Multicellular coupling fermentation for 3'-sialyllactose conversion using N-acetyl-glucosamine and lactose / 生物工程学报

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker's yeast were added to the above fermentation broth to synthesize 3'-sialyllactose (3'-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker's yeast, 20 mmol/L Mg2+, the maximum yield of 3'-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3'-SL.

Glucosamine Hydrochloride and N-Acetylglucosamine Influence the Response of Bovine Chondrocytes to TGF-β3 and IGF in Monolayer and Three-Dimensional Tissue Culture

BACKGROUND: Glucosamine hydrochloride (GlcN·HCl) has been shown to inhibit cell growth and matrix synthesis, but not with N-acetyl-glucosamine (GlcNAc) supplementation. This effect might be related to an inhibition of critical growth factors (GF), or to a different metabolization of the two glucosamine derivatives. The aim of the present study was to evaluate the synergy between GlcN·HCl, GlcNAc, and GF on proliferation and cartilage matrix synthesis. METHOD: Bovine chondrocytes were cultivated in monolayers for 48 h and in three-dimensional (3D) chitosan scaffolds for 30 days in perfusion bioreactors. Serum-free (SF) medium was supplemented with either growth factors (GF) TGF-β (5 ng mL₋₁) and IGF-I (10 ng mL₋₁), GlcN·HCl or GlcNAc at 1mM each or both. Six groups were compared according to medium supplementation: (a) SF control; (b) SF + GlcN·HCl; (c) SF + GlcNAc; (d) SF + GF; (e) SF + GF + GlcN·HCl; and (f) SF + GF + GlcNAc. Cell proliferation, proteoglycan, collagen I (COL1), and collagen II (COL2) synthesis were evaluated. RESULTS: The two glucosamines showed opposite effects in monolayer culture: GlcN·HCl significantly reduced proliferation and GlcNAc significantly augmented cellular metabolism. In the 30 days 3D culture, the GlcN·HCl added to GF stimulated cell proliferation more than when compared to GF only, but the proteoglycan synthesis was smaller than GF. However, GlcNAc added to GF improved the cell proliferation and proteoglycan synthesis more than when compared to GF and GF/GlcN·HCl. The synthesis of COL1 and COL2 was observed in all groups containing GF. CONCLUSION: GlcN·HCl and GlcNAc increased cell growth and stimulated COL2 synthesis in long-time 3D culture. However, only GlcNAc added to GF improved proteoglycan synthesis.

Effects of fermentation conditions on valuable products of ethanolic fungus Mucor indicus

Background: Mucor indicus is a dimorphic fungus used in the production of ethanol, oil, protein, and glucosamine. It can ferment different pentoses and hexoses; however, the yields of products highly depend on the nutrients and cultivation conditions. In this study, the effects of different morphologic forms, cultivation time and temperature, presence or absence of oxygen, carbon sources, and concentration of nitrogen source on the products of M. indicus were investigated. Results: The fungus with all morphologies produced high yields of ethanol, in the range of 0.32­0.43 g/g, on glucose. However, the fungus with filamentous morphology produced higher amounts of oil, protein, phosphate, and glucosamine together with ethanol, compared with other morphologies. A higher amount of oil (0.145 g/g biomass) was produced at 28°C, while the best temperature for protein and glucosamine production was 32 and 37°C, respectively. Although ethanol was produced at a higher yield (0.44 g/g) under anaerobic conditions compared with aerobic conditions (yield of 0.41 g/g), aerobic cultivation resulted in higher yields of protein (0.51 g/g biomass), glucosamine (0.16 g/g alkali insoluble material, AIM), and phosphate (0.11 g/g AIM). Conclusions: It is not possible to have the maximum amounts of the products simultaneously. The fermentation conditions and composition of culture media determine the product yields. Carbon source type and the addition of nitrogen source are among the most influencing factors on the product yields. Moreover, all measured products were made with higher yields in cultivation on glucose, except glucosamine, which was produced with higher yields on xylose.

Factors Related to Increased CA19-9 andLewis Antigen in Pancreatic Cancer Cell Lines

PURPOSE: The 8 pancreatic cancer cell lines (BxPC-3, Capan-2, CFPAC-1, HPAC, Capan-1, AsPC-1, MIA PaCa-2, and PANC-1) were investigated to identify the factors which would increase CA19-9 related to the Lewis antigen. CA19-9 in serum is a well-known tumor marker, and is frequently used for the clinical diagnosis of pancreatic cancer. The oligosaccharide on the CA19-9 epitope is a sialylated Lewis A blood group antigen. METHODS: beta3Gal-T was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The phenotypes and genotypes of Lewis antigen were determined by flow cytometry analysis and restriction fragment length polymorphism (RFLP), respectively. The phenotypes of sLe(a) were assessed by flow cytometry analysis and the sLe(a) on supernatants was detected by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). CA19-9 and DUPAN-2 on supernatants were measured by enzyme immunoassay. RESULTS: CA19-9 productions were possible from all cell lines since they all had beta3Gal-T and there were no genotypical Lewis negative (le/le). The elevation of CA19-9 was noted on Capan-2 and CFPAC-1, which were phenotypically Lewis positive (Le(a+b+)), as expected. Interestingly, it was also elevated in BxPC-3 even though the line was known to be phenotypically Lewis negative (Le(a-b-)). Sialyl Le(a) appeared to play an important role in this phenomenon. Although CA 19-9 was not detected in the phenotypically Lewis negative pancreatic cell line without sialyl Le(a), the levels of DUPAN-2 were variable. CONCLUSION: It was revealed that an elevated CA19-9 was related with increased expression of Lewis gene, not merely the existence of the gene. Further investigations on the role of ST3Gal are warranted to explain the mechanisms of the variable levels of DUPAN-2 in Le(a-b-) cell lines.

Preliminary studies on the antitumor activities of D-glucosamine and its derivatives / 中国海洋药物

Objective The growth inhibitory effects of GlcNH_2?HCl,GlcNH_2 and NAG on human hepatoma SMMC-7721 cells in vitro was investigated.The antitumor activity of GlcNH_2?HCl against Sarcoma_(180) in KM mice was also investigated.Methods The cell viability in vitro was examined with MTT assay.DNA isolation and cell-cycle analyses were also performed.GlcNH_2?HCl was ig administered to Sarcoma_(180) KM mice.The inhibition rates,spleen and thymus index were calculated.Results GlcNH_2?HCl and GlcNH_2 resulted in a concentration-dependent reduction in hepatoma cell growth.The inhibition rates against SMMC-7721 cell of GlcNH_2?HCl and GlcNH_2 at concentration of 500?g?mL~(-1) were(50.24)% and 52.19%.As to the concentration of 1000?g?mL~(1), the rates were(82.21%) and 83.20%.This effect was accompanied by a marked increase in the proportion of S phase cells.Compared with the control,there was no significant difference among various concentrations of NAG,GlcNH_(2)?HCl exhibited inhibitiory effect against Sarcoma_(180) in mice at the dosage of 125～500 mg?kg~(-1),and the inhibition rate was about 27.84%～34.02%.The optimal inhibitory effect was 250 mg?kg~(-1).GlcNH_2?HCl could enhance the weights of thymus and spleen.In addition,it could promote lymphocyte transformation.Conclusion It is therefore postulated that the antitumor effect of GlcNH_2?HCl is probably host-mediated and cytocidal.