Enzymatic Crosslinking Reaction Research Articles

From leather wastes to leather: enhancement of low quality leather using collagen recovered from leather tanned wastes

Leather industry produces huge amounts of solid wastes. In the last decade, several methods for the recovery and valorization of these wastes were developed, mainly focused on the extraction of collagen using chemical methods. The extracted collagen, due to its poor quality, is mostly used in agriculture as a nitrogen source ingredient of fertilizers. This study aims to apply collagen, extracted from leather tanned solid wastes using a recently reported new process based on enzymatic hydrolysis, as filling agent for low quality leather. Thanks to the enzymatic hydrolysis, collagen can be extracted without affecting its integrity and, therefore, its quality. In order to use the extracted collagen as filler for low quality leather, an enzymatic mediated cross-linking reaction between collagen and casein was developed. The enzymatic cross-linking reaction was added as an additional phase of the re-tanning process or as a replacement of one of the re-tanning steps. To evaluate the filling effect, thickness of the treated leather was measured and infrared and microscopy analyses were performed, comparing the new methods to the traditional standard one. The mechanical properties of the filled leather were tested and the sensorial features, such as fullness and touch feelings, were estimated through a panel test. Results suggest the high potential of extracted collagen to be employed back in leather processing both as additive and as substitutive filler.Graphical abstractAims and goals of the research study: comparison between re-designed (green workflow) and traditional re-tanning process (grey workflow).

Bioprinted Gelatin-Recombinant Type III Collagen Hydrogel Promotes Wound Healing.

Artificial skins are biomaterials that can replace the lost skin or promote the regeneration of damaged skin. Skin regenerative biomaterials are highly applauded because they can exempt patients with severe burns from the painful procedure of autologous skin transplantation. Notwithstanding decades of research, biocompatible, degradable, and printable biomaterials that can effectively promote skin regeneration as a transplantation replacement in clinical use are still scarce. Here, we report one type of all-protein hydrogel material as the product of the enzymatic crosslinking reaction of gelatin and a recombinant type III collagen (rColIII) protein. Doping the rColIII protein in gelatin reduces the inflammatory response as an implant underneath the skin. The all-protein hydrogel can be bioprinted as scaffolds to support the growth and proliferation of 3T3 fibroblast cells. The hydrogel used as a wound dressing promotes wound healing in a rat model of skin damage, showing a faster and healthier recovery than the controls. The rColIII protein in the hydrogel has been shown to play a critical role in skin regeneration. Altogether, this work manifests the development of all-protein gelatin-rColIII hydrogel and demonstrates its use in wound healing. The gelatin-collagen hydrogel wound dressing thereby may become a promising treatment of severe wounds in the future.

Injectable gelatin-poly(ethylene glycol) adhesive hydrogels with highly hemostatic and wound healing capabilities

Although numerous poly(ethylene glycol) (PEG)-based bioadhesives have been developed and commercialized for various biomedical applications, their biocompatibility and bioactivity performances are generally lacking. In addition, the development of adhesive hydrogels with rapid gelation and high tissue adhesion are desirable to promote the surgical procedures and enhance patient compliance. To overcome these problems, we designed a series of chemically crosslinked gelatin-PEG adhesive hydrogels (GP) through the enzymatic crosslinking reaction of horseradish peroxidase (HRP). The resultant GP hydrogels were rapidly formed in situ within seconds to minutes after injection, and their mechanical as well as adhesive properties were adjustable in a wide range by changing the molecular weight and content of PEG. Notably, the hybrid hydrogels composed of 20 kDa PEG with ratio of 2.5/7.5 gelatin/PEG (wt/wt) showed 11 times greater tissue adhesiveness than commercially available fibrin glues. From in vitro cell studies, the hybrid adhesive hydrogels are nontoxic and improve the cell proliferation. Importantly, the hydrogels exhibited excellent hemostatic capability and accelerated the wound healing in vivo, compared to fibrin glue. These injectable GP hydrogels effectively improved the tissue adhesiveness and bioactivity of PEG-based adhesives, and hold great potential for hemostatic and wound healing treatments.

Production of edible film based on catfish (Clarias batrachus) protein results of enzymatic cross-linking by transglutaminase

The existence of plastic is useful for various kinds of packaging purposes in our activities. However, it takes around 500 years to decompose because these plastics are made from petroleum. Thus the production of plastic is in high dependency on oil stock. Due to this problem, an edible film can be an alternative packaging because it is biodegradable. Protein is the most potential biopolymer to be an edible film but still needs improvement. The enzymatic cross-linking reaction can be selected as a good method to improve protein-based edible film’s mechanical properties. In this study, catfish meat (Clarias batrachus) was used as a protein source to produce an edible film through enzymatic cross-linking reaction with The Transglutaminase enzyme (TG-ase). The results of this protein-based edible film showed an improvement in thickness where TG-05 is the thickest film. The solubility decreases from 42.86 % to 20 %. Tensile Strength (TS) and Elongation at Break (EAB) results have range value from 1.630 ± 0.19-10.032 ± 1.316 MPa and 10.733 ± 0.6798-31.9 ± 6.531 %, respectively. FTIR analysis showed the change in Amide II. The biodegradable test showed that edible film could degrade in 6 days.

Injectable hydrogels functionalized with antibody fragments as intra-articular cytokine sinks neutralizing pro-catabolic cytokines

Purpose: Intra-articular (IA) injection with biologics like cytokine neutralizing antibodies to treat osteoarthritis (OA) have been explored, but so far success is limited. One of the key obstacle for the efficacy of intra-articular therapies is the short retention time of these biologics in the joint space due to rapid clearing by the synovial membrane. To overcome these limitations, we developed a new concept that potentially can increase the therapeutic value of pro-inflammatory cytokine neutralizing antibody fragments in the management of OA. The concept is based on the use of injectable hydrogels functionalized with the variable domain of single chain heavy chain only antibodies (VHHs) for cytokine capture. VHHs can be easily isolated using phage-display technology, recombinantly engineered and produced in yeast. These hydrogels can either be pre-crosslinked in microgels before injection or injected as polymer-VHH conjugates that cross link in a hydrogel in situ. Here, hyaluronic acid was used as model polymer after substitution with tyramine and maleimide groups. The tyramine moiety is used in an enzymatic cross-linking reaction to yield stable macromolecular hydrogel networks. The maleimide group is used for thiol-maleimide chemistry coupling a recombinantly engineered VHH with a free Cysteine in its C-terminal tail to the polymer backbone. We hypothesized that this strategy to functionalize hydrogels with the VHH can retain biological activity of neutralizing VHH. To proof the concept of these so-called cytokine sinks we have selected an anti-TNFα VHH that effectively neutralizes TNFα, which plays an essential role in cartilage degradation in inflammatory arthritis. Methods: To achieve directed conjugation of the VHH to the polymer, we have introduced an unpaired cysteine by using recombinant DNA technology in the C-terminus of the anti-TNFα VHH. Afterwards, the modified VHH was produced in yeast and purified. The conjugation of the VHH to the hyaluronic acid was performed by incubating the VHH with tyramine and maleimide functionalized hyaluronic acid. Characterization of binding affinity for both the non-conjugated VHH and conjugated VHH were measured by ELISA. hydrogels functionalized with the VHH was prepared by mixing the hyaluronic acid-VHH conjugates with enzyme peroxidase and H2O2. Biological activity of the conjugated VHH and hydrogels functionalized with the VHH was measured using an NFκb responsive luciferase reporter cell line after stimulation with TNFα. An equimolar concentration of non-conjugated VHH was used as a control. Results: We show successful conjugation of the VHH to the polymer backbone. The VHH functionalized polymer could be used for making stable hydrogels after tyramine mediated cross linking. This modification did not affect the biological activity of the VHH. Using an NFκb responsive luciferase reporter cell line we demonstrated that hydrogels functionalized with the VHH efficiently inactivated TNFα and that this inhibition was comparable to inhibition by an equimolar concentration of non-conjugated VHH. Conclusions: We successfully developed a biocompatible and efficient way to couple VHH to hyaluronic acid. These conjugates could be used for in situ generation of cytokine sinks capable of capturing different pro-catabolic cytokines in treatment of OA. Our Results demonstrate that cytokine sinks have great potential for neutralizing inflammatory pro-catabolic cytokines after intra-articular injection by increasing the retention time of neutralizing antibody fragments in the joint cavity.

Tunable and high tissue adhesive properties of injectable chitosan based hydrogels through polymer architecture modulation

Chitosan-based hydrogels have been widely used for various biomedical applications due to their versatile properties such as biocompatibility, biodegradability, muco-adhesiveness, hemostatic effect and so on. However, the inherent rigidity and brittleness of pure chitosan hydrogels are still unmanageable, which has limited their potential use in biomaterial research. In this study, we developed in situ forming chitosan/PEG hydrogels with improved mechanical properties, using the enzymatic crosslinking reaction of horseradish peroxidase (HRP). The effect of PEG on physico-chemical properties of hybrid hydrogels was thoroughly elucidated by varying the content (0–100 %), molecular weight (4, 10 and 20 kDa) and geometry (linear, 4-arm) of the PEG derivatives. The resulting hydrogels demonstrated excellent hemostatic ability and are highly biocompatible in vivo, comparable to commercially available fibrin glue. We suggest these chitosan/PEG hybrid hydrogels with tunable physicochemical and tissue adhesive properties have great potential for a wide range of biomedical applications in the near future.

Transglutaminase-mediated crosslinking of a host defence peptide derived from human apolipoprotein B and its effect on the peptide antimicrobial activity

BackgroundMicrobial transglutaminase (mTG) has been successfully used to produce site-specific protein conjugates derivatized at the level of Gln and/or Lys residues for different biotechnological applications. Here, a recombinant peptide identified in human apolipoprotein B sequence, named r(P)ApoBL and endowed with antimicrobial activity, was studied as a possible acyl acceptor substrate of mTG with at least one of the six Lys residues present in its sequence.MethodsThe enzymatic crosslinking reaction was performed in vitro using N,N-dimethylcasein, substance P and bitter vetch (Vicia ervilia) seed proteins, well known acyl donor substrates in mTG-catalyzed reactions. Mass spectrometry analyses were performed for identifying the Lys residue(s) involved in the crosslinking reaction. Finally, bitter vetch protein-based antimicrobial films grafted with r(P)ApoBL were prepared and, their biological activity evaluated.Resultsr(P)ApoBL was able to be enzymatically modified by mTG. In particular, it was demonstrated the highly selective crosslinking of the peptide under study by mTG at level of Lys-18. Interestingly, the biological activity of the peptide when grafted into protein-based films was found to be lost following mTG-catalyzed crosslinking.Conclusionsr(P)ApoBL was shown to be an effective acyl acceptor substrate of mTG. The involvement of Lys-18 in the enzymatic reaction was demonstrated. In addition, films grafted with r(P)ApoBL in the presence of mTG lost antimicrobial property.General significanceA possible role of mTG as biotechnological tool to modulate the r(P)ApoBL antimicrobial activity was hypothesized, and a potential use in food packaging of protein-based films grafted with r(P)ApoBL was suggested.

Enzyme-Cross-linked Gelatin Hydrogel Enriched with an Articular Cartilage Extracellular Matrix and Human Adipose-Derived Stem Cells for Hyaline Cartilage Regeneration of Rabbits.

Hyaline cartilage regeneration remains clinically challenging. In this study, microbial transglutaminase was used to cross-link gelatin. The articular cartilage extracellular matrix (cECM), mainly comprising collagen type II and glycosaminoglycans (GAGs), which can support chondrogenesis, was enclosed in this enzyme-catalyzed hydrogel. After human adipose-derived stem cells (hASCs) were encapsulated in the hydrogel enriched with the cECM, the results demonstrated that the enzymatic cross-linking reaction is of low cytotoxicity. Moreover, the stem cells showed great proliferation and chondrogenic differentiation potential in the hydrogel. Most importantly, we assessed the therapeutic effects of applying a hydrogel enriched with the cECM and hASCs to repair a full-thickness osteochondral defect. At 8 weeks after surgery, the GCC group (hydrogel encapsulating cells and the cECM) exhibited a smooth articular surface with transparent new hyaline-like tissue macroscopically. According to histological analysis, inflammatory responses were hardly observed, and sound chondrocytes were aligned in the newly formed chondral layer. In addition, the GCC group exhibited significant improvement in the GAG content between weeks 4 and 8. In summary, the implantation of a gelatin hydrogel enriched with the cECM and hASCs could facilitate the hyaline cartilage regeneration significantly in rabbit knee joint models.

Mussel-Inspired Dual-Cross-linking Hyaluronic Acid/ε-Polylysine Hydrogel with Self-Healing and Antibacterial Properties for Wound Healing.

Physicians have long been calling for an inherent antimicrobial wound dressing, which will be a great progress for treating complicated infections. Here, we report a novel bioadhesive hydrogel with inherent antibacterial properties prepared by mixing modified hyaluronic acid (HA) and ε-polylysine (EPL). This hydrogel can effectively kill Gram (+) and (-) bacteria for its high positive charge density on the surface. The sol-gel transition occurs within seconds via horseradish peroxidase enzymatic cross-linking and Schiff base reaction, which also allows the hydrogel to recover completely from destruction quickly within 5 min. In an infected rat wound model, histological studies indicated that the hydrogels effectively killed bacteria on the surface of wounds and accelerated wound healing. Histological analysis indicated that the thickness of the newborn skin, the density of the newborn microvascular, granulation tissue, and the collagen of rats treated with hydrogel dressings were twice as high as those treated by commercial fibrin glue. These results indicate that the HA/EPL hydrogel has great potential as an antibacterial wound dressing for future clinical applications.

A covalently crosslinked silk fibroin hydrogel using enzymatic oxidation and chemoenzymatically synthesized copolypeptide crosslinkers consisting of a GPG tripeptide motif and tyrosine: control of gelation and resilience

A covalently crosslinked silk fibroin hydrogel was successfully formedviaan enzymatic crosslinking reaction using copolypeptides, which consist of a glycine–proline–glycine tripeptide motif and tyrosine, as linker molecules.

In situ formation of silver nanoparticles-contained gelatin-PEG-dopamine hydrogels via enzymatic cross-linking reaction for improved antibacterial activities

Hydrogels containing silver nanoparticles (AgNPs) were recently found to exhibit excellent antibacterial properties against both gram-negative/positive bacteria and fungi. In this study, we reported the synthesis of AgNPs-contained gelatin-polyethylene glycol-dopamine (AgNP-GPD) hydrogels via the in situ formation of AgNPs in GPD polypeptide solution, followed by an enzymatic cross-linking reaction to form hydrogels. The experimental results showed that the reducing reaction exerted by GPD polypeptides under physiological conditions can afford the formation of AgNPs in situ in the polypeptide solution without the need for additional reducing agents and/or processes such as UV or thermal treatments and then the hydrogelation of GPD polypeptide solution containing AgNPs were preceeded via enzymatic cross-linking reaction. It was found that the gelation time, hydrogel mechanical property, degree of swelling and degree of enzymatic degradation for both GPD and AgNP-GPD hydrogels can be tuned by varying enzyme/oxidative agent concentration, catechol content, and reducing reaction conditions such as reaction time and silver ion concentration. Importantly, AgNP-GPD hydrogels exhibit excellent antibacterial properties against gram-negative and gram-positive bacteria. This type of hydrogel is a promising biomaterial for biomedical applications including wound healing and tissue engineering.

Materials from trees assembled by 3D printing – Wood tissue beyond nature limits

Materials from trees have the potential to replace fossil based and other non-sustainable materials in everyday products, thus transforming the society back to a bioeconomy. This paper presents a 3D printing platform which mimics wood biogenesis for the assembly of wood biopolymers into wood-like hierarchical composites. The genome was substituted with G-code, the programming language which controls how the 3D printer assembles material. The rosette was replaced by the printer head for extrusion of cellulose. Instead of microtubules guiding the alignment of cellulose, the printing direction was guided by an x/y stage, thus mimicking the microfibril angle. The printed structures were locked by an enzymatic crosslinking reaction similar to what occurs in the cell wall upon lignification. Hierarchical structures characteristic for wood were designed and printed with control of density, swelling and directional strength. Accelerating the development of the 3D printing technology helps realize the circular bioeconomy where garments, packaging, furniture and entire houses are manufactured by 3D printing wood.

Assembly of Protein–Polysaccharide Complexes for Delivery of Bioactive Ingredients: A Perspective Paper

Protein-polysaccharide complexes can be created in various ways (physical mixing, enzymatic cross-linking, chemical cross-linking, and Maillard reaction), and diverse protein-polysaccharide complexes are generally grouped into non-covalent and covalent complexes. Delivery systems constructed through assembly of protein-polysaccharide complexes (DSAPC) consist of emulsion-based delivery systems, capsule-based delivery systems, molecular complexes, nanogels, core-shell particles, composite nanoparticles, and micelles. DSAPC are effective delivery vehicles in enhancing the overall efficacy of bioactive ingredients, and DSAPC may possess multiple advantages over other delivery vehicles in bioactive ingredient delivery. However, designing and applying DSAPC are still faced with some challenges, such as low loading of bioactive ingredients. Efforts are required to reconsider and improve efficiency of DSAPC in many aspects, such as controlled release and targeted delivery. On the basis of more comprehensive and deeper understandings, DSAPC can be designed more rationally for delivery of bioactive ingredients.

Enhancement of human adipose-derived stem cell spheroid differentiation in an in situ enzyme-crosslinked gelatin hydrogel.

Human adipose-derived stem cells (hASCs) can differentiate into multiple lineages and be harvested abundantly. However, the expression of pluripotency markers, which is important for the renewal and differentiation capabilities of hASCs, decreases during monolayer culture. Increasing evidence has proven that cells aggregated to form cell spheroids in 3D cell cultures better mimic the in vivo microenvironment and can enhance the expression of stemness markers. In this study, uniform hASC spheroids were formed by seeding cells in agarose microwell plates, and the size of the spheroids could be adjusted. Most importantly, the stemness expression of the spheroids increased significantly. Additionally, we utilized microbial transglutaminase (mTG), which is an enzyme that exhibits highly specific activity over a wide range of temperature and pH, to crosslink gelatin. The enzymatic crosslinking reaction is milder than physical and chemical methods, which may lead to cell death. The properties of the gelatin/mTG hydrogel were evaluated in detail. In addition, the spheroids were encapsulated in the 3D hydrogel successfully. The results showed that the hydrogel has low toxicity to the cells, which significantly proliferated in the 3D hydrogel. Moreover, the analysis of the differentiation potential indicated that the cell spheroids in the 3D hydrogel exhibited good activity, especially adipogenesis and chondrogenesis, compared to the cell suspension group. Furthermore, the in vivo data confirmed the excellent injectability and biocompatibility of the 3D hydrogel.

Gelation behaviors of denaturated pea albumin and globulin fractions during transglutaminase treatment

The behavior of pea albumins (Alb) and globulins (Glob) in a denatured state toward microbial transglutaminase (MTGase) treatment was studied by SDS-PAGE analysis, free amine group determination, dynamic rheology and confocal laser scanning microscopy. The denaturation of pea proteins by chemical reduction with dithiothreitol (DTT) or by thermal treatment at 80 °C enhanced the enzymatic crosslinking degree of both protein isolates with greater crosslinking for the Glob fraction. The chemical denaturation affected preferentially the participation in crosslinking reaction of legumin acid subunits (40 kDa) for Glob sample and albumin polypeptides PA2 (26 kDa) for Alb sample, whereas the heat treatment led to complete polymerization of 55, 35 and 30 kDa vicilin polypeptides for pea globulins as shown by SDS-PAGE analysis. Up to 10 wt% concentration, the Alb fraction was not able to form MTGase crosslinked gels whatever the initial native or denaturated state. Compared to the native state, chemical and thermal denaturation of Glob fraction before enzymatic treatment led to the formation of weaker and stronger viscoelastic gels respectively. These contradictory results indicated that the enzymatic crosslinking reaction is highly related to polypeptides composition and conformation of proteins and the use of denaturation as a strategy to enhance gel forming properties by transglutaminase treatment has to be used with caution in the case of plant proteins.

Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture.

Growing cells within an extracellular matrix-like 3D gel is required for, or can improve, the growth of many cell types ex vivo. Here, we describe a protocol for the generation of well-defined matrices for the culture of intestinal stem cells (ISCs) and intestinal organoids. These matrices comprise a poly(ethylene glycol) (PEG) hydrogel backbone functionalized with minimal adhesion cues including RGD (Arg-Gly-Asp), which is sufficient for ISC expansion, and laminin-111, which is required for organoid formation. As such, the hydrogels present a defined and reproducible, but also tunable, environment, allowing researches to manipulate physical and chemical parameters, and examine their influence on ISC and organoid growth. Hydrogels are formed by an enzymatic cross-linking reaction of multiarm PEG precursors bearing glutamine- and lysine-containing peptides. PEG precursors containing either stable or hydrolytically degradable moieties are used to produce mechanically softening hydrogels, which are used for the expansion of ISCs or the formation of organoids, respectively. We also provide protocols for immunofluorescence analysis of cellular structures grown within these matrices, as well as for their dissociation and retrieval of cells for downstream use. Hydrogel precursors can be produced and their mechanical properties characterized to ascertain stiffness within 5-7 d. Hydrogel formation for ISC expansion or organoid formation takes 1-2 h. The materials described here can be readily adapted for the culture of other types of normal or transformed organoid structures.

Cloning, Expression, and the Effects of Processing on Sarcoplasmic-Calcium-Binding Protein: An Important Allergen in Mud Crab

Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. However, the allergen information is incomprehensive in crab. This study aimed to identify a novel allergen in crab, show its potential in diagnosis and reduce the allergenicity by food processing. A 21-kDa protein was purified from Scylla paramamosain and confirmed as sarcoplasmic calcium binding protein (SCP) by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Total RNA was isolated from crab muscle, and a rapid amplification of cDNA was performed to obtain an ORF of 579 bp that coded for 193 amino acid residues. According to the results of circular dichroism analysis and ELISA assay, the recombinant SCP (rSCP) expressed in Escherichia coli showed similar physicochemical and immunoreactive properties to native SCP (nSCP). Additionally, the extensive cross reactivity of SCP among different species and the bidirectional IgE cross-reactivity between nSCP and rSCP were detected by iELISA. The allergenicity of rSCP was reduced via Maillard reaction or enzymatic cross-linking reaction, which was confirmed by the results of scanning electron microscopy, dot blot, and digestion assay. A straightforward and reproducible way was developed to obtain high yields of rSCP that maintains structural integrity and full IgE reactivity, which could compensate the low specific IgE-titers of most patient sera for future diagnosis. Furthermore, the Maillard reaction and enzymatic cross-linking reaction were effective approaches for the production of hypoallergenic seafood.

Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering

Glycopolypeptides are an emerging class of bioinspired polymers that mimic naturally occurring glycopeptides or glycoproteins, and therefore are expected to exhibit great potential for biomedical applications. In this study, a glycopolypeptide was synthesized by conjugation of poly(γ-propargyl-l-glutamate) (PPLG) with azido-modified mannose and 3-(4-hydroxyphenyl) propanamide (HPPA), via click chemistry. Injectable hydrogels based on the glycopolypeptide were developed through enzymatic crosslinking reaction in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The physicochemical properties of the hydrogels, such as gelation time, storage modulus, swelling and degradation time, could be controlled by varying the concentrations of HRP and H2O2. The glycopolypetide copolymer as well as the extracts of the glycopolypetide hydrogels displayed good cytocompatibility in vitro. After subcutaneous injection into rats, the glycopolypeptide hydrogels were rapidly formed in situ, and exhibited acceptable biocompatibility accompanying the degradation of the hydrogels in vivo. The rabbit chondrocytes inside the glycopolypeptide hydrogels showed spherical morphology with high viability during the incubation period of 3 weeks in vitro, and exhibited a higher proliferation rate than within the hydrogel counterparts of PPLG grafted with 2-(2-(2-methoxyethoxy)ethoxy)ethane (MEO3) and HPPA. Biochemical analysis demonstrated that the production of glycosaminoglycans (GAG) and type II collagen were significantly enhanced after incubation for 2 and 3 weeks in vitro. Moreover, the chondrocyte-containing glycopolypeptide hydrogels in subcutaneous model of nude mice maintained chondrocyte phenotype and produced the cartilaginous specific matrix. These results indicated that the biomimetic glycopolypeptide-based hydrogels hold potential as three-dimensional scaffolds for cartilage tissue engineering.

Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions.

Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0 %; GluDH-A*/GluDH = 105.2 %). Additionally, in the coupled reaction system with glycerol and α-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.

Preparation of carboxymethyl cellulose based microgels for cell encapsulation

Biocompatible and biodegradable carboxymethyl cellulose (CMC) has been modified with 4-hydroxybenzy- lamine (CMC-Ph) in order to prepare CMC-based microgels through the horseradish peroxidise/hydrogen peroxide enzy- matic reaction. CMC-Ph was identified as a blend, and the amount of the grafted 4-hydroxybenzylamine per 100 units of CMC was between 17 and 23 according to the molecular weight of CMC. Through a special designed co-flowing microflu- idic device, CMC-Ph microgels were prepared with the radius from 100 to 500 µm via adjusting the flow rates of the dis- perse phase and the continuous phase, respectively. The chondrocytic cell line ATDC5 was encapsulated in the CMC-Ph microgels. The cell-laden microgels were cultured for up to 40 days, illustrating the biocompatibility of CMC-Ph and the microfluidic approach through the enzymatic crosslinking reaction primarily. CMC-Ph showed a great promise to encapsu- late the cells for further fabrication of the injectable scaffolds.