Conversion of carbon dioxide to oxaloacetate using integrated carbonic anhydrase and phosphoenolpyruvate carboxylase

The development and implementation of strategies for CO₂ mitigation are necessary to counteract the greenhouse gas effect of carbon dioxide emissions. To demonstrate the possibility of simultaneously capturing CO₂ and utilizing four-carbon compounds, an integrated system using CA and PEPCase was developed, which mimics an in vivo carbon dioxide concentration mechanism. We first cloned the PEPCase 1 gene of the marine diatom Phaeodactylum tricornutum and produced a recombinant PtPEPCase 1. The affinity column purified PtPEPCase 1 exhibited specific enzymatic activity (5.89 U/mg). When the simultaneous and coordinated reactions of CA from Dunaliella sp. and the PtPEPCase 1 occurred, more OAA was produced than when only PEPCase was present. Therefore, this integrated CA-PEPCase system can be used not only to capture CO₂ but also for a new technology to produce value-added four-carbon platform chemicals.     

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.     

Convenient one-step purification and immobilization of lipase using a genetically encoded aldehyde tag

To avoid the unwanted and random covalent linkage between the cross-linker and enzyme's active site in covalent immobilization, a genetically encoded “aldehyde tag” was introduced into recombinant lipase and applied for the one-step purification and covalent immobilization of this enzyme. The effects of the immobilization time, temperature and the amount of enzyme were investigated, and the thermo-stability of immobilized lipase was also examined. The specific activity and the k_cat/K_m of the immobilized lipase using aldehyde tag (IL-AT) were 2.50 and 3.02 fold higher, respectively, than those of the traditionally immobilized lipase using glutaraldehyde (IL-GA). The newly immobilized lipase also presented better thermo-stability than the traditionally immobilized one. The results show that the recombinant enzyme could be conveniently immobilized without glutaraldehyde and that the enzyme's active site was well protected. This is a new immobilization method able to avoid glutaraldehyde or 2,4,6-trichloro-1,3,5-triazine as an activating agent. The greener method without hazardous chemicals for the one-step purification and immobilization of an enzyme using a genetically encoded “aldehyde tag” can be exploited for numerous other enzyme purification and immobilization applications.     

Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis

Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and purity. These features make enzyme-immobilized MNPs applicable to organic synthesis.     

Non-porous magnetic supports for cell immobilization

A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/l · h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.     

Immobilization of carbonic anhydrase in alginate and its influence on transformation of CO2 to calcite

Present investigation entails carbonic anhydrase (CA) immobilization and its influence on transformation of CO₂ to calcite. CA enzyme was immobilized in alginate beads, subsequently maintained its catalytic efficiency after sequential operational cycles. The immobilized beads showed better operational stability by retaining nearly 67% of its initial activity even after six cycles. Batch scale studies with free and immobilized enzyme revealed that the entrapped CA hydrates CO₂ to bicarbonate and/or carbonate which was then made to react with Ca²⁺ ions to transform into calcite. Calcite was characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The entrapped CA was employed for the performance evaluation with respect to several operational parameters including the influence of enzyme concentration in free and immobilized condition. It was concluded that immobilized CA in alginate beads would have the potential for CO₂ sequestration by biomimetic route.     

Polymer materials for enzyme immobilization and their application in bioreactors

Enzymatic catalysis has been pursued extensively in a wide range of important chemical processes for their unparalleled selectivity and mild reaction conditions. However, enzymes are usually costly and easy to inactivate in their free forms. Immobilization is the key to optimizing the in-service performance of an enzyme in industrial processes, particularly in the field of non-aqueous phase catalysis. Since the immobilization process for enzymes will inevitably result in some loss of activity, improving the activity retention of the immobilized enzyme is critical. To some extent, the performance of an immobilized enzyme is mainly governed by the supports used for immobilization, thus it is important to fully understand the properties of supporting materials and immobilization processes. In recent years, there has been growing concern in using polymeric materials as supports for their good mechanical and easily adjustable properties. Furthermore, a great many work has been done in order to improve the activity retention and stabilities of immobilized enzymes. Some introduce a spacer arm onto the support surface to improve the enzyme mobility. The support surface is also modified towards biocompatibility to reduce non-biospecific interactions between the enzyme and support. Besides, natural materials can be used directly as supporting materials owning to their inert and biocompatible properties. This review is focused on recent advances in using polymeric materials as hosts for lipase immobilization by two different methods, surface attachment and encapsulation. Polymeric materials of different forms, such as particles, membranes and nanofibers, are discussed in detail. The prospective applications of immobilized enzymes, especially the enzyme-immobilized membrane bioreactors (EMBR) are also discussed.     

Activity and stability of immobilized carbonic anhydrase for promoting CO2 absorption into a carbonate solution for post-combustion CO2 capture

An Integrated Vacuum Carbonate Absorption Process (IVCAP) currently under development could significantly reduce the energy consumed when capturing CO2 from the flue gases of coal-fired power plants. The biocatalyst carbonic anhydrase (CA) has been found to effectively promote the absorption of CO2 into the potassium carbonate solution that would be used in the IVCAP. Two CA enzymes were immobilized onto three selected support materials having different pore structures. The thermal stability of the immobilized CA enzymes was significantly greater than their free counterparts. For example, the immobilized enzymes retained at least 60% of their initial activities after 90 days at 50 °C compared to about 30% for their free counterparts under the same conditions. The immobilized CA also had significantly improved resistance to concentrations of sulfate (0.4 M), nitrate (0.05 M) and chloride (0.3 M) typically found in flue gas scrubbing liquids than their free counterparts.     

Carbon dioxide fixation by immobilized chloroplasts

Chloroplasts of chinese mustard (Brassica campestris L.) were immobilized in polyacrylamide gel. A 8% polymer concentration was suitable for the immobilization. The activity of the carbon dioxide fixation of immobilized chloroplasts was 65% of that of free chloroplasts. The optimum conditions for the carbon dioxide fixation of immobilized chloroplasts were similar to that of native chloroplasts. However, immobilized chloroplasts were more stable under alkaline conditions and high temperatures than native chloroplasts. Light penetration of the gel was not a limiting parameter of the carbon dioxide fixation. The lifetime of immobilized chloroplasts was three times longer than that of free chloroplasts. 3-Phosphoglyceraldehyde and other compounds were produced continuously by immobilized chloroplasts.     

Polyvinylamine‐coated polyester fibers as a carrier matrix for the immobilization of peroxidases

Biocatalytic transformations that employ immobilized enzymes become increasingly important for industrial applications. Synthetic or natural textile fiber materials such as polyester, polyamide or viscose are support materials that are comparatively inexpensive. Contrary to traditional support materials, their flexibility enables their use in reactors of any geometry and a fast and residue‐free removal from batch reactors. In this study a permanently immobilized peroxidase (Baylase®) has been investigated on polyester felt as a solid support as a new heterogeneous catalyst system. The polyester felt was functionalized by coating with polyvinylamine and subsequent activation with glutaraldehyde as a crosslinking agent. The enzyme load on the textile surface, the activity of the immobilized protein after repeated use as well as the storage stability was evaluated. Scanning electron micrographs and UV Vis spectroscopy made it possible to verify the enzyme immobilization on the textile surface. Furthermore, the load of immobilized peroxidase was determined by ICP OE spectrometry to be 9–12 mg per gram of textile. The activity of immobilized Baylase® remained high over 35 reaction cycles and a storage period of 8 weeks.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

Clinical uses of enzyme electrode probes

Clinical applications of enzyme electrochemical sensors are reported; they are based on the coupling of enzymes with potentiometric membrane electrodes (pH, iodide) or potentiometric probes (ammonia, carbon dioxide) or amperometric devices (oxygen, hydrogen peroxide). The most popular and successful immobilization procedures for enzymes are reviewed, namely physical entrapments and chemical methods for binding enzymes to solid support like collagen and nylon net; procedures specifically developed for clinical uses of enzyme probes.The simplicity of the apparatus is evidenced, and it is explained how a single instrument can be useful for all kind of measurements. Practical suggestions for constructing a typical probe are given. Single paragraphs are devoted to the determination of urea, cholesterol, creatinine, amino acids, glucose, lactate, protein, choline and acetylcholine to clarify the sequence of enzymatic and electrochemical reactions in order to elucidate the application range the sensitivity and the selectivity as well as the relevant interferences for each metabolite either in the enzymatic or in the electrochemical step. The applications performed in vitro, in vivo and ex vivo and the commercial availability of some instruments are reported.     

Structured fiber supports for gas phase biocatalysis

Pseudomonas cepaciae lipase adsorbed onto non-porous structured fiber supports in the form of woven fabrics, was used to catalyze hydrolysis and transesterification reactions in the gas phase. The enzyme adsorbed onto carbon fiber support exhibited much higher catalytic activity compared to the enzyme immobilized onto glass fiber carrier. The effect of temperature and relative humidity on reactions catalyzed by P. cepaciae lipase adsorbed onto structured fiber carbon support was studied in the gas system. Under the conditions investigated (up to 60 °C and 80% relative humidity), the immobilized enzyme showed a high thermostability and could be efficiently used to catalyze hydrolytic and transesterification reactions in continuous mode. Structured fiber supports, with a high specific surface area and a high mechanical resistance, showed a low-pressure drop during the passage of reactants through a reactor. The approach proposed in this study could be suitable for immobilization of a wide variety of enzymes.     

Production,purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum

Carbon dioxide capture technologies have the potential to become an important climate change mitigation option through sequestration of gaseous CO₂. A new concept for CO₂ capture involves use of immobilized carbonic anhydrase (CA) that catalyzes the reversible hydration of CO₂ to HCO₃^? and H⁺. Cost‐efficient production of the enzyme and an inexpensive immobilization system are critical for development of economically feasible CA‐based CO₂ capture processes. An artificial, bifunctional enzyme containing CA from Neisseria gonorrhoeae and a cellulose binding domain (CBD) from Clostridium thermocellum was constructed with a His₆ tag. The chimeric enzyme exhibited both CA activity and CBD binding affinity. This fusion enzyme is of particular interest due to its binding affinity for cellulose and retained CA activity, which could serve as the basis for improved technology to capture CO₂ from flue gasses. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Further facile immobilization of enzymes on hydrous metal oxides and use of their immobilization reversibility phenomena for the recovery of peptide antibiotics

Practical aspects of the facile immobilization of enzymes on hydrous metal oxides, a well-established means of enzyme-movement restriction, are described. Various enzymes (e.g. glucoamylase, peroxidase, dextranase) have been immobilized by chelation of several hydrous metal oxides, those of titanium(IV) and zirconium(IV) proving to be the most satisfactory for practical purposes. Localization of the gel into a granular form could be achieved successfully with good enzyme-immobilization characteristics by using ion-exchange resin as an internal matrix. The immobilization process was highly efficient for the relative proportions of hydrous oxide to enzyme used, with usually >90% of the available protein being insolubilized. Retention of enzyme activity was generally very good and was stable to reuse and to conventional buffer conditions. Activities of the immobilized enzymes were partially stable to lyophilization or drying of the hydrous oxide gels. Modification of the hydrous metal oxide surface by drying or treatment with phosphate or carbonate led to a decrease in complexing ability. The effect of carbonate can be circumvented by lowering the pH of the solution to around 5 and removing any carbon dioxide formed, by aeration. Such treatment allowed compounds to chelate to hydrous zirconium oxide(IV) in the presence of carbonate and therefore the hydrous oxide could be applied successfully to the concentration of peptide antibiotics from the fermentation medium in which they are being produced, including production at low concentrations.     

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。     

Biocatalysis with immobilized Escherichia coli

Immobilization is one of the great tools for developing economically and ecologically available biocatalysts and can be applied for both enzymes and whole cells. Much research dealing with the immobilization of Escherichia coli has been published in the past two decades. E. coli in the form of immobilized biocatalyst catalyzes many interesting reactions and has been used mainly in laboratories, but also on an industrial scale, leading to the production of valuable substances. It has the potential to be applied in many fields of modern biotechnology. This paper aims to give a general overview of immobilization techniques and matrices suitable mostly for entrapment, encapsulation, and adsorption, which have been most frequently used for the immobilization of E. coli. An extensive analysis reviewing the history and current state of immobilized E. coli catalyzing different types of biotransformations is provided. The review is organized according to the enzymes expressed in immobilized E. coli, which were grouped into main enzyme classes. The industrial applications of immobilized E. coli biocatalyst are also discussed.     

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 CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.     

Immobilization of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> β-galactosidase on low-pressure plasma-modified cellulose acetate membrane using polyethyleneimine for production of galactooligosaccharide

The aim of this study was to produce galactooligosaccharides (GOS) from lactose using β-galactosidase from Aspergillus oryzae immobilized on a low-pressure plasma-modified cellulose acetate (CA) membrane. Specifically, a novel method was developed for multilayer enzyme immobilization involving polyethyleneimine (PEI)-enzyme aggregate formation and growth on a CA membrane. A large amount of enzyme (997 μg/cm² membrane) was immobilized with 66% efficiency. The K _m value for the immobilized enzyme was estimated to be 48 mM, which indicates decreased affinity for the substrate, whereas the Vmax value was smaller. The immobilized enzyme showed good storage and operational stability. The half-life of the immobilized enzyme on the membrane was about 1 month at 30°C and ∼ 60 h at 60°C. Maximum GOS production of 27% (w/w) was achieved with 70% lactose conversion from 320 g/L of lactose at pH 4.5 and 60°C. Trisaccharides were the major types of GOS formed and accounted for about 75% of the total GOS produced. Based on these results, immobilized enzyme technology could be applied to GOS production from lactose.