Zhilei Tan

Mesoporous phenylalanine ammonia lyase microspheres with improved stability through calcium carbonate templating

Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising method for enzyme immobilization due to its simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications in some cases. Here, monodisperse spherical CLEAs of phenylalanine ammonia lyase (PAL microspheres) were prepared based on CaCO3 microtemplates. The preparation procedure involves filling porous CaCO3 microtemplates with the protein by salt precipitation, glutaraldehyde crosslinking, and dissolution of the microtemplates. The formulation of CaCO3 templates with controlled size was studied in detail. Characterization of the prepared PAL microspheres was investigated. The results showed that the PAL microspheres with high immobilization efficiency (79%) exhibited excellent stability, including increased tolerance to proteolysis, low pH, and denaturants, and excellent mechanical properties. For example, free PAL almost lost all activity after they were incubated in the presence of trypsin for 2min, whereas PAL microspheres still retained 95% of their initial activity. Moreover, scanning electron microscope, transmission electron microscope, and N2 adsorption-desorption isotherms revealed that the resultant PAL microspheres possessed good monodispersity and mesoporous structure instead of the amorphous clusters of conventional CLEAs with few pores. Compared with conventional CLEAs, the monodisperse PAL microspheres with mesoporous make them more potentially useful for biomedical and biotechnological applications.

Analysis of metabolic responses of Dunaliella salina to phosphorus deprivation

It has been reported that phosphorus deprivation can induce β-carotene and triacylglycerol accumulation in Dunaliella salina cells. In this study, we aimed to elucidate the metabolic responses of D. salina to phosphorus deprivation, using gas chromatography-mass spectrometry as analytical tool. A total of 79 metabolites were identified in cells cultured in either phosphorus-deprived or replete media, including 18 amino acids, 28 other acids, 16 sugars, 12 alcohols, and 5 amino compounds. Hierarchical clustering was used to sort these metabolites into three groups with different change trends. Most amino acids and sugars, including the abiotic stress-related metabolites lysine, proline, trehalose, talose, and tagatose, increased, whereas N,N-dimethylglycine, L-serine, D-erythro-pentose, and D-ribose remained constant upon phosphorus deprivation. Multivariate statistical partial least squares and principal component analyses indicated that metabolite profiles were significantly changed upon phosphorus deprivation, and 18 biomarkers which can be used to distinguish the two culture conditions were identified. Stress-related polyamines such as cadaverine, antioxidants such as L-ascorbic acid, and L-methionine, as well as the osmolytes proline, mannitol, and arabitol, also increased. Furthermore, phosphorus deprivation resulted in increases of both saturated and unsaturated fatty acids in D. salina cells. These results suggest that phosphorus deprivation triggers comprehensive metabolic responses in D. salina which may be useful for future bioprocesses.

The relationship between monosaccharide composition of extracellular polysaccharide and activities of related enzymes in Nostoc flagelliforme under different culture conditions

The relationship between monosaccharide composition of Nostoc flagelliforme extracellular polysaccharide (EPS) and activities of EPS synthesis enzymes under various carbon sources, nitrogen sources and light culture condition was investigated. Culture conditions showed significant influences on both monosaccharide composition and related enzyme activities. Under both carbon and nitrogen sources conditions, mannose mole percentage was increased with the increase of initial mole ratio of C/N and positively related to fructose-1, 6-bisphosphatase activity, and glucuronic acid and galactose mole percentages were positively correlated with UDP-glucose dehydrogenase, while arabinose and rhamnose mole percentages were negatively associated with UDP-glucose pyrophosphorylase. Different correlation between monosaccharide composition and enzymes activity from carbon and nitrogen sources conditions was found under light condition. These findings will be helpful to establish a novel fermentation process aimed to produce the N. flagelliforme EPS with desired monosaccharide composition.

The physiological responses of terrestrial cyanobacterium Nostoc flagelliforme to different intensities of ultraviolet-B radiation

Nostoc flagelliforme is a pioneer organism in the desert and exerts important ecological functions. The habitats of N. flagelliforme are characterized by intense solar radiation, while the ultraviolet B (UV-B) tolerance has not been fully explored yet. To evaluate the physiological responses of N. flagelliforme to UV-B radiation, three intensities (1 W m−2, 3 W m−2 and 5 W m−2) were used, and the changes in photosynthetic pigments, cell morphology, mycosporine-like amino acids (MAAs) synthesis and cell metabolism were comparatively investigated. Under high UV-B intensity or long term radiation, chlorophyll a, allophycocyanin and phycocyanin were greatly decreased; scanning electron microscope observations showed that cell morphology significantly changed. To reduce the damage, cells synthesized a large amount of carotenoid. Moreover, three kinds of MAAs were identified, and their concentrations varied with the changes of UV-B intensity. Under 1 W m−2 radiation, cells synthesized shinorine and porphyra-334 against UV-B, while with the increase of intensity, more shinorine turned into asterine-330. Metabolite profiling revealed the contents of some cytoprotective metabolites were greatly increased under 5 W m−2 radiation. The principal component analysis showed cells exposed to UV-B were metabolically distinct from the control sample, and the influence on metabolism was particularly dependent on intensity. The results would improve the understanding of physiological responses of N. flagelliforme to UV-B radiation and provide an important theoretical basis for applying this organism to control desertification.

Effects of ε-Poly-l-lysine on the cell wall of Saccharomyces cerevisiae and its involved antimicrobial mechanism

ε-Poly-l-lysine (ε-PL) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the antimicrobial mechanism of ε-PL against Saccharomyces cerevisiae (S. cerevisiae) is only vaguely described. Especially, it is widely accepted that membrane disruption is its main antimicrobial mode of action, but its effect on the cell wall remains unclear. In this study, the effects of ε-PL on cell wall of S. cerevisiae were investigated, and the possible action mode of ε-PL on the cell wall was discussed. The results showed that ε-PL affected significantly the cell wall composition such as β-1, 3-glucan, mannosylphosphate and chitin, and caused cell wall more fragile. The cell wall permeability was significantly increased. Furthermore, ε-PL induced the intracellular accumulation of reactive oxygen species (ROS), as well as lead to DNA fragmentation. These results indicate that ε-PL may have a complicated antimicrobial mode of action with multi-target mechanisms against S. cerevisiae cells.

Effect of Yeast Extract on Production of ε-poly-L-lysine by Streptomyces diastatochromogenes

In order to discuss the effect of yeast extract on e-poly-L-lysine (e-PL) fermentation, the effect of yeast extract which are from different factories on fermentation of Streptomyces diastatochromogenes 6#-7 was performed. The result shows that there is a significant difference when adding yeast extract which are from different factories (p 0.05).In addition, the effect of different concentration of yeast extract in medium on fermentation of strain 6#-7 was investigated. When the concentration of yeast extract is 15 g/L, the e-PL yield of strain 6#-7 can be up to 1.34 g/L and has a 6-fold enhancement compared to the group without yeast extract. The study of the key enzyme activities indicated that the improvement of e-PL may be due to the concentration of yeast extract and the growing status of cell can be evaluated by the value of aspartokinase (Ask), e-PL synthetase (Pls) and homoserine dehydrogenase (HSD).

Breeding of Streptomyces diastatochromogenes for Mass-Producing ε-Poly-L-Lysine by Composite Mutation

In order to improve the productivity of e-Poly-l-Lysine (e-PL) by Streptomyces diastatochromogenes Cbγ4, the selection and breeding of mass-producing strains were carried out through the single spore suspension treated with several groups of the composite mutation of NTG and UV and LiCl. The result showed that a high yield producing strain 6#−7 was obtained through 0.8 g/L NTG for 45 min, followed with UV irradiation for 45 s under magnetic stirring, and then through a series of screening, such as resistant plate, methylene blue plate, shake-flask screening. With the good genetic stability, the yield of e-PL could reach 0.775 ± 0.046 g/L at 72 h, 42.2 % higher than the initial strain. Moreover, the mutant displayed a great potential for increasing productivity in later period of fermentation.