-glucosidase from Thermotoga maritima and lipase A from Candida antarctica (CalA) co-immobilized on chitosan-coated magnetic nanoparticles. This nanobiocatalyst can be efficiently applied for the biotransformation of oleuropein to hydroxytyrosol, a reaction of increased biotechnological interest. Several techniques, as well as methodologies that are required for the characterization of the structure and the activity of such systems are also comprehensively described.


Assuntos
Candida , Enzimas Imobilizadas , Biotransformação , Enzimas Imobilizadas/química , Glucosídeos Iridoides , Álcool Feniletílico/análogos & derivados
12.
Molecules ; 27(11)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35684413

RESUMO

As for ligand fishing, the current immobilization approaches have some potential drawbacks such as the small protein loading capacity and difficult recycle process. The core-shell metal-organic frameworks composite (Fe3O4-COOH@UiO-66-NH2), which exhibited both magnetic characteristics and large specific surface area, was herein fabricated and used as magnetic support for the covalent immobilization of porcine pancreatic lipase (PPL). The resultant composite Fe3O4-COOH@UiO-66-NH2@PPL manifested a high loading capacity (247.8 mg/g) and relative activity recovery (101.5%). In addition, PPL exhibited enhanced tolerance to temperature and pH after immobilization. Then, the composite Fe3O4-COOH@UiO-66-NH2@PPL was incubated with the extract of Scutellaria baicalensis to fish out the ligands. Eight lipase inhibitors were obtained and identified by UPLC-Q-TOF-MS/MS. The feasibility of the method was further confirmed through an in vitro inhibitory assay and molecular docking. The proposed ligand fishing technique based on Fe3O4-COOH@UiO-66-NH2@PPL provided a feasible, selective, and effective platform for discovering enzyme inhibitors from natural products.


Assuntos
Lipase , Estruturas Metalorgânicas , Animais , Enzimas Imobilizadas/química , Ligantes , Lipase/química , Fenômenos Magnéticos , Estruturas Metalorgânicas/química , Simulação de Acoplamento Molecular , Ácidos Ftálicos , Extratos Vegetais/farmacologia , Scutellaria baicalensis , Suínos , Espectrometria de Massas em Tandem
13.
J Agric Food Chem ; 70(23): 7139-7147, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35648591

RESUMO

Enzymes are important catalysts for biological processes due to their high catalytic activity and selectivity. However, their low thermal stability limited their industrial applications. The present work demonstrates a simple and effective method for enzyme immobilization via spray drying. Alginate was used as a support material. Phytase, an important enzyme in the animal feed industry, was selected to study the effect of enzyme immobilization using alginate particles on its thermal stability. The physicochemical properties of alginate particles such as size, surface morphology, and heat resistance were studied. Successful immobilization of phytase was confirmed by confocal microscopy, and the immobilized phytase retained 58% of its original activity upon heating at 95 °C, compared to 4% when the alginate support material was absent. Phytase was released promptly in a simulated gastrointestinal tract with >95% of its original activity recovered. The spray drying method for phytase immobilization is scalable and applicable to other enzymes for various applications.


Assuntos
6-Fitase , Alginatos , 6-Fitase/química , Alginatos/química , Animais , Estabilidade Enzimática , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Secagem por Atomização
14.
Carbohydr Polym ; 291: 119483, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35698322

RESUMO

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for ß-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized ß-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.


Assuntos
Alginatos , Aspergillus oryzae , Aspergillus oryzae/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Gelatina , Hidrólise , Iridoides , Lactose/química , Espalhamento a Baixo Ângulo , Difração de Raios X , beta-Galactosidase/metabolismo
15.
Anal Chim Acta ; 1215: 339906, 2022 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-35680335

RESUMO

Full characterization of the attached carbohydrate moieties of glycoproteins is of high importance for both the rapidly growing biopharmaceutical industry and the biomedical field. In this paper we report the design and production of three important 6HIS-tagged exoglycosidases (neuraminidase, ß-galactosidase and hexosaminidase) to support rapid solid phase N-glycan sequencing with high robustness using immobilized enzymes. The exoglycosidases were generated in bacterial expression systems with high yield. Oriented immobilization via the 6HIS-tag portion of the molecules supported easy accessibility to the active sites and consequently high digestion performance. The three exoglycosidases were premixed in an appropriate matrix format and processed in a low-salt buffer to support long term storage. The digestion efficiencies of the immobilized enzymes were demonstrated by using solid phase sequencing in conjunction with capillary electrophoresis analysis of the products on a commercial glycoprotein therapeutic (palivizumab) and human serum derived fluorophore labeled glycans.


Assuntos
Enzimas Imobilizadas , Glicosídeo Hidrolases , Eletroforese Capilar/métodos , Glicoproteínas/química , Glicosídeo Hidrolases/metabolismo , Humanos , Polissacarídeos/análise
16.
Sci Rep ; 12(1): 9297, 2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35662266

RESUMO

A novel approach termed the "concentrated method" was developed for the instant fabrication of laccase@Co3(PO4)2•hybrid nanoflowers (HNFs). The constructed HNFs were obtained by optimizing the concentration of cobalt chloride and phosphate buffer to reach the highest activity recovery. The incorporation of 30 mM CoCl2 and 160 mM phosphate buffer (pH 7.4) resulted in a fast anisotropic growth of the nanomaterials. The purposed method did not involve harsh conditions and prolonged incubation of precursors, as the most reported approaches for the synthesis of HNFs. The catalytic efficiency of the immobilized and free laccase was 460 and 400 M-1S-1, respectively. Also, the enzymatic activity of the prepared biocatalyst was 113% of the free enzyme (0.5 U mL-1). The stability of the synthesized HNFs was enhanced by 400% at pH 6.5-9.5 and the elevated temperatures. The activity of laccase@Co3(PO4)2•HNFs declined to 50% of the initial value after 10 reusability cycles, indicating successful immobilization of the enzyme. Structural studies revealed a 32% increase in the α-helix content after hybridization with cobalt phosphate, which improved the activity and stability of the immobilized laccase. Furthermore, the fabricated HNFs exhibited a considerable ability to remove moxifloxacin as an emerging pollutant. The antibiotic (10 mg L-1) was removed by 24% and 75% after 24 h through adsorption and biodegradation, respectively. This study introduces a new method for synthesizing HNFs, which could be used for the fabrication of efficient biocatalysts, biosensors, and adsorbents for industrial, biomedical, and environmental applications.


Assuntos
Lacase , Nanoestruturas , Catálise , Cobalto , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Lacase/química , Nanoestruturas/química , Fosfatos/química
17.
Talanta ; 248: 123592, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-35671549

RESUMO

In clinical practice, sera creatinine level is regarded as a crucial biomarker for the diagnosis, staging and monitoring of kidney disease. An amperometric biosensor is rapid, accurate, and cost-effective, with a portability and a simple operation. Herein, we report for the firsttime a disposable, printed amperometric biosensor for the clinical evaluation of creatinine in renal function detection. The sensor is constructed based on Prussian blue/carbon-graphite paste as the working electrode and the immobilization of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase. The creatinine biosensor shows a linear detection range from 0.05 to 1.4 mM with a detection time of about 3 min. In addition, the sensor shows a high stability that can maintain above 86% of the initial activity after being stored for over 4 months. Moreover, the sensor shows almost the same results as those with the Jaffe method for measuring the real blood samples. We anticipate that the creatinine biosensor could be widely used in the medical and healthcare areas, especially for at-home testing and onsite medical examinations.


Assuntos
Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Creatinina , Eletrodos , Enzimas Imobilizadas , Rim/fisiologia , Sarcosina Oxidase
18.
Microb Cell Fact ; 21(1): 108, 2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35655182

RESUMO

BACKGROUND: Catalytically active inclusion bodies (CatIBs) are known for their easy and cost efficient production, recyclability as well as high stability and provide an alternative purely biological technology for enzyme immobilization. Due to their ability to self-aggregate in a carrier-free, biodegradable form, no further laborious immobilization steps or additional reagents are needed. These advantages put CatIBs in a beneficial position in comparison to traditional immobilization techniques. Recent studies outlined the impact of cooperative effects of the linker and aggregation inducing tag on the activity level of CatIBs, requiring to test many combinations to find the best performing CatIB variant. RESULTS: Here, we present the formation of 14 glucose dehydrogenase CatIB variants of Bacillus subtilis, a well-known enzyme in biocatalysis due to its capability for substrate coupled regeneration of reduced cofactors with cheap substrate glucose. Nine variants revealed activity, with highest productivity levels for the more rigid PT-Linker combinations. The best performing CatIB, BsGDH-PT-CBDCell, was characterized in more detail including long-term storage at -20 °C as well as NADH cofactor regeneration performance in repetitive batch experiments with CatIB recycling. After freezing, BsGDH-PT-CBDCell CatIB only lost approx. 10% activity after 8 weeks of storage. Moreover, after 11 CatIB recycling cycles in repetitive batch operation 80% of the activity was still present. CONCLUSIONS: This work presents a method for the effective formation of a highly active and long-term stable BsGDH-CatIB as an immobilized enzyme for robust and convenient NADH regeneration.


Assuntos
Enzimas Imobilizadas , NAD , Biocatálise , Enzimas Imobilizadas/química , Corpos de Inclusão/metabolismo , NAD/metabolismo , Oxirredução
19.
Int J Biol Macromol ; 213: 516-533, 2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35636531

RESUMO

Type of cross-linking agents influence the stability and active cross-linked enzyme aggregates (CLEA) immobilization. The information of molecular interaction between enzyme-cross linker is not well explored thus screening wide numbers of cross-linker is crucial in CLEA development. This study combined the molecular modeling and experimental optimization to investigate the influences of different cross-linking agents in developing CLEA of cyclodextrin glucanotranferase G1 (CGTase G1) for cyclodextrins (CDs) synthesis. Seven types of cross-linkers were tested and CGTase G1 cross-linked with chitosan (CS-CGTG1-CLEA) displayed the highest activity recovery (84.6 ± 0.26%), aligning with its highest binding affinity, radius of gyration and flexibility through in-silico analysis towards CGTase G1. CS-CGTG1-CLEA was characterized and showed a longer half-life (30.06 ± 1.51 min) and retained a greater thermal stability (52.73 ± 0.93%) after 30 min incubation at optimal conditions compared to free enzyme (10.30 ± 1.34 min and 5.51 ± 2.10% respectively). CS-CGTG1-CLEA improved CDs production by 33% and yielded cumulative of 52.62 g/L CDs after five cycles for 2 h of reaction. This study reveals that abundant of hydroxyl group on chitosan interacted with CGTase G1 surface amino acid residues to form strong and stable CLEA thus can be a promising biocatalyst in CDs production.


Assuntos
Quitosana , Ciclodextrinas , Bacillus , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glucosiltransferases/metabolismo , Temperatura , Termodinâmica
20.
ACS Synth Biol ; 11(5): 1881-1896, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35500299

RESUMO

In industries, enzymes are often immobilized to obtain stable preparations that can be utilized in batch and flow processes. In contrast to traditional immobilization methods that rely on carrier binding, various immobilization strategies have been recently presented that enable the simultaneous production and in vivo immobilization of enzymes. Catalytically active inclusion bodies (CatIBs) are a promising example for such in vivo enzyme immobilizates. CatIB formation is commonly induced by fusion of aggregation-inducing tags, and numerous tags, ranging from small synthetic peptides to protein domains or whole proteins, have been successfully used. However, since these systems have been characterized by different groups employing different methods, a direct comparison remains difficult, which prompted us to benchmark different CatIB-formation-inducing tags and fusion strategies. Our study highlights that important CatIB properties like yield, activity, and stability are strongly influenced by tag selection and fusion strategy. Optimization enabled us to obtain alcohol dehydrogenase CatIBs with superior activity and stability, which were subsequently applied for the first time in a flow synthesis approach. Our study highlights the potential of CatIB-based immobilizates, while at the same time demonstrating the robust use of CatIBs in flow chemistry.


Assuntos
Benchmarking , Escherichia coli , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Biocatálise , Enzimas Imobilizadas/química , Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Proteínas/metabolismo
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