Biosynthesis of ß-nicotinamide mononucleotide from glucose via a new pathway in Bacillus subtilis.

Introduction: ß-nicotinamide mononucleotide (ß-NMN) is an essential precursor of nicotinamide adenine dinucleotide (NAD+) and plays a key role in supplying NAD+ and maintaining its levels. Existing methods for NMN production have some limitations, including low substrate availability, complex synthetic routes, and low synthetic efficiency, which result in low titers and high costs. Methods: We constructed high-titer, genetically engineered strains that produce NMN through a new pathway. Bacillus subtilis WB600 was used as a safe chassis strain. Multiple strains overexpressing NadE, PncB, and PnuC in various combinations were constructed, and NMN titers of different strains were compared via shake-flask culture. Results: The results revealed that the strain B. subtilis PncB1-PnuC exhibited the highest total and extracellular NMN titers. Subsequently, the engineered strains were cultured in a 5-L fermenter using batch and fed-batch fermentation. B. subtilis PncB1-PnuC achieved an NMN titer of 3,398 mg/L via fed-batch fermentation and glucose supplementation, which was 30.72% higher than that achieved via batch fermentation. Discussion: This study provides a safe and economical approach for producing NMN on an industrial scale.

Co-production of 7-chloro-tryptophan and indole pyruvic acid based on an efficient FAD/FADH2 regeneration system.

Efficient FAD/FADH2 regeneration is vital for enzymatic biocatalysis and metabolic pathway optimization. Here, we constructed an efficient and simple FAD/FADH2 regeneration system through a combination of L-amino acid deaminase (L-AAD) and halogenase (CombiAADHa), which was applied for catalyzing the conversion of an L-amino acid to halide and an α-keto acid. For cell-free biotransformation, the optimal activity ratio of L-AAD and halogenase was set between 1:50 and 1:60. Within 6 h, 170 mg/L of 7-chloro-tryptophan (7-Cl-Trp) and 193 mg/L of indole pyruvic acid (IPA) were synthesized in the selected mono-amino acid system. For whole-cell biotransformation, 7-Cl-Trp and IPA synthesis was enhanced by 15% (from 96 to 110 mg/L) and 12% (from 115 to 129 mg/L), respectively, through expression fine-tuning and the strengthening of FAD/FADH2 supply. Finally, ultrasound treatment was applied to improve membrane permeability and adjust the activity ratio, resulting in 1.6-and 1.4-fold higher 7-Cl-Trp and IPA yields. The products were then purified. This system could also be applied to the synthesis of other halides and α-keto acids. KEY POINTS: • In this study, a whole cell FAD/FADH2 regeneration system co-expressing l-AAD and halogenase was constructed • This study found that the activity and ratio of enzyme and the concentration of cofactors had a significant effect on the catalytic process for the efficient co-production of 7-chlorotryptophan and indole pyruvate.

Metabolomic Analysis of Biosynthesis Mechanism of ε-Polylysine Produced by Streptomyces diastatochromogenes.

ε-Polylysine (ε-PL), a natural preservative with broad-spectrum antimicrobial activity, has been widely used as a green food additive, and it is now mainly produced by Streptomyces in industry. In the previous study, strain 6#-7 of high-yield ε-PL was obtained from the original strain TUST by mutagenesis. However, the biosynthesis mechanism of ε-PL in 6#-7 is still unclear. In this study, the metabolomic analyses of the biosynthesis mechanism of ε-PL in both strains are investigated. Results show that the difference in metabolisms between TUST and 6#-7 is significant. Based on the results of both metabolomic and enzymatic activities, a metabolic regulation mechanism of the high-yield strain is revealed. The transport and absorption capacity for glucose of 6#-7 is improved. The enzymatic activity benefits ε-PL synthesis, such as pyruvate kinase and aspartokinase, is strengthened. On the contrary, the activity of homoserine dehydrogenase in the branched-chain pathways is decreased. Meanwhile, the increase of trehalose, glutamic acid, etc. makes 6#-7 more resistant to ε-PL. Thus, the ability of the mutagenized strain 6#-7 to synthesize ε-PL is enhanced, and it can produce more ε-PLs compared with the original strain. For the first time, the metabolomic analysis of the biosynthesis mechanism of ε-PL in the high-yield strain 6#-7 is investigated, and a possible mechanism is then revealed. These findings provide a theoretical basis for further improving the production of ε-PL.

Effects of Amino Acids and Overexpression of dapA Gene on the Production of ε-Poly-L-lysine by Streptomyces diastatochromogenes Strains.

In this study, the strain Streptomyces diastatochromogenes 6#-7, which efficiently synthesizes ε-Poly-L-lysine, was studied and the effects of 18 amino acids and overexpression of dapA gene on the fermentation efficiency of ε-PL by S. diastatochromogenes were investigated. It was shown that L-proline, L-lysine, L-isoleucine, and L-threonine could promote the production of ε-PL. Moreover, the overexpression of the dihydrodipicolinate synthase gene (dapA) helped improve the fermentation performance of S. diastatochromogenes. The maximum ε-PL yield of the overexpressing strain (S. diastatochromogenes 12#-2) increased by 17.5% compared with the original strain in 500 mL shake flask. When the fermentation was conducted in a 5-L fermenter, the fermentation duration was extended by 48 h, and ε-PL yield reached 30.54 g/L, which was a 19.8% increase compared to the original strain. The results of this study offered a promising approach to augment the production of ε-PL from Streptomyces, thus paving the way to reduce the cost of product ε-PL and enhance the fermentation efficiency of ε-PL production.

Combination of multi-enzyme expression fine-tuning and co-substrates addition improves phenyllactic acid production with an Escherichia coli whole-cell biocatalyst.

The aim of this study was to develop an environmentally safe and efficient method for phenyllactic acid (PLA) production using whole-cell cascade catalysis with l-amino acid deaminase (l-AAD), lactate dehydrogenase (LDH), and formate dehydrogenase (FDH). The PPA titer was low due to relatively low expression of LDH, intermediate accumulation, and lack of cofactors. To address this issue, ribosome binding site regulation, gene duplication, and induction optimization were performed to increased the PLA titer to 43.8 g/L. Then co-substrates (glucose, yeast extract, and glycerol) were used to increase NADH concentration and cell stability, resulting that the PLA titer was increased to 54.0 g/L, which is the highest reported production by biocatalyst. Finally, glucose was replaced with wheat straw hydrolysate as co-substrate to decrease the cost. Notably, the strategies reported herein may be generally applicable to other whole-cell cascade biocatalysts.

Comparisons of Functional Properties of Polysaccharides from Nostoc flagelliforme under Three Culture Conditions.

Nostoc flagelliforme is an edible cyanobacterium with excellent food and herbal values. It has been used as food in China for more than 2000 years. Many studies have been focused on improving the yield and bioactivity of Nostoc flagelliforme polysaccharides although these have ignored the functional properties. In this study, we extracted and purified three polysaccharides (WL-CPS, NaCl-CPS and Glu-CPS) from Nostoc flagelliforme under normal, salt stress and mixotrophic culture conditions, respectively, in order to change the physicochemical properties of polysaccharides with the aim of obtaining better functional properties. Both salt stress and mixotrophic culture conditions increased the specific yield of polysaccharides. Their functional properties were comparatively investigated and the results showed that NaCl-CPS exhibited the highest emulsification activity and flocculation capability, which was also higher than that of some commercial products. In contrast, Glu-CPS exhibited the highest water and oil holding capacities, foaming property, intrinsic viscosity and bile acids binding capacity. Our results indicated that both NaCl-CPS and Glu-CPS could be considered to be functional polysaccharides according to their respective characteristics, which have great potential in numerous applications, such as food, pharmaceutical, cosmetic, chemical and mineral industries. These findings also demonstrated the potential application of the proper regulation of culture conditions in the development of polysaccharides with desired functional properties.

Carbonic Anhydrase@ZIF-8 Hydrogel Composite Membrane with Improved Recycling and Stability for Efficient CO2 Capture.

In this study, carbonic anhydrase (CA, EC 4.2.1.1) molecules were embedded into metal-organic frameworks (MOFs) via co-precipitation (CA@ZIF-8), and then these CA@ZIF-8 nanocomposites were encapsulated in the poly(vinyl alcohol) (PVA)-chitosan (CS) hydrogel networks to prepare CA@ZIF-8-PVA-CS composite hydrogels (PVA/CS/CA@ZIF-8) with high activity, stability, and reusability. The immobilization efficiency of CA was greater than 70%, suggesting the high immobilization efficiency. The prepared PVA/CS/CA@ZIF-8 composite membranes displayed excellent higher stability against a high temperature, denaturants, and acid than free CA and CA@ZIF-8. Furthermore, these membranes exhibited an excellent performance for CO2 capture. The amount of calcium carbonate obtained by PVA/CS/CA@ZIF-8 hydrogel membranes was 20- and 1.63-fold than free CA and CA@ZIF-8 composites, respectively. Furthermore, the hydrogel membranes exhibited superior reusability and mechanical strength. The hydrogel membrane maitained 50% of its original activity after 11 cycles. However, CA@ZIF-8 completely lost activity. These results indicated that the PVA/CS/CA@ZIF-8 membranes can be efficiently applied to capture CO2 sequestration.

Biodegradation of polyvinyl alcohol using cross-linked enzyme aggregates of degrading enzymes from Bacillus niacini.

In this study, polyvinyl alcohol (PVA)-degrading bacteria were screened from sludge samples using PVA as a sole source of carbon. A novel strain was obtained and identified as Bacillus niacini based on the analysis of a partial 16S rDNA nucleotide sequence and morphological characteristics. PVA-degrading enzyme (PVAase) from Bacillus niacini was immobilized as cross-linked enzyme aggregates (CLEAs) via precipitation with ammonium sulfate followed by glutaraldehyde cross-linking. The effects of precipitation and cross-linking on PVAase-CLEAs activity were investigated and characterized. 70% ammonium sulfate and 1.5% glutaraldehyde were used for precipitation and 1-h cross-linking reaction. The activity recovery of PVAase-CLEAs was approximately 90% starting from free PVAase, suggesting non-purification steps are required for extended use. No significant differences in optimum pH and temperature values of the PVAase were recorded after immobilization. The PVAase-CLEAs showed a ball-like morphology and enhanced PVA degradation efficiency in comparison with the free PVAase in solution. Furthermore, the PVAase-CLEAs exhibited excellent thermal stability, pH stability and storage stability compared to free PVAase. The PVAase-CLEAs retained about 75% of initial PVAase activity after 4 cycles of use. These results suggest that this CLEA is potentially usable for PVA degradation in industrial applications.

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 ß-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.

Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.

Enzymes@ZIF-8 Nanocomposites with Protection Nanocoating: Stability and Acid-Resistant Evaluation.

Zeolitic imidazole framework-8 (ZIF-8) with tunable pore sizes and high surface areas have recently emerged as a promising support for immobilizing enzymes. However, the instability in the aqueous acidic environment and difficulty of recovery has limited their practical applications in some cases. In this study, catalase/ZIF-8 composites with a protective nanocoating were prepared by the controlled self-assembly of silanes or coordination complexes (tannic acid (TA) and Fe3+). The properties of the catalase (CAT)/ZIF-8 composites with a protective nanocoating were also determined. The recovered activity of CAT/ZIF-8 and CAT/ZIF-8 with protective nanocoating was 70% and 65%, respectively. Compared with the conventional CAT/ZIF-8 composites, CAT/ZIF-8 with protective nanocoating exhibited excellent acid resistance. For example, after treatment for 60 min in phosphate buffer solution (pH 3.0), CAT/ZIF-8 composites only maintained 20% of their initial activity (about 12 U/mg). However, CAT/ZIF-8 with a protective nanocoating could still retain about 50% of its initial activity (about 10 U/mg). Meanwhile, the thermostability and storage stability of the CAT/ZIF-8 composites was enhanced significantly due to the presence of nanocoating compared with conventional CAT/ZIF-8. More importantly, the CAT/ZIF-8 with a protective nanocoating retained 40% of its initial activity after 7 cycles, whereas CAT/ZIF-8 only retained 8% of the initial activity. The approach in this study could be an efficient strategy to prepare enzyme/ZIF-8 composites with both high acid resistance and excellent recyclability.

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.

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.

Enzyme Shielding in a Large Mesoporous Hollow Silica Shell for Improved Recycling and Stability Based on CaCO3 Microtemplates and Biomimetic Silicification.

We report a novel "anchor-shield" approach for synthesizing a yolk-shell-structured biocatalytic system that consists of a phenylalanine ammonia lyase (PAL) protein particle core and a hollow silica shell with large mesopores by a combination of CaCO3 microtemplates and biomimetic silicification. The method is established upon filling porous CaCO3 cores with PAL via co-precipitation, controlled self-assembly and polycondensation of silanes, cross-link of the PAL molecules, and subsequent CaCO3 dissolution. During this process, the self-assembled layer of cetyltrimethylammonium bromide served as a structure-directing agent of the mesostructure and directed the overgrowth of the mesostructured silica on the external surface of PAL/CaCO3 hybrid microspheres; after CaCO3 dissolution, the cross-linked PAL particles were encapsulated in the hollow silica shell. The hollow silica shell around the enzyme particles provided a "shield" to protect from biological, thermal, and chemical degradation for the enzyme. As a result, the recycling of the PAL enzyme was improved remarkably in comparison to adsorbed PAL on CaCO3. PAL particles with a hollow silica shell still retained 60% of their original activity after 13 cycles, whereas adsorbed PAL on CaCO3 microparticles lost activity after 7 cycles. Moreover, immobilized PAL exhibited higher stability against a proteolytic agent, denaturants, heat, and extreme pH than adsorbed PAL on CaCO3 microparticles. These results demonstrated that the "anchor-shield" approach is an efficient method to obtain a stable and recycled biocatalyst with a yolk-shell structure.

Mesoporous Metal-Organic Framework with Well-Defined Cruciate Flower-Like Morphology for Enzyme Immobilization.

Metal-organic frameworks (MOFs) have recently emerged as a promising candidates for the immobilization of enzymes due to their diversified structures and porosity. However, a lack of good size and morphological control over the as-prepared MOFs has limited their practical applications in some cases. Herein, instead of zeolitic imidazolate framework-8 (ZIF-8) with the standard rhombic dodecahedral morphology, we successfully synthesize a novel mesoporous catalase@ZIF composite with cruciate flower-like morphology by embedding catalase molecules into uniformly sized ZIF crystals. With extraordinarily large mesopore size and high protein loading capacity, the catalase@ZIF composites with cruciate flower-like morphology exhibit 400% higher activity than that of catalase@ZIF composites with conventional rhombic dodecahedral morphology, and show higher reusability than conventional rhombic dodecahedral morphology. More importantly, we demonstrate for the first time that the biomacromolecules (proteins) can not directly regulate the crystal size, morphology, and crystallinity of ZIF-8. Moreover, the crystal morphology of ZIF has primary dependence on concentrations of 2-methylimidazole and Zn2+ ions, and can be directly controlled by adjusting concentrations of Zn2+ ions while keeping the high concentration of 2-methylimidazole.

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.

Encapsulation of Spherical Cross-Linked Phenylalanine Ammonia Lyase Aggregates in Mesoporous Biosilica.

Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising tool for enzyme immobilization because of their simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications. For example, the prepared CLEAs exhibit amorphous large clusters that would cause significant mass-transfer limitations, which lead to a low catalytic efficiency. Here, inspired by biomineralized core-shell structures in nature, we develop a novel mesoporous spherical CLEA with a biosilica shell by using phenylalanine ammonia lyase based on CaCO3 microtemplates and biomimetic mineralization. The resultant CLEAs exhibited a spherical structure with good monodispersity instead of the amorphous clusters of conventional CLEAs and showed activity higher than that of conventional CLEAs. Moreover, the thermostability, tolerance against denaturants, and mechanical stability of the spherical CLEAs with a biosilica shell were enhanced significantly compared with those of conventional CLEAs. In particular, the spherical CLEAs with a biosilica shell retained 70% of their original activity after 13 cycles, whereas the conventional CLEAs retained only 35% of their original activity. This approach could be an efficient strategy for improving the catalytic properties of CLEAs.

Comparison of bacterial community structures of terrestrial cyanobacterium Nostoc flagelliforme in three different regions of China using PCR-DGGE analysis.

Filamentous Nostoc flagelliforme form colloidal complex, with beaded cells interacting with other bacteria embedded in the complex multilayer sheath. However, the species of bacteria in the sheath and the interaction between N. flagelliforme and associated bacteria remain unclear. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of N. flagelliforme from three regions of China. DGGE patterns showed variations in all samples, exhibiting 25 discrete bands with various intensities. The diversity index analysis of bands profiles suggested the high similarity of bacterial communities to each other but also the dependence of microbial composition on each location. Phylogenetic affiliation indicated that the majority of the sequences obtained were affiliated with Actinobacteria, Cyanobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, of which Cyanobacteria was dominant, followed the Proteobacteria. Members of the genus Nostoc were the most abundant in all samples. Rhizobiales and Actinobacteria were identified, whereas, Craurococcus, Caulobacter, Pseudomonas, Terriglobus and Mucilaginibacter were also identified at low levels. Through comparing the bacterial composition of N. flagelliforme from different regions, it was revealed that N. flagelliforme could facilitate the growth of other microorganisms including both autotrophic bacteria and heterotrophic ones and positively contributed to their harsh ecosystems. The results indicated N. flagelliforme played an important role in diversifying the microbial community composition and had potential application in soil desertification.

Antibacterial action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shell nanoparticles against Escherichia coli correlated with molecular chain conformation.

The action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shell nanoparticles (CM-HTCC/PAMAM) against Escherichia coli (E. coli) was investigated via a combination of approaches including measurements of cell membrane integrity, outer membrane (OM) and inner membrane (IM) permeability, and scanning electron microscopy (SEM). CM-HTCC/PAMAM dendrimer nanoparticles likely acted in a sequent event-driven mechanism, beginning with the binding of positively charged groups from nanoparticle surface with negative cell surface, thereby causing the disorganization of cell membrane, and subsequent leakage of intracellular components which might ultimately lead to cell death. Moreover, the chain conformation of polymers was taken into account for a better understanding of the antibacterial action mode by means of viscosity and GPC measurements. High utilization ratio of positive charge and large specific surface area generated from a compacted conformation of CM-HTCC/PAMAM, significantly different from the extended conformation of HTCC, were proposed to be involved in the antibacterial action.

[Effects of carbon and nitrogen sources on 5-keto-gluconic acid production].

Gluconobacter oxydans is known to oxidize glucose to gluconic acid (GA), and subsequently, to 2-keto-gluconic acid (2KGA) and 5-keto-gluconic acid (5KGA), while 5KGA can be converted to L-(+)-tartaric acid. In order to increase the production of 5KGA, Gluconobacter oxydans HGI-1 that converts GA to 5KGA exclusively was chosen in this study, and effects of carbon sources (lactose, maltose, sucrose, amylum and glucose) and nitrogen sources (yeast extract, fish meal, corn steep liquor, soybean meal and cotton-seed meal) on 5KGA production were investigated. Results of experiment in 500 mL shake-flask show that the highest yield of 5KGA (98.20 g/L) was obtained using 100 g/L glucose as carbon source. 5KGA reached 100.20 g/L, 109.10 g/L, 99.83 g/L with yeast extract, fish meal and corn steep liquor as nitrogen source respectively, among which the optimal nitrogen source was fish meal. The yield of 5KGA by corn steep liquor is slightly lower than that by yeast extract. For the economic reason, corn steep liquor was selected as nitrogen source and scaled up to 5 L stirred-tank fermentor, and the final concentration of 5KGA reached 93.80 g/L, with its maximum volumetric productivity of 3.48 g/(L x h) and average volumetric productivity of 1.56 g/(L x h). The result obtained in this study showed that carbon and nitrogen sourses for large-scale production of 5KGA by Gluconobacter oxydans HGI-1 were glucose and corn steep liquor, respectively, and the available glucose almost completely (85.93%) into 5KGA.