enzyme reusability
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OBJECTIVES: To improve the storage stability and reusability of various yeast strains and species by immobilization in polyvinyl alcohol (PVA) hydrogel particles. RESULTS: Debaryomyces hansenii, Pichia sorbitophila, Saccharomyces cerevisiae, Yarrowia lipolytica, and Zygosaccharomyces rouxii were immobilized in PVA particles using LentiKats technology and stored in sterile water at 4 °C. The immobilization improved the survival of all species; however, the highest storage stability was achieved for S. cerevisiae and Y. lipolytica which survived more than 1 year, in contrast to free cells that survived for only 3 months. Tests of the reusability of immobilized recombinant laccase-secreting S. cerevisiae revealed that the cells were suitable for repetitive use (55 cycles during 15 months) even after storage in water at 4 °C for 9 months. A suitable method for killing immobilized laccase-secreting cells without affecting the produced enzyme activity was also developed. CONCLUSIONS: The immobilization of yeasts in PVA hydrogel enables long-term, cheap storage with very good cell viability and productivity, thus becoming a promising approach for industrial applications.
- MeSH
- biotechnologie MeSH
- buněčné kultury MeSH
- imobilizované buňky * cytologie enzymologie metabolismus MeSH
- lakasa chemie metabolismus MeSH
- mikrobiální viabilita MeSH
- ochrana biologická metody MeSH
- opakované použití vybavení MeSH
- polyvinylalkohol chemie MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- Saccharomyces cerevisiae * cytologie enzymologie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Capillary electrophoresis is a modern separation technique characterized by many benefits, which qualify it also for enzyme assays and the study of enzyme kinetics during drug development. Homogeneous or heterogeneous approaches can be followed for the enzymatic incubation. In this study, an immobilization procedure of aldehyde oxidase on magnetic particles was developed considering their integration with capillary electrophoresis. A number of magnetic nano/microparticle types were tested for this purpose, showing that aldehyde oxidase was most active when immobilized on bare silica magnetic nanoparticles. Primarily, the reusability of the enzyme immobilized on bare silica nanoparticles was tested. Three consecutive incubations with substrate could be performed, but the activity considerably dropped after the first incubation. One reason could be an enzyme detachment from the nanoparticles, but no release was detected neither at 4°C nor at 37°C during 5 h. The drop in enzymatic activity observed in consecutive incubations, could also be due to inactivation of the enzyme over time at given temperature. For the immobilized enzyme stored at 4°C, the activity decreased to 83% after 5 h, in contrast with a steep decrease at 37°C to 37%.
Imobilizace enzymů a mikroorganismů se stala nedílnou součástí mnoha biotechnologických procesů. V dnešní době je pro imobilizaci k dispozici celá řada anorganických, organických i syntetických materiálů a způsobů imobilizace, pomocí kterých je možné zvýšit stabilitu biokatalyzátorů, zajistit možnost jejich opětovného použití, a tím snížit náklady výrobního procesu. Zda je pro daný proces vhodné biokatalyzátor imobilizovat či nikoliv, závisí na řadě parametrů, mimo jiné na typu biokatalyzátoru, jeho vlastnostech a na aplikaci výsledného produktu.
Immobilization of enzymes and microorganisms has become an important part of many biotechnological processes. Different types of inorganic, organic, and synthetic materials and immobilization methods are used for increasing the biocatalyst stability, reusability and reduction of operating costs. Suitability of biocatalyst immobilization in the process depends on many parameters, such as biocatalyst properties, type of product and its application.
- MeSH
- biokatalýza * MeSH
- biotechnologie metody MeSH
- enzymy * MeSH
- imobilizace MeSH
- Publikační typ
- práce podpořená grantem MeSH
Nanozymes (NZs) are nanomaterials that mimic enzyme-like catalytic activity. They have attracted substantial attention due to their inherent physicochemical properties for use as promising alternatives to natural enzymes (NEs) in a variety of research fields. Particularly, in biosensing and bioassays, NZs have opened a new horizon to eliminate the intrinsic limitations of NEs, including their denaturation at extreme pH values and temperatures, poor reusability and recyclability, and high production costs. Moreover, the catalytic activity of NZs can be modulated in the preparation step by following an appropriate synthesis strategy. This review aims to gain insight into the potential substitution of NEs by NZs in biosensing and bioassays while considering both the pros and cons.
- MeSH
- biosenzitivní techniky * MeSH
- biotest MeSH
- enzymy MeSH
- katalýza MeSH
- nanostruktury * MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Ligninolytic enzymes from white-rot fungi are widely used in biotechnological processes. However, the application of these enzymes as free enzymes is limited due to their instability and lack of reusability. Enzyme stabilization is therefore a major challenge in biocatalytic process research, and immobilization methods are desirable. Using cross-linked enzyme aggregates (CLEAs) such as magnetic CLEAs, porous-CLEAs and combi-CLEAs is a promising technique for overcoming these issues. Cross-linking methods can stabilize and immobilize enzymes by interconnecting enzyme molecules via multiple bonds using cross-linking agents such as glutaraldehyde. The high catalyst density and microporous assembly of CLEAs guarantee high catalyst activity, which, together with their long shelf life, operational stability, and reusability, provide a cost-efficient alternative to matrix-assisted immobilization approaches. Here, we review current progress in ligninolytic enzyme immobilization and provide a comprehensive review of CLEAs. Moreover, we summarize the use of these CLEAs for biocatalysis processes, bioremediation such as dye decolourization, wastewater treatment or pharmaceutically active compound elimination.
The key to obtaining an optimum performance of an enzyme is often a question of devising a suitable enzyme and optimisation of conditions for its immobilization. In this study, laccases from the native isolates of white rot fungi Fomes fomentarius and/or Trametes versicolor, obtained from Czech forests, were used. From these, cross-linked enzyme aggregates (CLEA) were prepared and characterised when the experimental conditions were optimized. Based on the optimization steps, saturated ammonium sulphate solution (75 wt.%) was used as the precipitating agent, and different concentrations of glutaraldehyde as a cross-linking agent were investigated. CLEA aggregates formed under the optimal conditions showed higher catalytic efficiency and stabilities (thermal, pH, and storage, against denaturation) as well as high reusability compared to free laccase for both fungal strains. The best concentration of glutaraldehyde seemed to be 50 mM and higher efficiency of cross-linking was observed at a low temperature 4 °C. An insignificant increase in optimum pH for CLEA laccases with respect to free laccases for both fungi was observed. The results show that the optimum temperature for both free laccase and CLEA laccase was 35 °C for T. versicolor and 30 °C for F. fomentarius. The CLEAs retained 80% of their initial activity for Trametes and 74% for Fomes after 70 days of cultivation. Prepared cross-linked enzyme aggregates were also investigated for their decolourisation activity on malachite green, bromothymol blue, and methyl red dyes. Immobilised CLEA laccase from Trametes versicolor showed 95% decolourisation potential and CLEA from Fomes fomentarius demonstrated 90% decolourisation efficiency within 10 h for all dyes used. These results suggest that these CLEAs have promising potential in dye decolourisation.
- MeSH
- azosloučeniny chemie MeSH
- barva MeSH
- barvicí látky chemie MeSH
- bromthymolová modř chemie MeSH
- enzymy imobilizované chemie MeSH
- glutaraldehyd chemie MeSH
- katalýza MeSH
- lakasa chemie MeSH
- Polyporales enzymologie MeSH
- reagencia zkříženě vázaná chemie MeSH
- rosanilinová barviva chemie MeSH
- síran amonný chemie MeSH
- teplota MeSH
- Trametes enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1-10 °C in Luria-Bertani (LB) broth or 1-30 °C in Reasoner's 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1-10 °C in LB medium. About 70-80% of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60% of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10-20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C2-C18). The highest hydrolytic activity was obtained with p-NP-butyrate (C4) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C12) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.
- MeSH
- enzymy imobilizované chemie metabolismus MeSH
- hydrolýza MeSH
- kultivační média chemie MeSH
- lipasa chemie izolace a purifikace metabolismus MeSH
- metabolismus lipidů MeSH
- nízká teplota MeSH
- půdní mikrobiologie MeSH
- Rhodococcus enzymologie růst a vývoj izolace a purifikace MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.
- MeSH
- elektroforéza MeSH
- enzymy imobilizované metabolismus MeSH
- glykopeptidasa metabolismus MeSH
- glykoproteiny chemie metabolismus MeSH
- glykosylace MeSH
- imunoglobulin G chemie metabolismus MeSH
- mikrosféry * MeSH
- oligosacharidy metabolismus MeSH
- polysacharidy metabolismus MeSH
- ribonukleasy chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Enzymes have a wide range of applications in different industries owing to their high specificity and efficiency. Immobilization is often used to improve biocatalyst properties, operational stability, and reusability. However, changes in the structure of biocatalysts during immobilization and under process conditions are still largely uncertain. Here, three microscopy techniques - bright-field, confocal and electron microscopy - were applied to determine the distribution and structure of an immobilized biocatalyst. Free enzyme (haloalkane dehalogenase), cross-linked enzyme aggregates (CLEAs) and CLEAs entrapped in polyvinyl alcohol lenses (lentikats) were used as model systems. Electron microscopy revealed that sonicated CLEAs underwent morphological changes that strongly correlated with increased catalytic activity compared to less structured, non-treated CLEAs. Confocal microscopy confirmed that loading of the biocatalyst was not the only factor affecting the catalytic activity of the lentikats. Confocal microscopy also showed a significant reduction in the pore size of lentikats exposed to 25% tetrahydrofuran and 50% dioxane. Narrow pores appeared to provide protection to CLEAs from the detrimental action of cosolvents, which significantly correlated with higher activity of CLEAs compared to free enzyme. The results showed that microscopy can provide valuable information about the structure and properties of a biocatalyst during immobilization and under process conditions.
- MeSH
- biokatalýza MeSH
- dioxany farmakologie MeSH
- furany farmakologie MeSH
- hydrolasy chemie metabolismus MeSH
- mikroskopie metody MeSH
- proteiny červů chemie metabolismus MeSH
- Schistosoma japonicum enzymologie MeSH
- stabilita enzymů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The purpose of the current study was to evaluate the functional activity and storage viability (at 4 °C and 35 °C) of an immobilized as well as lyophilized multienzyme, viz., pectinase, cellulase, and amylase (PCA) that was produced by Bacillus subtilis NG105 under solid state fermentation (SSF) at 35 °C for 10 days using mosambi peel as a substrate. After SSF, the culture media was divided into two aliquots. From the first aliquot, the produced ME was extracted, precipitated, and further immobilized on calcium alginate beads (MEICA). In order to immobilize on mosambi peel matrix, the second aliquot was mixed with acetone and subsequently lyophilized (MELMP). Thus, ready MEICA and MELMP extracted 87.5 and 91.5% juice from mango pulp, respectively. In the reusability study, after 5 cycles, MEICA exhibited 23.8%, 24.4%, and 36.5% PCA activity, respectively. The PCA activity of MEICA and MELMP was examined after 60 days of storage at 4 °C. The result revealed that the PCA for MEICA declined from 100 to 66%, 58.2%, and 64.5%, respectively, while for MELMP, it dropped from 100 to 84.2%, 82.1%, and 69.7%, respectively. Further, after 60 days of storage, the reduction of total protein content (TPC) in free multienzyme (FME), MEICA, and MELMP was 92.2%, 91.5%, and 36.3% observed, respectively. In the localization study, the maximum levels of multienzyme activity were found in cell exudates. This study demonstrated that immobilizing of multienzyme through lyophilization on waste substrates like mosambi peel boosted its stability and shelf-life along with greatly reducing the cost of products.
