Glucose‐6‐phosphate dehydrogenase encapsulated in silica‐based hydrogels for operation in a microreactor

The future of hydrogen as fuel strongly depends on the possibility to produce it in an economic and clean way. Hydrogen can be produced from carbohydrates and water under mild conditions by means of a multistep synthetic pathway (13 enzymes) with very high yield. Crossover inhibitions and different optimal conditions of involved enzymes hinder the use of one‐pot approach. Immobilization of enzymes in coupled individual reactors may avoid this problem. This work deals with the immobilization in silica‐based hydrogels of one key enzyme of this pathway: glucose 6‐phosphate dehydrogenase from Leuconostoc mesenteroides. The carriers were prepared with an ethylene glycol‐modified silane, two polymers (polyethylene oxide and Pluronic®) and amino groups created by 3‐aminopropyltriethoxysilane. These parameters influenced the enzymatic activity after immobilization. Gels prepared by addition of polyethylene oxide gave the best results and were used as monoliths in microreactors with two different geometries. The systems showed a high operational stability but a low effective enzyme activity. Enzyme leaching and a nonideal flow pattern may account for the low activity observed. This work is possibly the first one dealing with the immobilization of glucose 6‐phosphate dehydrogenase in silica‐based gels for its application in flow‐through microreactors.     

Enzymes immobilized on carbon nanotubes

Enzyme immobilizations on carbon nanotubes for fabrication of biosensors and biofuel cells and for preparation of biocatalysts are rapidly emerging as new research areas. Various immobilization methods have been developed, and in particular, specific attachment of enzymes on carbon nanotubes has been an important focus of attention. The method of immobilization has an effect on the preservation of the enzyme structure and retention of the native biological function of the enzyme. In this review, we focus on recent advances in methodology for enzyme immobilization on carbon nanotubes.     

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process.Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications.     

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, ?thus not reusable- and their performance under real operational conditions is uncertain.Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts.In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel “immobilized biocatalyst engineering” research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.     

酶的分子设计、改造与工程应用

酶工程的研究已经发展到分子水平 ,在体外通过基因工程、化学、物理等手段改造酶分子结构与功能 ,大幅提高了酶分子的进化效率和催化效率 ,生产有价值的非天然酶。对酶工程学若干“热点”和前沿课题的研究、应用进行了概述 ,分析了国际上酶工程研究及应用技术、手段、方法 ,包括体外分子进化、核酶和抗体酶的设计、酶分子的定向固定化技术、酶蛋白分子的化学修饰、融合酶、人工合成及模拟酶等技术 ,并展望了酶工程的技术进步和应用的新进展。     

Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme–support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions).This method could be extended to any other multimeric enzyme expressed in mesophilic hosts.     

Multi-faceted strategy based on enzyme immobilization with reactant adsorption and membrane technology for biocatalytic removal of pollutants: A critical review

In the modern era, the use of sustainable, environmentally friendly alternatives for removal of recalcitrant pollutants in streams resulting from industrial processes is of key importance. In this context, biodegradation of phenolic compounds, pharmaceuticals and dyes in wastewater by using oxidoreductases offers numerous benefits. Tremendous research efforts have been made to develop novel, hybrid strategies for simultaneous immobilization of oxidoreductase and removal of toxic compounds. The use of support materials with the options for combining enzyme immobilization with adsorption technology focused on phenolic pollutants and products of biocatalytic conversion seems to be of particular interest. Application of enzymatic reactors based on immobilized oxidoreductases for coupling enzyme-aided degradation and membrane separation also attract still growing attention. However, prior selection of the most suitable support/sorbent material and/or membrane as well as operational mode and immobilization technique is required in order to achieve high removal efficiency. Thus, in the framework of this review, we present an overview of the impact of support/sorbent material on the catalytic properties of immobilized enzymes and sorption of pollutants as well as parameters of membranes for effective bioconversion and separation. Finally, future perspectives of the use of processes combining enzyme immobilization and sorption technology as well as application of enzymatic reactors for removal of environmental pollutants are discussed.     

Characterization of CIM monoliths as enzyme reactors

The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis-Menten constant K(m) and the turnover number k(3) of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K(m) values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.     

An automated method for measuring the operational stability of biocatalysts with carbonic anhydrase activity

The operational stability of an enzyme can be quantified by its half-life, or the length of time after which 50% of its original activity has degraded. Ideally, continuous methods for measuring half-lives are preferred but they can be expensive and relatively low throughput. Batch methods, while simple, cannot be used for all enzymes. For example, batch reactions can be difficult when there is a gas phase reactant or when there is significant product or substrate inhibition. Here we describe a repeated-batch method for measuring the half-life of carbonic anhydrase (CA)-based biocatalysts by automated periodic switching between a forward and reverse reaction. This method is inexpensive and can be multiplexed for high-throughput analysis of enzyme variants. Several purified CA enzymes as well as whole-cell biocatalysts with engineered CA activity were evaluated with this method. The results indicate a significant increase in operational stability is achieved upon immobilization of CA in the cellular periplasm of Escherichia coli.     

Intensification of biokinetics of enzymes using ultrasound-assisted methods: a critical review

The use of low intensity ultrasound has gotten surprising consideration over the last decade as a method for enhancing the catalytic activity of enzyme. Ultrasounds have the potential to significantly influence the activity of the enzymatic processes, provided that the energy input is not so high as to inactivate the enzyme. By providing the variation in parameters, various physical and chemical effects can be attained that can enhance the enzymatic reaction. Ultrasonic reactors are known for their application in bioprocesses. However, the potential of their applications is still limited broadly due to the lack of proper information about their operational and performance parameters. In this review, the detailed information about ultrasonic reactors is provided by defining the different types of reactors and number and position of ultrasonic transducers. Also, it includes mechanism of intensification and influence of ultrasonic parameters (intensity, duty cycle, and frequency) and enzymatic factors (enzyme concentration, temperature, and pH) on the catalytic activity of enzyme during ultrasound treatment.     

Anionic exchange nylon laminated membranes for enzyme immobilization

Summary This work presents the optimization of the chemical steps involved in nylon modification with dimethyl sulphate, polyethyleneimine, glutaraldehyde and 2-diethyl aminoethylamine to obtain a weak basic anion exchange support. Activated nylon laminated membranes were utilized for aminoacylase immobilization, allowing an ionically adsorbed enzyme derivative with high activity (0.16 U/mg E·cm²) and low removed activity (<1%). Optimum immobilization conditions and kinetic parameters were also determined. This immobilized enzyme can be used in laminated enzyme membrane reactors.     

Enzyme immobilization on electrospun polymer nanofibers: An overview

Enzyme immobilization has attracted continuous attention in the fields of fine chemistry, biomedicine, and biosensor. The performance of immobilized enzyme largely depends on the structure of supports. Nanostructured supports are believed to be able to retain the catalytic activity as well as ensure the immobilization efficiency of enzyme to a high extent. Electrospinning provides a simple and versatile method to fabricate nanofibrous supports. Compared with other nanostructured supports (e.g. mesoporous silica, nanoparticles), nanofibrous supports show many advantages for their high porosity and interconnectivity. This review mainly discusses the recent advances in using nanofibers as hosts for enzyme immobilization by two different methods, surface attachment and encapsulation. Surface attachment refers to physical adsorption or covalent attachment of enzymes on pristine or modified nanofibrous supports, and encapsulation means electrospinning a mixture of enzyme and polymer. We make a detailed comparison between these two immobilization approaches and highlight their distinct characteristics. The prospective applications of enzyme immobilized electrospun nanofibers in the development of biosensors, biofuel cells and biocatalysts are also discussed.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Immobilization strategies to develop enzymatic biosensors

Immobilization of enzymes on the transducer surface is a necessary and critical step in the design of biosensors. An overview of the different immobilization techniques reported in the literature is given, dealing with classical adsorption, covalent bonds, entrapment, cross-linking or affinity as well as combination of them and focusing on new original methods as well as the recent introduction of promising nanomaterials such as conducting polymer nanowires, carbon nanotubes or nanoparticles. As indicated in this review, various immobilization methods have been used to develop optical, electrochemical or gravimetric enzymatic biosensors. The choice of the immobilization method is shown to represent an important parameter that affects biosensor performances, mainly in terms of sensitivity, selectivity and stability, by influencing enzyme orientation, loading, mobility, stability, structure and biological activity.     

Understanding the pH-dependent immobilization efficacy of feruloyl esterase-C on mesoporous silica and its structure–activity changes

The purpose of the present investigation was to study the pH dependence of both the immobilization process and the enzyme activity of a feruloyl esterase (FoFaeC from Fusarium oxysporum) immobilized in mesoporous silica. This was done by interpreting experimental results with theoretical molecular modeling of the enzyme structure. Modeling of the 3D structure of the enzyme together with calculations of the electrostatic surface potential showed that changes in the electrostatic potential of the protein surface were correlated with the pH dependence of the immobilization process. High immobilization yields were associated with an increase in pH. The transesterification activity of both immobilized and free enzyme was studied at different values of pH and the optimal pH of the immobilized enzyme was found to be one unit lower than that for the free enzyme. The surface charge distribution around the binding pocket was identified as being a crucial factor for the accessibility of the active site of the immobilized enzyme, indicating that the orientation of the enzyme inside the pores is pH dependent. Interestingly, it was observed that the immobilization pH affects the specific activity, irrespective of the changes in reaction pH. This was identified as a pH memory effect for the immobilized enzyme. On the other hand, a change in product selectivity of the immobilized enzyme was also observed when the transesterification reaction was run in MOPS buffer instead of citrate phosphate buffer. Molecular docking studies revealed that the MOPS buffer molecule can bind to the enzyme binding pocket, and can therefore be assumed to modulate the product selectivity of the immobilized enzyme toward transesterification.     

Concanavalin A: a useful ligand for glycoenzyme immobilization--a review.

Concanavalin A is finding increasing applications as a useful ligand in glycoenzyme immobilization. An attempt therefore, has been made to summarize the work available in the area. Glycoenzymes that are recalcitrant to immobilization procedures involving covalent coupling to solid supports can be immobilized in high yields by binding to matrices precoupled with concanavalin A. In addition, glycoenzymes associated with concanavalin A matrices usually exhibit high retention of activity and enhanced stability against various forms of inactivation. Binding of the glycoenzymes on the concanavalin A supports, being noncovalent, can be reversed by incubating the preparation with a high concentration of sugars/glycosides or at acidic pH. The association can be, however, rendered covalent by crosslinking the preparations with bifunctional reagents like glutaraldehyde. Crosslinking may be accompanied by further increase in stability, albeit at the expense of the loss of some enzyme activity. Several laboratory-size reactors containing concanavalin A matrix-bound glycoenzyme have been successfully operated for reasonably long durations with only small losses in catalytic activity. Insoluble glycoenzyme preparation can also be obtained by precipitating them from solution as concanavalin A complexes. Such complexes have small particle dimensions but can be successfully used in column reactors after a subsequent immobilization step. Insoluble concanavalin A-flocculates containing various microorganisms and glycoenzymes that successfully carry out multistep transformations have also been obtained by several investigators.     

Immobilization of dextranase on bentonite

Dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) from Penicillium aculeatum culture has been immobilized on a bentonite support. The matrix-bound enzyme could be stored as acetone-dried powder or as a suspension in acetate buffer, pH 5.6, for about three weeks at 4°C without any loss of activity. There was no change in the specific activity of the enzyme on immobilization and the enzyme yield was 0.1–0.6 mg/g bentonite matrix. In the presence of sucrose, thermal stability of the immobilized enzyme was high and the bound enzyme could be used for about six cycles.     

Enzyme stabilization by nano/microsized hybrid materials

Immobilization is a key technology for successful realization of enzyme‐based industrial processes, particularly for production of green and sustainable energy or chemicals from biomass‐derived catalytic conversion. Different methods to immobilize enzymes are critically reviewed. In principle, enzymes are immobilized via three major routes (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross‐linking (carrier free). As a result, immobilizing enzymes on certain supports can enhance storage and operational stability. In addition, recent breakthroughs in nano and hybrid technology have made various materials more affordable hosts for enzyme immobilization. This review discusses different approaches to improve enzyme stability in various materials such as nanoparticles, nanofibers, mesoporous materials, sol–gel silica, and alginate‐based microspheres. The advantages of stabilized enzyme systems are from its simple separation and ease recovery for reuse, while maintaining activity and selectivity. This review also considers the latest studies conducted on different enzymes immobilized on various support materials with immense potential for biosensor, antibiotic production, food industry, biodiesel production, and bioremediation, because stabilized enzyme systems are expected to be environmental friendly, inexpensive, and easy to use for enzyme‐based industrial applications.     

Immobilization of papain on cotton fabric by sol–gel method

A method has been developed to immobilize papain on cotton fabric by means of sol–gel technique. The activity of free papain and papain in silica sol under sonication was studied. Scanning electron microscopy, energy dispersive spectrometer and the Bradford method were used to characterize papain immobilization. The efficiency of the immobilization was investigated by examining the relative enzymatic activity of free and immobilized papain, respectively. The results show that the optimum pH value in the medium for immobilized papain is shifted to alkaline side. In addition, the adaptability of papain to environmental acidity is significantly increased. The thermostability of immobilized papain shows no significant change compared to the free enzyme. The papain immobilized on fabric by sol–gel technique retains more than 30% of the original activity after six reuses continuously.     

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。