Hong Wu

Methods for the regeneration of nicotinamide coenzymes

Oxidoreductase is the largest class of enzymes and has broad applications in biotechnology since a number of bioconversions involve oxidation/reduction reactions. Coenzymes are always required in oxidoreductase-catalyzed reactions, where nicotinamide coenzymes, NAD(P)H/NAD(P)+, are the most commonly used. They undergo reactions with substrates in biocatalytic processes by converting into their reductive or oxidative forms. The efficient and economical regeneration of nicotinamide coenzymes is therefore of particular significance for industrial applications due to their high cost and large usage. The principal methods used for the regeneration of nicotinamide coenzymes, including enzymatic, chemical, electrochemical and photochemical regeneration methods are surveyed with emphasis on the crucial issues and the state-of-art research relevant to each method. Screening and improving the performance of the enzymes, designing and implementing efficient regeneration routes as well as retaining/recycling coenzymes are the three key issues for the enzymatic method. Development of efficient catalysts with high selectivity is the top priority of the chemical regeneration method. For the electrochemical regeneration method, improvement of the electrode by modification of either the nano-materials or electron mediators is the major concern. The focus of the photochemical regeneration method lies in the exploitation of efficient visible-light photosensitizers.

Metal–Organic Coordination-Enabled Layer-by-Layer Self-Assembly to Prepare Hybrid Microcapsules for Efficient Enzyme Immobilization

A novel layer-by-layer self-assembly approach enabled by metal-organic coordination was developed to prepare polymer-inorganic hybrid microcapsules. Alginate was first activated via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) coupling chemistry, and subsequently reacted with dopamine. Afterward, the dopamine modified alginate (Alg-DA) and titanium(IV) bis(ammonium lactato) dihydroxide (Ti(IV)) were alternatively deposited onto CaCO3 templates. The coordination reaction between the catechol groups of Alg-DA and the Ti(IV) allowed the alternative assembly to form a series of multilayers. After removing the templates, the alginate-titanium hybrid microcapsules were obtained. The high mechanical stability of hybrid microcapsules was demonstrated by osmotic pressure experiment. Furthermore, the hybrid microcapsules displayed superior thermal stability due to Ti(IV) coordination. Catalase (CAT) was used as model enzyme, either encapsulated inside or covalently attached on the surface of the resultant microcapsules. No CAT leakage from the microcapsules was detected after incubation for 48 h. The encapsulated CAT, with a loading capacity of 450-500 mg g(-1) microcapsules, exhibited desirable long-term storage stability, whereas the covalently attached CAT, with a loading capacity of 100-150 mg g(-1) microcapsules, showed desirable operational stability.

Constructing inorganic shell onto LBL microcapsule through biomimetic mineralization: A novel and facile method for fabrication of microbioreactors

Inspired by the biological cell structure, a novel and facile method for preparing organic–inorganic composite microcapsules is developed by a synergy of layer-by-layer (LBL) self-assembly and biomimetic mineralization. The LBL microcapsule is fabricated by the alternative assembly of a negatively charged polyelectrolyte, poly(styrene sulfonate) (PSS), and a positively charged biomacromolecule, protamine (Pro), onto calcium carbonate microparticles. An inorganic silica layer is then constructed through a biomimetic mineralization process induced by the outer protamine layer. Catalase is captured in the CaCO3 templates via co-precipitation and encapsulated in the composite capsules followed by dissolution of the templates. TEM and confocal laser scanning microscopy (CLSM) observations show that the microcapsules possess a hollow structure and the enzyme inside exists in a free state. The morphology and chemical composition of the microcapsules are characterized by SEM, FTIR and NMR. The dual role of protamine as both a positive layer component and an inducer for silicification provides a simple and efficient approach to form a microbioreactor with a complete, uniform and robust outer shell. Compared to the Pro/PSS LBL microcapsule, the encapsulation efficiency, harsh condition tolerance and long-term storage stability of the encapsulated enzyme are all notably improved due to the shielding effect of the inorganic shell. The fabrication method presented here may provide a general strategy for the preparation of composite materials whose structure of organic membrane and inorganic shell could be easily tuned by varying the LBL and mineralization conditions.

Facile One-Pot Preparation of Chitosan/Calcium Pyrophosphate Hybrid Microflowers

Flower-like chitosan/calcium pyrophosphate hybrid microparticles (microflowers) are prepared using a facile one-pot approach by combining ionotropic gelation with biomimetic mineralization. Chitosan-tripolyphosphate (CS-TPP) nanocomplexes are first synthesized through ionotropic gelation; meanwhile, excess TPP is partly hydrolyzed into pyrophosphate ions (P2O7(4-)). Upon addition of CaCl2, CS-TPP nanocomplexes serve as a versatile template, inducing in situ mineralization of Ca2P2O7 and directing its growth and assembly into microflowers. The whole preparation process can be completed within half an hour. The as-prepared microflowers are composed of 23.0% CS-TPP nanocomplexes and 77.0% Ca2P2O7 crystals. Mesopores (3.7 and 11.2 nm) and macropores coexist in the microflowers, indicating porous and hierarchical structures. The microflowers exhibit high efficiency in dye adsorption and enzymatic catalysis. Specifically, a high adsorption capacity of 520 mg g(-1) for Congo red is achieved. And the immobilized enzyme retains about 85% catalytic activity compared with that of the free enzyme. The facile one-pot preparation process ensures the broad applications of the porous hybrid microflowers.

Coordination-Enabled One-Step Assembly of Ultrathin, Hybrid Microcapsules with Weak pH-Response

In this study, an ultrathin, hybrid microcapsule is prepared though coordination-enabled one-step assembly of tannic acid (TA) and titanium(IV) bis(ammonium lactate) dihydroxide (Ti-BALDH) upon a hard-templating method. Briefly, the PSS-doped CaCO3 microspheres with a diameter of 5-8 μm were synthesized and utilized as the sacrificial templates. Then, TA-Ti(IV) coatings were formed on the surface of the PSS-doped CaCO3 templates through soaking in TA and Ti-BALDH aqueous solutions under mild conditions. After removing the template by EDTA treatment, the TA-Ti(IV) microcapsules with a capsule wall thickness of 15 ± 3 nm were obtained. The strong coordination bond between polyphenol and Ti(IV) conferred the TA-Ti(IV) microcapsules high structural stability in the range of pH values 3.0-11.0. Accordingly, the enzyme-immobilized TA-Ti(IV) microcapsules exhibited superior pH and thermal stabilities. This study discloses the formation of TA-Ti(IV) microcapsules that are suitable for use as supports in catalysis due to their extensive pH and thermal stabilities.

Nanotube-doped alginate gel as a novel carrier for BSA immobilization.

In this paper different dopants, including carbon nanotubes (CNTs), silica nanotubes (SiNTs), graphite and silica gel, were incorporated into alginate (ALG) gel to form nanotube/alginate or nanoparticle/alginate composites used for bovine serum albumin (BSA) immobilization carrier. During encapsulation, first BSA was adsorbed on the dopants, and then BSA and dopants were suspended in alginate solution, followed by being added dropwise into the CaCl2 solution to form the biocomposites. The BSA leakage decreased significantly in these biocomposites as the following order: BSA-ALG-SiNT > BSA-ALG-CNT > BSA-ALG-SiO2 > BSA-ALG-graphite, which was mainly due to firstly the protein molecule adsorption on the dopants which could not be washed out easily and secondly, during the biocomposites formation, water loss in BSA-ALG-dopant biocomposites became less than that in BSA-ALG biocomposite. In addition, the mechanical properties of these biocomposites were remarkably reinforced in the same order as BSA leakage decrease.

Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres

In this study, a novel approach combing the chelation and covalent binding was explored for facile and efficient enzyme immobilization. The unique capability of titania to chelate with catecholic derivatives at ambient conditions was utilized for titania surface functionalization. The functionalized titania was then used for enzyme immobilization. Titania submicrospheres (500-600 nm) were synthesized by a modified sol-gel method and functionalized with carboxylic acid groups through a facile chelation method by using 3-(3,4-dihydroxyphenyl) propionic acid as the chelating agent. Then, catalase (CAT) was covalently immobilized on these functionalized titania submicrospheres through 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. The immobilized CAT retained 65% of its free form activity with a loading capacity of 100-150 mg/g titania. The pH stability, thermostability, recycling stability and storage stability of the immobilized CAT were evaluated. A remarkable enhancement in enzyme stability was achieved. The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 successive cycles of decomposition of hydrogen peroxide, respectively. Both the Km and the Vmax values of the immobilized CAT (27.4 mM, 13.36 mM/min) were close to those of the free CAT (25.7 mM, 13.46 mM/min).

Immobilization of β -glucuronidase in lysozyme-induced biosilica particles to improve its stability

Mesoporous silica particles were prepared for efficient immobilization of the β-glucuronidase (GUS) through a biomimetic mineralization process, in which the solution containing lysozyme and GUS were added into the prehydrolyzed tetraethoxysilane (TEOS) solution. The silica particles were formed in a way of biomineralization under the catalysis of lysozyme and GUS was immobilized into the silica particles simultaneously during the precipitation process. The average diameter of the silica particles is about 200 nm with a pore size of about 4 nm. All the enzyme molecules are tightly entrapped inside the biosilica nanoparticles without any leaching even under a high ionic strength condition. The immobilized GUS exhibits significantly higher thermal and pH stability as well as the storage and recycling stability compared with GUS in free form. No loss in the enzyme activity of the immobilized GUS was found after 30-day’s storage, and the initial activity could be well retained after 12 repeated cycles.

Conferring Natural-Derived Porous Microspheres with Surface Multifunctionality through Facile Coordination-Enabled Self-Assembly Process

In this study, multifunctional chitin microspheres are synthesized and utilized as a platform for multiple potential applications in enzyme immobilization, catalytic reduction and adsorption. Porous chitin microspheres with an average diameter of 111.5 μm and a porous architecture are fabricated through a thermally induced phase separation method. Then, the porous chitin microspheres are conferred with surface multifunctionality through facile coordination-enabled self-assembly of tannic acid (TA) and titanium (Ti(IV)) bis(ammonium lactate)dihydroxide (Ti-BALDH). The multipoint hydrogen bonds between TA and chitin microspheres confer the TA-Ti(IV) coating with high adhesion capability to adhere firmly to the surface of the chitin microspheres. In view of the biocompatibility, porosity and surface activity, the multifunctional chitin microspheres are used as carriers for enzyme immobilization. The enzyme-conjugated multifunctional porous microspheres exhibit high catalytic performance (102.8 U·mg(-1) yeast alcohol dehydrogenase). Besides, the multifunctional chitin microspheres also find potential applications in the catalytic reduction (e.g., reduction of silver ions to silver nanoparticles) and efficient adsorption of heavy metal ions (e.g., Pb(2+)) taking advantages of their porosity, reducing capability and chelation property.

Facile Fabrication of Organic–Inorganic Hybrid Beads by Aminated Alginate Enabled Gelation and Biomimetic Mineralization

Abstract Inspired by biomineralization, design and preparation of biomimetic organic–inorganic composites have become a hot issue and a research frontier in many areas, including enzyme engineering. In this research, a unique and facile method for fabricating organic–inorganic hybrid beads is proposed. Modified alginate with a dual function of gelation and mineralization was synthesized for fabrication of hybrid carriers for enzyme immobilization. With the aid of EDC/NHS conjugation chemistry, the amine groups from diethylene triamine were grafted onto alginate in a controllable way. The resultant aminated alginate served manifold functions: forming a hydrogel via Ca2+-cross-linking, inducing the biomimetic silicification and manipulating the distribution of silica nanoparticles. Owing to the compact polymer network structure and the homogeneous silica nanoparticle dispersion, the as-prepared NH2-alginate/silica hybrid beads displayed superior swelling resistance and mechanical stability to pure alginate beads. The hybrid beads were subsequently utilized for encapsulation of yeast alcohol dehydrogenase (YADH). It was found that the thermal stability, pH tolerance and storage stability of the immobilized enzyme were all improved without significantly lowering the catalytic activity.