Multi-point enzyme immobilization,surface chemistry,and novel platforms: a paradigm shift in biocatalyst design

Engineering enzymes with improved catalytic properties in non-natural environments have been concerned with their diverse industrial and biotechnological applications. Immobilization represents a promising but straightforward route, and immobilized biocatalysts often display higher activities and stabilities compared to free enzymes. Owing to their unique physicochemical characteristics, including the high-specific surface area, exceptional chemical, electrical, and mechanical properties, efficient enzyme loading, and multivalent functionalization, nano-based materials are postulated as suitable carriers for biomolecules or enzyme immobilization. Enzymes immobilized on nanomaterial-based supports are more robust, stable, and recoverable than their pristine counterparts, and are even used for continuous catalytic processes. Furthermore, the unique intrinsic properties of nanomaterials, particularly nanoparticles, also confer the immobilized enzymes to be used for their broader applications. Herein, an effort has been made to present novel potentialities of multi-point enzyme immobilization in the current biotechnological sector. Various nano-based platforms for enzyme/biomolecule immobilization are discussed in the second part of the review. In summary, recent developments in the use of nanomaterials as new carriers to construct robust nano-biocatalytic systems are reviewed, and future trends are pointed out in this article.     

多孔纳米材料固定化酶研究进展

酶是一种天然生物催化剂,有催化效率高、底物选择性强和绿色环保等优点,但酶结构不稳定且重复利用率低,制约了其产业化应用。随着技术的发展,酶的固定化可以提高酶的活性和稳定性,为生物酶的工程化应用带来了新的机遇。多孔纳米材料具有比表面积大、孔隙率高、机械和化学性能稳定等特点和优异的成本效益,是理想的固定化酶载体。本文综述了近些年来金属有机框架、共价有机框架和多孔微球等纳米材料固定化酶的研究进展和应用,重点介绍了载体固定酶的方式,并总结了每种载体的特点,最后讨论了多孔纳米材料固定化酶面临的挑战和发展趋势。     

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Recent advances in nano-carrier immobilized enzymes and their applications

Enzymes have been widely used because of their catalytic properties, and immobilization is a promising technique to improve their catalytic activity and stability. Due to their large specific surface areas, exceptional chemical, mechanical, thermal and cost effective characteristics, nanomaterials should be ideal carriers for the immobilization of enzymes. Enzymes immobilized on nano-carriers are more robust and stable, and can be recycled and reused. This review focuses on the nanomaterial immobilized enzymes and their applications. The introduction addresses the advantages of immobilized enzymes and the features of enzyme immobilization nanocarriers. The next section covers carbonaceous nanomaterials used in enzymes immobilization, with subsections on carbon nanotube, graphene, graphene oxide and reduced graphene oxide. The third section treats metallic nanomaterials for enzymes immobilization, with subsections on metal (gold), metal oxide (titanium dioxide, zinc oxide) and metal hydroxide (layered double hydroxide) nanomaterials. Then, the next section summarizes the applications of nanomaterial immobilized enzymes. A concluding section discusses the challenges and prospects of nanomaterial immobilized enzymes.     

Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery

Abstract

Topical or transdermal drug delivery is challenging because the skin acts as a natural and protective barrier. Therefore, several methods have been examined to increase the permeation of therapeutic molecules into and through the skin. One approach is to use the nanoparticulate delivery system. Starting with liposomes and other vesicular systems, several other types of nanosized drug carriers have been developed such as solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanoparticles and magnetic nanoparticles for dermatological applications. This review article discusses how different particulate systems can interact and penetrate into the skin barrier. In this review, the effectiveness of nanoparticles, as well as possible mode of actions of nanoparticles, is presented. In addition to nanoparticles, cell-penetrating peptide (CPP)-mediated drug delivery into the skin and the possible mechanism of CPP-derived delivery into the skin is discussed. Lastly, the effectiveness and possible mechanism of CPP-modified nanocarriers into the skin are addressed.     

Nanoparticulate drug delivery systems for cancer chemotherapy

Abstract

Nanoparticles (NPs) are, in general, colloidal particles, less than 1000 nm, that can be used for better drug delivery and prepared either by encapsulating the drug within a vesicle and or by dispersing the drug molecules within a matrix. Nanoparticulate drug delivery systems have been extensively studied in recent years for spatial and temporal delivery, especially in tumour and brain targeting. NPs have great promise for better drug delivery as found in both pharmaceutical and clinical research. As a drug carrier, NPs have significant advantages like better bioavailability, systemic stability, high drug loading, long blood circulation time and selective distribution in the organs/tissues with longer half life. The selective targeting of NPs can be achieved by the enhanced permeability and retention effect (EPR-effect), attaching specific ligands, or by making selective distribution due to change of the physiological conditions of specific systems like nature, pH, temperature, etc. It has been observed that drug-loaded NPs can have selective distribution to organs/tissues using different types of and proportions of polymers. The current aim of researchers is to prepare NPs that are long-lived with and that demonstrate the appropriate selective distribution for better therapy and thus improved clinical outcomes. Nanoparticulate drug delivery systems have the potential to deliver a drug to the target site with specificity and to maintain the desired concentration at the site for the intended time without untoward effects. In this review article, the methods for the preparation of NPs, their characterization, biodistribution, and pharmacokinetic characteristics are discussed.     

CO2生物转化关键酶固定体系构筑研究进展与挑战

酶催化CO₂还原制备高值化学品对缓解全球环境和能源危机具有重要意义,利用甲酸脱氢酶(formate dehydrogenase,FDH)或多酶级联还原CO₂制备甲酸/甲醇具有选择性高、条件温和的优势,但关键酶活性低、稳定性差和重复利用率低的问题限制了其规模化应用,酶的固定化为这些问题提供了有效解决方案。本文总结了近年来利用膜、无机材料、金属有机框架和共价有机框架等载体对酶进行固定化的研究进展,阐释了不同固定材料和固定方式的特点和优势;进一步总结了固定化酶与电催化或光催化耦联反应体系对CO₂还原的协同效果及应用,同时指出酶固定化技术和耦联反应体系目前存在的问题并对其发展前景进行了展望。     

Einfluß einiger Faktoren in Abhängigkeit von den Methoden bei der Immobilisierung eines bakteriellen Enzympräparats mit Milchkoagulationswirkung

The influence of different factors (binding time, pH, temperature, and enzyme/carrier ratio) on the efficiency of immobilization of enzyme preparation with milk clotting activity from Bacillus mesentericus M strain by use of different carriers and methods of binding was investigated. It has been established that the dependence of immobilization from pH is most pronounced and is mainly influenced by the nature of the carrier. The binding time is of essential importance only by immobilization through adsorption. The temperature has not any influence on the efficiency of immobilization of the indicate preparation. Saturation of many of the used carriers by the different methods for immobilization was achieved at enzyme/carrier ratio 1 : 10, with exception of the cases where glutaraldehyde was used as binding reagent. In this case then the ratio was 1 : 4.     

Application of immobilized enzyme technologies for the textile industry: a review

Abstract

A number of enzymes have applications in the textile sector, as several natural fibres (e.g. cotton, wool and flax) can be subjected to processing using such natural biocatalysts. In the latest two decades, demand for industrial enzymes has increased considerably; however, the textile industry requires highly stable enzymes, and good performance at extreme values of pH and temperature. New strategies are continuously emerging for the immobilization of enzymes with superior efficiency and usage. Enzymatic immobilization can stabilize enzymes and extend their useful life. Additionally, reduction in effluent treatment costs and improvement of efficiency is possible. This paper briefly reviews the most recent research efforts pertaining to immobilization of enzymes with potential application in the textile industry.     

Immobilization of inulinase obtained by solid-state fermentation using spray-drying technology

Abstract

This work focuses on the immobilization of a crude inulinase extract obtained by solid-state fermentation using spray-drying technology. Maltodextrin and arabic gum were used as immobilizing agents. The effects of inlet air temperature, maltodextrin/arabic gum ratio and mass fraction of crude enzyme extract on the activity of immobilized inulinase were assessed using a central composite rotatable design (CCRD) (2³). The optimum operational conditions for the immobilization of inulinase by spray-drying was obtained at an inlet air temperature of 200°C, mass fraction of crude enzyme extract of 0.5 wt% and using only arabic gum as immobilizing agent. The immobilized enzyme had good thermostability, comparable with other inulinases obtained from different microorganisms. The method used gave good enzyme activity after immobilization and could be applied to other enzymes which have good thermal stability.     

Immobilized enzymes – valuable tools for the indication of temperature events

Abstract

The enzymes trypsin and urease were covalently tethered to cellulose to utilize their ability to produce colored products as a consequence of enzymatic activity. Therefore, cellulose had to be chemically modified first in order to generate appropriate chemical functionalities. Different approaches including periodate supported oxidation followed by immobilization via reductive amination, insertion of a reactive polymer interface, and cross-linking inside the cellulose matrix were utilized for the immobilization. The success of immobilization was assessed by the quantification of surface-bound protein as well as by recording of enzymatic activities under different conditions. The enzymatic activity of trypsin and urease was maintained best when a hydrophilic intermediate polymer layer was used for immobilization. The applicability of immobilized enzymes as temperature indicators was demonstrated using cross-linked urease.     

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

Background

Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.

Methods and findings

We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.

Conclusions

Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.     

In-vitro evaluation of copper/copper oxide nanoparticles cytotoxicity and genotoxicity in normal and cancer lung cell lines

BackgroundNanotoxicology is a major field of study that reveals hazard effects of nanomaterials on the living cells.MethodsIn the present study, Copper/Copper oxide nanoparticles (Cu/CuO NPs) were prepared by the chemical reduction method and characterized by different techniques such as: X-Ray Diffraction, Transmission and Scanning Electron Microscopy. Evaluation of the toxicity of Cu/CuO NPs was performed on 2 types of cells: human lung normal cell lines (WI-38) and human lung carcinoma cell (A549). To assess the toxicity of the prepared Cu/CuOs NPs, the two cell types were exposed to Cu/CuO NPs for 72 h. The half-maximal inhibitory concentration IC₅₀ of Cu/CuO NPs for both cell types was separately determined and used to examine the cell genotoxicity concurrently with the determination of some oxidative stress parameters: nitric oxide, glutathione reduced, hydrogen peroxide, malondialdehyde and superoxide dismutase.ResultsCu/CuO NPs suppressed proliferation and viability of normal and carcinoma lung cells. Treatment of both cell types with their IC₅₀’s of Cu/CuO NPs resulted in DNA damage besides the generation of reactive oxygen species and consequently the generation of a state of oxidative stress.ConclusionOverall, it can be concluded that the IC₅₀'s of the prepared Cu/CuO NPs were cytotoxic and genotoxic to both normal and cancerous lung cells.     

Nanoparticles and cancer therapy: Perspectives for application of nanoparticles in the treatment of cancers

Rapid growth in nanotechnology toward the development of nanomedicine agents holds massive promise to improve therapeutic approaches against cancer. Nanomedicine products represent an opportunity to achieve sophisticated targeting strategies and multifunctionality. Nowadays, nanoparticles (NPs) have multiple applications in different branches of science. In recent years, NPs have repetitively been reported to play a significant role in modern medicine. They have been analyzed for different clinical applications, such as drug carriers, gene delivery to tumors, and contrast agents in imaging. A wide range of nanomaterials based on organic, inorganic, lipid, or glycan compounds, as well as on synthetic polymers has been utilized for the development and improvement of new cancer therapeutics. In this study, we discuss the role of NPs in treating cancer among different drug delivery methods for cancer therapy.     

Microbial Enzymes for Oxidation of Organic Molecules

Abstract

Enzymatic systems employed by microorganisms for oxidative transformation of various organic molecules include laccases, ligninases, tyrosinases, monooxygenases, and dioxygenases. Reactions performed by these enzymes play a significant role in maintaining the global carbon cycle through either transformation or complete mineralization of organic molecules. Additionally, oxidative enzymes are instrumental in modification or degradation of the ever-increasing man-made chemicals constantly released into our environment. Due to their inherent stereo- and regioselectivity and high efficiency, oxidative enzymes have attracted attention as potential biocatalysts for various biotechnological processes. Successful commercial application of these enzymes will be possible through employing new methodologies, such as use of organic solvents in the reaction mixtures, immobilization of either the intact microorganisms or isolated enzyme preparations on various supports, and genetic engineering technology.     

Biochemical alterations induced by nickel oxide nanoparticles in female Wistar albino rats after acute oral exposure

Context: Nickel oxide (NiO) nanoparticles (NPs) with appropriate surface chemistry have been widely used for their potential new applications in biomedical industry. Increased usage of these NPs enhances the chance of exposure of personnel involved in the work place.

Objective: This study was designed to assess the ability of NiO NPs to cause biochemical alterations post-acute oral exposure in female Wistar rats.

Materials and methods: Rats were administered with 125, 250, and 500?mg/kg doses of NiO NPs for haematological, biochemical, and histopathological studies. Biodistribution patterns of NiO NPs in female Wistar rats were also monitored.

Results: NiO NPs caused significant (p?Conclusions: This study revealed that exposure to nanosized NiO particles at acute doses may cause adverse changes in animal biochemical profiles. Further, the in vivo studies on toxicity evaluation help in biomonitoring of the potential contaminants.     

Enzymatic reactors for the removal of recalcitrant compounds in wastewater

Abstract

Enzymatic treatments based on oxidative enzymes, such as peroxidases, laccases and tyrosinases, have been proposed as an alternative to conventional methods to remove a broad range of contaminants present in wastewater. The aim of this study is to discuss existing technologies for the removal of pollutants based on the use of oxidative enzymes, including a discussion on the most important factors affecting the efficiency of the proposed systems. Factors involved in the catalytic cycle of the enzyme (biocatalyst, substrates and mediators), the addition of certain components to the reaction medium (additives, surfactants or solvents) as well as operational parameters (temperature, pH or agitation) will be discussed. Finally, two types of reactors: one-stage and two-stage enzymatic membrane reactors, especially designed for the treatment of micropollutants present in secondary effluents, will be described in detail.     

纳米材料固定化酶的研究进展

近年来,纳米技术为酶固定化提供了多种纳米级材料,纳米材料固定化酶不仅具有高的酶负载量,而且具有良好的酶稳定性。本文基于纳米材料固定化酶,对纳米材料的种类进行了总结,分析了纳米材料对固定化酶性能的影响,并介绍了纳米级固定化方法及纳米材料固定化酶在生物转化、生物传感器、生物燃料电池等领域的应用。     

Modified chitosan as a spacer for protein immobilization

Abstract

Three new, water-soluble, N-modified chitosan derivatives containing poly(ethylene glycol), dextran or inulin side chains were used as spacers for enzyme immobilization on a natural silk carrier. Amylolytic enzymes Maltogenase L and Promozyme D2, lipolytic enzyme Resinase HT and a complex of proteolytic enzymes from Streptomyces flavus 197 were immobilized. The activity of the immobilized enzymes and their stability during storage were similar to that obtained with synthetic polyamine—poly(ethylene imine) as a spacer. High operational stability of co-immobilized amylolytic enzymes Maltogenase L and Promozyme D2 in a continuous flow mini-reactor was demonstrated.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.