Yuhong Ren

Facile, high efficiency immobilization of lipase enzyme on magnetic iron oxide nanoparticles via a biomimetic coating.

BackgroundImmobilization of lipase on appropriate solid supports is one way to improve their stability and activity, and can be reused for large scale applications. A sample, cost- effective and high loading capacity method is still challenging.ResultsA facile method of lipase immobilization was developed in this study, by the use of polydopamine coated magnetic nanoparticles (PD-MNPs). Under optimal conditions, 73.9% of the available lipase was immobilized on PD-MNPs, yielding a lipase loading capacity as high as 429 mg/g. Enzyme assays revealed that lipase immobilized on PD-MNPs displayed enhanced pH and thermal stability compared to free lipase. Furthermore, lipase immobilized on PD-MNPs was easily isolated from the reaction medium by magnetic separation and retained more than 70% of initial activity after 21 repeated cycles of enzyme reaction followed by magnetic separation.ConclusionsImmobilization of enzyme onto magnetic iron oxide nanoparticles via poly-dopamine film is economical, facile and efficient.

Magnetic Catechol-Chitosan with Bioinspired Adhesive Surface: Preparation and Immobilization of ω-Transaminase

The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed. Here we report a simple catechol modified protocol for preparing a novel catechol-chitosan (CCS) - iron oxide nanoparticles (IONPs) composites carrying adhesive moieties with strong surface affinity. The ω-transaminase (ω-TA) was immobilized onto this magnetic composite via nucleophilic reactions between catechol and ω-TA. Under optimal conditions, 87.5% of the available ω-TA was immobilized on the composite, yielding an enzyme loading capacity as high as 681.7 mg/g. Furthermore, the valuation of enzyme activity showed that ω-TA immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme. Importantly, the immobilized ω-TA retained more than 50% of its initial activity after 15 repeated reaction cycles using magnetic separation and 61.5% of its initial activity after storage at 4°C in phosphate buffered saline (PBS) for 15 days. The results suggested that such adhesive magnetic composites may provide an improved platform technology for bio-macromolecules immobilized.

Enhancement of the activity of enzyme immobilized on polydopamine-coated iron oxide nanoparticles by rational orientation of formate dehydrogenase.

Immobilization of enzymes onto nanoparticles and retention of their structure and activity, which may be related to the orientation of enzymes on nanoparticles, remain a challenge. Here, we developed a novel enzyme-orientation strategy to enhance the activity of formate dehydrogenase immobilized on polydopamine-coated iron oxide nanoparticles via site-directed mutation. Seven mutants were constructed based on homology modeling of formate dehydrogenase and immobilized on polydopamine-coated iron oxide nanoparticles to investigate the influence of these mutations on immobilization. The immobilized mutant C242A/C275V/C363V/K389C demonstrated the highest immobilization yield and retained 90% of its initial activity, which was about 3-fold higher than that of wild-type formate dehydrogenase. Moreover, co-immobilization of formate dehydrogenase and leucine dehydrogenase was performed for the synthesis of l-tert-leucine. The catalytic efficiency of the co-immobilized mutant C242A/C275V/C363V/K389C and leucine dehydrogenase increased by more than 4-fold compared to that of co-immobilized wild-type formate dehydrogenase and leucine dehydrogenase.

Fe3+-induced oxidation and coordination cross-linking in catechol–chitosan hydrogels under acidic pH conditions

Mussel byssus is rich in Fe3+ and catechol-containing proteins; chemical interactions between these components vary widely with respect to changes in pH during byssal maturation. Previous studies have indicated the key role played by Fe3+–catechol interactions in regulating many attributes of biological materials, such as toughness, extensibility, and self-assembly. In this study, a platform based on a highly substituted catechol-modified chitosan (70%, CCS) was used to investigate the effect of pH on the reactions between Fe3+ and catechols. This study demonstrated that the Fe3+-induced CCS hydrogel is essentially a dual cross-linking system composed of covalent and coordination crosslinks, under acidic pH conditions. Variations in the Fe3+–catechol molar ratios could strongly affect the gelation time, physical properties, and UV-vis and Raman spectra. These changes represent different balance states between oxidation and coordination mechanisms in the hydrogel network. In addition, the system was subjected to optical microscopy and SEM in order to obtain a visual description of the dual-crosslinking mechanism.

A novel technique for in situ aggregation of Gluconobacter oxydans using bio‐adhesive magnetic nanoparticles

Here, we present a novel technique to immobilize magnetic particles onto whole Gluconobacter oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine‐coated nanoparticles to G. oxydans which resulted in cell aggregation. After optimization, 21.3 mg (wet cell weight) G. oxydans per milligram of nanoparticle was aggregated and separated with a magnet. Importantly, the G. oxydan aggregates showed high specific activity and good reusability. The facile approach offers the potential advantages of low cost, easy cell separation, low diffusion resistance, and high efficiency. Furthermore, the approach is a convenient platform technique for magnetization of cells in situ by direct mixing of nanoparticles with a cell suspension. Biotechnol. Bioeng. 2012; 109: 2970–2977.

Efficient production of (R)-(-)-mandelic acid in biphasic system by immobilized recombinant E. coli.

The recombinant Escherichia coli M15/BCJ2315 which harbored a mandelonitrilase from Burkholderia cenocepacia J2315 was immobilized via catecholic chitosan and functionalized with magnetism by iron oxide nanoparticles. The immobilized cells showed high activity recovery, enhanced stability and good operability in the enantioselective hydrolysis of mandelonitrile to (R)-(-)-mandelic acid. Furthermore, the immobilized cells were reused up to 15 cycles without any activity loss in completely hydrolyzing mandelonitrile (100 mM) within 1h in aqueous solution. The ethyl acetate-water biphasic system was built and optimized. Under the optimal conditions, as high as 1M mandelonitrile could be hydrolyzed within 4h with a final yield and ee value of 99% and 95%, respectively. Moreover, the successive hydrolysis of mandelonitrile was performed by repeated use of the immobilized cells for 6 batches, giving a final productivity (g L⁻¹ h⁻¹) and relative production (g g⁻¹) of 40.9 and 38.9, respectively.

One-step purification and immobilization of his-tagged protein via Ni2+-functionalized Fe3O4@polydopamine magnetic nanoparticles

Ni2+-functionalized Fe3O4@polydopamine magnetic nanoparticles (Ni2+-PD-MNPs) were designed and synthesized by in situ coating of magnetic nanoparticles with polydopamine, followed by conjugation of Ni2+ to the polydopamine film. The Ni2+-PD-MNPs were used to purify His-tagged red fluorescent protein (His-RFP) via affinity interaction between Ni2+ and the His-tag. The results showed that the Ni2+-PD-MNPs had extraordinary selectivity for His-RFP purification. In addition, a Histagged transaminase (ω-transaminase BJ110) was selectively immobilized onto the Ni2+-PD-MNPs without purification, and the immobilized enzyme showed improved specific activity, as well as enhanced stability and reusability.

Enhancing Biosynthesis of a Ginsenoside Precursor by Self-Assembly of Two Key Enzymes in Pichia pastoris.

Ginsenosides from the edible and medicinal plant ginseng have demonstrated various pharmacological activities. However, producing ginsenoside efficiently remains a challenge. Engineering metabolic pathways through protein assembly in yeast is a promising way for ginsenoside production. In the biosynthetic pathway of ginsenosides, dammarenediol-II synthase and squalene epoxidase are two key enzymes that determine the production rate of the dammarane-type ginsenoside precursor dammarenediol-II. In this work, a strategy to enhance the biosynthesis of dammarenediol-II in Pichia pastoris was developed by the self-assembly of the two key enzymes via protein-protein interaction. After being modified by interacting proteins, the two enzymes were successfully co-localized, resulting in a 2.1-fold enhancement in dammarenediol-II yields.

Enhanced itaconic acid production by self-assembly of two biosynthetic enzymes in Escherichia coli

Here, we described a novel strategy for the production of itaconic acid in Escherichia coli by self-assembly of aconitase (ACO) and cis-aconitate decarboxylase (CAD) existing in the metabolic pathway of itaconic acid via the protein-peptide interactions of PDZ domain and PDZ ligand. Co-expression of ACO and CAD in E. coli (uCA) resulted in low levels of itaconate (117.25 mg/L) after 48 h fermentation while the itaconate titre was significantly improved up to 222.15 mg/L by self-assembly of ACO-PDZ (APd) and CAD-PDZlig (CPl) in E. coli (sPP) under the same conditions. To further confirm the effect of self-assembly, the itaconate catalyzed from sodium citrate was determined. The sPP was extra efficacious in the early catalytic period, showing approximately threefold itaconate yields increased after 2 h catalysis, when compared to uCA. Furthermore, the itaconate production of sPP was increased from 5 to 8.7 g/L after 30 h of reaction compared to uCA. This self-assembly strategy showed remarkable potential for the further improvement of itaconate production. Biotechnol. Bioeng. 2017;114: 457-462.

Fe3+-induced bioinspired chitosan hydrogels for the sustained and controlled release of doxorubicin

In this study, a novel Fe3+-induced bioinspired chitosan hydrogel was developed to easily deliver the anticancer drug, doxorubicin (DOX). Catechol–chitosan conjugates (CCS) and an N-acetyl cysteine–chitosan conjugate (NACCS) were synthesized and used to prepare the hydrogels. The addition of NACCS accelerated the gelation rate and improved the mechanical strength of the hydrogels, due to the Michael addition reaction between NACCS and the oxidation product of CCS. This study demonstrated that the Fe3+-induced CCS–NACCS hydrogel was a dual covalent-coordination crosslinking system under acidic conditions. Release curves for DOX were evaluated at different pH values, and the release kinetics and mechanism were also investigated. The CCS–NACCS hydrogel showed no obvious toxicity and the DOX released from the hydrogel could effectively inhibit the proliferation of several kinds of tumor cells.