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Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.
Subject(s)
Biocatalysis , Chitin , Chitinases , Hydrogen-Ion Concentration , Polymerase Chain ReactionABSTRACT
Objective To investigate effects of xylo-oligosaccharides with chitooligosaccharides on alcohol-induced liver injury and immune function in mice. Methods Different doses of chitosan oligosaccharide (0.045 g/kg-0.26 g/kgB.W) and xylo-oligosaccaride (0.055 g/kg-0.32 g/kgB.W) were feed to the mice for 30 days. The mice live-injury model was induced by alcohol. MDA、 GSH、 TG level in liver and T lymphocyte proliferation of spleen cells induced by conA, the antibody-producing cells, and natural killer (NK) activity were detected. Results Compared with the live-injury model group, MDA level of low/middle dose group, TG level of middle dose group and pathological evaluation score of liver steatosis of high dose group in mice liver were decreased because of chitosan oligosaccharide and xylo-oligosaccaride feeding. Compared with the control group, the ability of T lymphocyte proliferation of mouse spleen induced by ConA and the antibody-producing cells were increased in mice of middle and high dose group. The differences had statistical significances (P<0.05) . Conclusion Under this experimental condition, xylo-oligosaccharides in combination with chitooligosaccharides could protect the mice from alcohol-induced liver injury and enhance immune function in spleen of normal mice synergistically.
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OBJECTIVE@#To observe the chitooligosaccharides (COS) effect on the proliferation inhibition and radiosensitivity of three types of human gastric cancer cell line.@*MOTHODS@#CCK-8 assay was employed to obtain the inhibition ratio of COS on BGC823 cells, MKN45 cells and SGC7901 cells at 48 h after treatment and the proliferation-inhibition curve was drawn with the inhibition ratio of COS on three types of cells. The clonogenic assay was used to detect the cell viability of 0, 1, 2, 4, 6 and 8 Gy (6 dose grades) in RAY group and RAY + COS group after X-ray, and the cell survival curve was used to analyze the sensitization enhancement ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY + COS group after 48 h treatment.@*RESULTS@#COS inhibited the proliferation of three types of cells. The inhibition rate was positively correlated with the concentration of COS, and the susceptibility of MKN45 cells, SGC7901 cells and BGC823 cells to COS decreased in turn. The cell viability decreased gradually with the increasing radiation dose in RAY group and RAY + COS group (P < 0.01). The cell viabilities of RAY + COS group were lower than those of RAY group at all the dose grades under X-ray exposure (P < 0.01), and the sensitization enhancement ratios of COS on BGC823 cells, MKN45 cells and SGC7901 cells were 1.06, 1.28 and 1.15, respectively. In controlled trials, apoptosis rate and percentage in the G2/M phase of three types of cells in RAY + COS group were higher than those in control group and RAY group, and percentage in the S phase and the G0/G1 phase in RAY + COS group were lower than those in the other two groups (P < 0.01).@*CONCLUSIONS@#COS can inhibit the proliferation of three types of human gastric cancer cells and enhance the radiosensitivity by inducing apoptosis and G2/M phase arrest.
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Objective To observe the chitooligosaccharides (COS) effect on the proliferation inhibition and radiosensitivity of three types of human gastric cancer cell line. Mothods CCK-8 assay was employed to obtain the inhibition ratio of COS on BGC823 cells, MKN45 cells and SGC7901 cells at 48 h after treatment and the proliferation-inhibition curve was drawn with the inhibition ratio of COS on three types of cells. The clonogenic assay was used to detect the cell viability of 0, 1, 2, 4, 6 and 8 Gy (6 dose grades) in RAY group and RAY + COS group after X-ray, and the cell survival curve was used to analyze the sensitization enhancement ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY + COS group after 48 h treatment. Results COS inhibited the proliferation of three types of cells. The inhibition rate was positively correlated with the concentration of COS, and the susceptibility of MKN45 cells, SGC7901 cells and BGC823 cells to COS decreased in turn. The cell viability decreased gradually with the increasing radiation dose in RAY group and RAY + COS group (P < 0.01). The cell viabilities of RAY + COS group were lower than those of RAY group at all the dose grades under X-ray exposure (P < 0.01), and the sensitization enhancement ratios of COS on BGC823 cells, MKN45 cells and SGC7901 cells were 1.06, 1.28 and 1.15, respectively. In controlled trials, apoptosis rate and percentage in the G
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OBJECTIVE@#To observe the anti-proliferation and radiosensitization effect of chitooligosaccharides (COS) on human lung cancer cell line HepG2.@*METHODS@#CCK-8 assay was employed to obtain the inhibition ratio of COS on HepG2 cells at 24 h after treatment. The clonogenic assay was used to analyze the cell viability of RAY group and RAY + COS group with X-ray of 0, 1, 2, 4, 6 and 8 Gy, and the cell survival curve was used to analyze the sensitization ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY + COS group after 24 h treatment.@*RESULTS@#COS inhibited the proliferation of HepG2 cells, and the inhibition rate positively correlated with the concentration of COS. The cell viability decreased with increasing exposure dose in RAY group and RAY + COS group. The cell viabilities of RAY + COS group were lower than those of RAY group at the dose of 4, 6 and 8 Gy (P < 0.05), and the sensitization ratio of COS was 1.19. There were higher percentage at G2/M phase and apoptosis rate, and lower percentage at S phase in RAY + COS group versus the other two groups (P < 0.01).@*CONCLUSIONS@#COS can inhibit the proliferation of HepG2 cells, and enhance the radiosensitization of HepG2 cells, induce apoptosis and G2/M phase arrest.
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Objective: To observe the anti-proliferation and radiosensitization effect of chitooligosaccharides (COS) on human lung cancer cell line HepG2. Methods: CCK-8 assay was employed to obtain the inhibition ratio of COS on HepG2 cells at 24 h after treatment. The clonogenic assay was used to analyze the cell viability of RAY group and RAY + COS group with X-ray of 0, 1, 2, 4, 6 and 8 Gy, and the cell survival curve was used to analyze the sensitization ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY + COS group after 24 h treatment. Results: COS inhibited the proliferation of HepG2 cells, and the inhibition rate positively correlated with the concentration of COS. The cell viability decreased with increasing exposure dose in RAY group and RAY + COS group. The cell viabilities of RAY + COS group were lower than those of RAY group at the dose of 4, 6 and 8 Gy (P < 0.05), and the sensitization ratio of COS was 1.19. There were higher percentage at G
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Objective To investigate the protective effects of chitooligosaccharides on chemical hepatic injury induced by carbon tetrachloride(CCl_4) in mice.Methods Liver injury model was established by administration of CCl_4 in mice.The activities of serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST),the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in hepatic tissue were measured.The hepatic histological changes were observed by optical microscope. Results Chitooligosaccharides(given at 167,500mg?kg~(-1)?d~(-1)for 7 days) remarkably inhibited the rises of serum ALT and AST,and decreased the content of MDA in hepatic tissues in mice,meanwhile increased the activities of SOD in hepatic tissues.Furthermore,the pathological changes were also significantly improved in the mice of treating group.Conclusion Chitooligosaccharides had protective effects on the acute hepatic injury induced by CCl_4 in mice.
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Objective To investigate the inhibition effects of chitooligosaccharides on the production of pro-angiogenesis factors of estradiol(E2) induced human MCF-7 cells.Methods The cell survival rate was demonstrated by MTT assay at 24 and 48 hours;and the mRNA level of some pro-angiogenesis factors such as MMP-9,TIMP-1,2 and VEGF were investigated by RT-PCR.Results There was no significant effect of chitooligosaccharides on proliferation of human MCF-7cells;but when induced by E_2,MMP-9 was significantly down-regulated by chitooligosaccharides.Conclusion Chitooligosaccharides had potent inhibition effects on the mRNA production of MMP-9 of human MCF-7 cells.