Preparation of magnetic polyacrylamide hydrogel with chitosan for immobilization of glutamate decarboxylase to produce γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA.

Multisite survey of bacterial contamination in ready-to-eat meat products throughout the cooking and selling processes in urban supermarket, Nanjing, China.

OBJECTIVE: Ready-to-eat (RTE) meat is a kind of popular instant food easily contaminated by microbes, which is one of the causes of foodborne diseases. This study analyzes the possible sources of RTE food bacterial contamination during processing and subsequent selling. METHOD: Samples of eight kinds of RTE meat were collected from four supermarkets in Nanjing, China. The knives, chopping boards, trays(containers of food), clamps, air, water, and hands of the sales staff were sampled, and the enumeration of aerobic plate count and total coliforms and pathogenic bacteria was performed. RESULTS: The survey revealed that poor hygienic levels was the causes that RTE meat products were contaminated by bacteria at different levels. With regard to pathogen, the incidences of Salmonella spp. and Staphylococcus aureus were 4.2% and 2.1%, respectively. These results also revealed that the bacterial contamination of RTE food was caused by the air, as well as clamps, chopping boards, knives, trays, and hands of the operators. The total number of aerobic colonies were positively correlated with the amount of RTE food in one pot (r = .87728, p = .0217), and negatively correlated with the maximum temperature in the center of the meat (r = -.81633, p = .0475). CONCLUSION: The high number of bacteria in RTE foods indicates potential food safety risks and the need to improve the health of supermarket sales staff. The most important thing is to determine how to raise hygiene awareness of employees through food safety education. Meanwhile, a comprehensive set of regulations on hand cleaning and disinfection should be developed to facilitate public health and reduce foodborne illness caused by the consumption of RTE food.

Attenuation of Pseudomonas aeruginosa biofilm by hordenine: a combinatorial study with aminoglycoside antibiotics.

Pseudomonas aeruginosa is a ubiquitous pathogen that is the leading cause of chronic infections. Bacterial biofilm formation facilitates CF development and restricts the anti-bacterial potential of many current antibiotics. The capacity of P. aeruginosa to form biofilms and resist antibiotics is closely correlated with quorum sensing (QS). Disrupting QS by QS inhibitors is a promising strategy for treating chronic infections. Here, we evaluated the effect of hordenine, a recently characterized QS inhibitor, on the susceptibility of aminoglycoside antibiotics against P. aeruginosa biofilms. Hordenine significantly enhanced the susceptibility of aminoglycoside antibiotics tobramycin, gentamycin, and amikacin against P. aeruginosa PAO1 biofilm formation. Combinations of hordenine and aminoglycoside antibiotics showed potent efficiency in disrupting the preformed biofilms of P. aeruginosa. Microscopic observations showed flat, scattered, and unstructured biofilm architecture after treatment with hordenine. Mechanistic study further revealed that hordenine treatment led to the downregulation of genes involved in QS and biofilm formation. Thus, our results suggest that hordenine has the potential to function as an antibiotic accelerant in treating P. aeruginosa infections.

Stereoselective biotransformation of racemic mandelic acid using immobilized laccase and (S)-mandelate dehydrogenase.

OBJECTIVES: (S)-Mandelate dehydrogenase (SMDH) and laccase were immobilized on chitosan. The bi-enzymatic system with immobilized SMDH and immobilized laccase was taken to catalyze the stereoselective transformation of racemic mandelic acid and (R)-mandelic acid was obtained from its racemic mixture. RESULTS: Characteristics of the immobilized enzymes were valuated. The optimum pH and temperature of the immobilized SMDH were found to be pH 3.4 and 45 °C, and these of the immobilized laccase were about pH 6.0 and 55 °C, respectively. The Km value of the immobilized SMDH for racemic mandelic acid was 0.27 mM and that of the immobilized laccase for ferrocyanide was 0.99 mM. The thermal and storage stabilities of these enzymes were improved with immobilization. The enantiomeric purity of the bi-enzymatically produced (R)-mandelic acid was determined to be over 99%. CONCLUSION: The immobilized bi-enzymatic system for the stereoselective transformation of racemic mandelic acid showed higher productivity, faster reaction velocity, and more stable catalytic ability.Graphical abstract.

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

OBJECTIVES: To find an efficient and cheap system for NAD(+) regeneration RESULTS: A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 µM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

Stimulation of the proliferation of human normal esophageal epithelial cells by fumonisin B1 and its mechanism.

Previous epidemiological studies have demonstrated a correlation between fumonisin B1 (FB1) and human esophageal cancer in China, Iran and South Africa. The purpose of this study was to investigate the effects of FB1 on the proliferation, cell-cycle and apoptosis of normal human esophageal epithelial cells (HEECs) and to explore the molecular mechanisms of these effects. The proliferation of HEECs treated with FB1 was assessed using a colorimetric assay, while analyses of the cell cycle and apoptosis were performed using flow cytometry and the measurement of the protein expressions of genes associated with the cell cycle was conducted using western blotting. The results showed that FB1 stimulated the proliferation of HEECs, decreased the percentage of cells in the G0/G1 phase and reduced apoptosis. The western blotting results showed that FB1 significantly increased the protein expression of cyclin D1 and significantly decreased the protein expression of cyclin E, p21 and p27. The results indicated that FB1 stimulated the proliferation of HEECs by affecting the cell cycle and apoptosis. This mechanism was associated with changes in cyclin D1, cyclin E, p21 and p27 expression.

Effect of fumonisin B1 on the cell cycle of normal human liver cells.

Fumonisin B1 (FB1) is a well-known liver and kidney carcinogen in rodents and humans. The aim of the present study was to investigate the effect of FB1 on the proliferation and cell cycle of the normal human liver cell line HL-7702 and to explore the underlying molecular mechanisms of action. The cells were treated with FB1 (0.0, 0.1, 1.0, 10.0 and 100.0 µmol/l) for 24, 48, 72 and 96 h. Cell proliferation was assessed by colorimetric assay. Cell cycle analysis was performed by flow cytometry. The mRNA and protein expression of cyclin E and P21 were determined by RT­PCR and western blot analysis, respectively. FB1 was initially demonstrated to significantly inhibit the proliferation of HL-7702 cells; however, cell proliferation increased with increasing treatment time. The percentage of cells in the G0/G1 phase was significantly increased by FB1; however, significantly decreased with an increasing concentration of FB1. The mRNA expression of cyclin E was upregulated and then gradually downregulated with increasing treatment time. The mRNA expression of P21 was significantly increased following treatment with 0.1 µmol/l FB1, and decreased following treatment with 10.0 and 100.0 µmol/l FB1 for different treatment durations. Western blot analysis showed that FB1 significantly increased the protein expression of cyclin E and significantly decreased the protein expression of P21 at various concentrations and treatment durations. Our results demonstrated that FB1 affects the cell cycle of normal human liver cells and that the underlying mechanism of action is associated with alterations in the expression levels of cyclin E and P21 induced by FB1.