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通过建立α-葡萄糖苷酶抑制剂筛选模型,对美国圣璜岛海域海绵共附生微生物发酵产物进行活性筛选.其中,α-葡萄糖苷酶活性测试反应体系是在96孔酶标板中进行,在α-葡萄糖苷酶浓度为0.008 U/cm3,pH值为6.8,反应温度为37℃及测定生成物波长为405 nm的基础上,优化筛选模型.结果表明,模型的最佳筛选条件:底物PNPG的浓度为10 mmol/dm3、活性测试反应时间为25min,样品溶剂DMSO含量为2%(V/V).以α-葡萄糖苷酶抑制剂阿卡波糖作为阳性对照样品,根据优化后的抑制活性筛选模型对212株海绵共附生微生物的粗提物样品进行筛选.结果表明:19株微生物的粗提物抑制率大于50%;其中,抑制活性最高的5株菌株的粗提物,在浓度为0.4、0.6、0.8 mg/cm3时,其抑制活性均在70%以上,远远高于阿卡波糖的最高抑制率58%(1.6 mg/cm3).其中菌株HY936粗提物浓度在0.4 mg/cm3时,仍具有很高的α-葡萄糖苷酶抑制率79.3%.通过16SrDNA序列测定与分析,鉴定该菌株为坚强芽孢杆菌(Bacillus firmus).坚强芽孢杆菌代谢产物能显著抑制α-葡萄糖苷酶活性,这为研制新型α-葡萄糖苷酶抑制剂提供了有力的线索,对坚强芽孢杆菌有待作进一步的研究.
Through the establishment of a-glucosidase inhibitor screening model, the San Juan Island in the United States sea sponge co-fermentation products were screened.Among them, the α-glucosidase activity test reaction system is carried out in a 96-well microplate, The screening model was optimized on the basis of α-glucosidase concentration of 0.008 U / cm3, pH value of 6.8, reaction temperature of 37 ℃ and wavelength of 405 nm.The results showed that the optimal screening conditions were as follows: The concentration of PNPG was 10 mmol / dm3, the reaction time was 25 min and the content of DMSO in the sample was 2% (V / V) .According to the optimized acarbose-α-glucosidase inhibitor, Inhibition activity screening model of 212 sponge co-epiphytic microorganisms crude extract samples were screened.The results showed that: 19 strains of crude extracts inhibition rate of more than 50%; of which, the highest inhibitory activity of the five strains of crude extracts , And its inhibitory activity was above 70% at concentrations of 0.4, 0.6, 0.8 mg / cm3, which was much higher than that of acarbose at the highest inhibition rate of 58% (1.6 mg / cm3) At a concentration of 0.4 mg / cm3, there was still a high α-glucosidase inhibition rate of 79.3% .This strain was identified as Bacillus firmus by 16S rDNA sequence analysis and analysis.The metabolites of B. firmus significantly inhibited the activity of α-glucosidase, which provided a powerful method for the development of novel α-glucosidase inhibitors Clues, for strong Bacillus need to be further studied.