Biocatalytic strategies for the production of ginsenosides using glycosidase: current state and perspectives.

Panax ginseng is a traditional Chinese medicine with significant pharmaceutical effects and broad application. Rare ginsenosides with high antitumor activities can be generated via oriented modification of their glycosyl moiety. For this purpose, suitable microorganisms and their enzymatic systems can be used. In this review, we address several issues associated with these systems. Under aerobic conditions, fungus biotransformation provides an efficient and inexpensive biotransformation process that can be easily scaled up. Considering the profound use of probiotics, wild strains generally recognized as safe have shown a potential through classical fermentation in food manufacturers of deglycosylated ginsenosides. Commonly applied recombinant enzymes from E. coli, especially recombinant hyperthermophilic enzymes, showed efficient conversion in biomedical or pharmaceutical industries. In this review, key genes dedicated to the production of ginsenosides (especially in Saccharomyces cerevisiae) are highlighted in relation to the large-scale production of ginsenosides. We also evaluate biocatalytic strategies that are aimed to improve product specificity and biocatalytic efficiency with industrial applications. Perspectives of protein engineering and solvent engineering in the development and large-scale preparation of ginsenosides in anticancer drugs, food and health care products are explored. KEY POINTS : • Modification of ginsenosides with food/engineered microorganisms is summarized. • Optimization of cell factories by protein engineering remains challenging. • Solvent engineering offers an attractive potential alternative.

Improving the production of human-like collagen by pulse-feeding glucose during the fed-batch culture of recombinant Escherichia coli.

To increase the target protein production and reduce acetic acid accumulation during fed-batch cultivation of recombinant Escherichia coli BL21 in a 30-L bioreactor, 12 different models of pulse feeding were performed to evaluate the effect of pulse feeding at different cultivation phases and pulse frequency on cell growth, acetic acid accumulation, and human-like collagen (HLC) synthesis. The results showed that the acetate concentration was kept at a low level (below 0.5 g/L) in all cases when pulse feeding was introduced before induction, whereas the pulse frequency affected cytoactivity significantly through cell growth rate, oxygen uptake rate, carbon dioxide evolution rate, and the synthesis of the target protein. The final biomass and HLC reached 75.46 and 7.26 g/L, respectively, in the model of 8-Sec feedings per 188 Sec. After induction, the pulse frequency had a great effect on HLC synthesis after high-temperature induction; low frequency was adverse to microorganisms. The model of 3-Sec feeding per 27 Sec was best and resulted in the highest biomass and HLC production. Compared to the pseudo-exponential feeding, pulse feeding reduced acetic acid accumulation effectively.

A genetic algorithm for the optimization of the thermoinduction protocol for high-level production of recombinant human-like collagen from Escherichia coli.

Production of recombinant human-like collagen (RHLC) by thermoinduction of recombinant Escherichia coli BL 21 during high cell density cultivation was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon-to-nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermoinduction protocol for RHLC production was determined by using a model coupling genetic algorithm and artificial neural networks. The optimal operating conditions were as follows: maintenance of induction temperature at 42°C for 3 H and then at 39.4°C until the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, whereas the experimental value was 12.1 g/L under the optimal induction conditions.

Magnetic nanoparticle-loaded polymer nanospheres as magnetic hyperthermia agents.

Uniform magnetic nanoparticle-loaded polymer nanospheres with different loading contents of manganese ferrite nanoparticles were successfully synthesized using a flexible emulsion process. The MnFe2O4-loaded polymer nanospheres displayed an excellent dispersibility in both water and phosphate buffer saline. The effect of loading ratio and size of MnFe2O4 nanoparticles within the nanospheres on the specific absorption rate (SAR) under an alternating magnetic field was investigated. Our results indicate that a large size (here 18 nm) and a low loading ratio are preferable for a high SAR. For a smaller particle size (6 nm), the low loading ratio did not result in an enhancement of the SAR value, while a very low SAR value is expected for 6 nm. In addition, the SAR of low-content MnFe2O4 (18 nm)-loaded polymer nanospheres in the agarose gel which is simulated for in vivo environment is the highest among the samples and does not change substantially in physiological environments. This differs largely from the behaviour of singly dispersed nanoparticles. Our results have paved the way for the design of MnFe2O4-loaded polymer nanospheres as magnetic hyperthermia agents for in vivo bio-applications.

Preparation of N,O-carboxymethyl chitosan coated alginate microcapsules and their application to Bifidobacterium longum BIOMA 5920.

In order to greatly improve vitality of probiotic bacteria within the application, a novel biocompatible vehicle, N,O-carboxymethyl chitosan (NOCs) with appropriate degrees of substitution coat alginate (ALg) microparticles, was prepared by electrostatic droplet generation. The amount of chitosan (Cs) and N,O-carboxymethyl chitosan (NOCs) coated on the ALg microparticles was determined by differential scanning calorimetry. The surface morphology of ALg microparticles, Cs coated ALg microparticles and NOCs coated ALg microparticles was determined using scanning electron microscopy. The coating thickness of Cs coated ALg microparticles and that of NOCs coated ALg microparticles was directly observed with confocal laser scanning microscopy. In order to assess pH sensitivity of microparticles, the bovine serum albumin release from the microspheres was tested in acid solution (pH 2.0) for 2 h and subsequently in alkaline solution (pH 7.0) for 2 h. The survival of Bifidobacterium longum BIOMA 5920 loaded in NOCs coated with ALg microparticle was improved in simulated gastric juice (pH 2.0, for 2 h) compared to that of B. longum BIOMA 5920 loaded in ALg microparticles and Cs coated ALg microparticles. After incubation in simulated intestinal juices (pH 7.0, 2 h), the release of microencapsulated B. longum BIOMA 5920 was investigated.

Coordination study of recombinant human-like collagen and zinc (II).

In the present investigation, the complex of recombinant human-like collagen (r-HLC) with zinc (II) has been synthesized in aqueous solution and was analyzed by UV-vis spectroscopy, FTIR spectroscopy, circular dichroism (CD) spectroscopy, thermo gravimetric (TG) and differential scanning calorimetry (DSC) analysis. It can be concluded from UV-vis spectra that there exists interaction between r-HLC and zinc, and the complex is a new chemical compound different from pure r-HLC. In the complex of Zn, recombinant human-like collagen acts as ligand, linking the zinc ion via both groups of C=O and N-H. Besides, the results of TG and DSC confirm that the complex was significantly different from ligand, and the former is more thermally stable in comparison with the latter. The results obtained from the current investigation are of crucial importance to understand the r-HLC-Zn complex and provide theoretical evidence for the further study.

Encapsulation of probiotic Bifidobacterium longum BIOMA 5920 with alginate-human-like collagen and evaluation of survival in simulated gastrointestinal conditions.

Alginate (ALg)-human-like collagen (HLC) microspheres were prepared by the technology of electrostatic droplet generation in order to develop a biocompatible vehicle for probiotic bacteria. Microparticles were spherical with mean particle size of 400µm. The encapsulation efficiency (EE) of ALg-HLC microspheres could reach 92-99.2%. Water-soluble and fibrous human-like collagen is combined with sodium alginate through intermolecular hydrogen bonding and electrostatic force which were investigated by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC), thus the matrix of ALg-HLC was very stable. Bifidobacterium longum BIOMA 5920, as a kind of probiotic bacteria, was encapsulated with alginate-human-like collagen to survive and function in simulated gastrointestinal juice. Microparticles were very easy to degradation in simulated intestinal juices. After incubation in simulated gastric (pH 2.0, 2h), the encapsulated B. longum BIOMA 5920 numbers were 4.81 ± 0.38 log cfu/g.

Breakthrough model of recombinant human-like collagen in immobilized metal affinity chromatography.

The adsorption of recombinant human-like collagen by metal chelate media was investigated in a batch reactor and in a fixed-bed column. The adsorption equilibrium and kinetics had been studied by batch adsorption experiments. Equilibrium parameters and protein diffusivities were estimated by matching the models with the experimental data. Using the parameters of equilibrium and kinetics, various models, such as axial diffusion model, linear driving force model, and constant pattern model, were used to simulate the breakthrough curves on the columns. As a result, the most suitable isotherm was the Langmuir-Freundlich model, and the ionic strength had no effect on the adsorption capacity of chelate media. In addition, the pore diffusion model fitted very well to the kinetic data. The pore diffusivities decreased with increasing the initial protein concentration, however had little change with the ionic strength. The results also indicated that the models predict breakthrough curves reasonably well to the experimental data, especially at low initial protein concentration (0.3 mg ml(-1)) and low flow rate (34 cm h(-1)). By the results, we optimized the experimental conditions of a chromatographic process using immobilized metal affinity chromatography to purify recombinant human-like collagen.