海绵共附生微生物中具有α-葡萄糖苷酶抑制活性菌株的筛选与初步鉴定

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.

Screening and identifying for strains with inhibitory activity against α-glucosidase from marine sponge associated microorganisms

Marine sponge associated microorganisms are important source of nature products for pharmaceuticals.It is a potential way for developing new diabetes drug（α-glycosidase inhibitor）.In this study,the α-glucosidase inhibitory activity screening model is built-up to search for the active crude extract from the microorganism metabolite samples prepared from marine sponges in Friday harbor of US.The reaction system of the α-glucosidase inhibitory activity screening is performed on a 96 wells panel.The reaction condition,including the concentration of enzymes,is set at 0.008U/cm3,pH=6.8,37℃ and wavelength 405 nm.Moreover,the screening model was optimized with reactant PNPG concentration 10 mmol/dm3,reaction time 25 min and the solvent DMSO content 2%（V/V）.Used acarbose as a positive controlled sample,212 crude extract samples from marine sponge associated microorganisms were screened with the optimized model.The results indicated that metabolites of 19 strains had high inhibitory activity（〉50%）.Among them,5 strains showed inhibitory effect higher than 70% against α-glucosidase at concentration 0.4,0.6,0.8 mg/cm3.The inhibitory effect was higher than that of Acarbose（58%） at concentration 1.6 mg/cm3.As result,the strain HY936 with high inhibitory activity（79.3%） at concentration 0.4 mg/cm3 was identified as Bacillus firmus by the means of 16S rDNA sequence.The metabolites from Bacillus firmus can inhibit the activity of α-glycosidase significantly.This provides potential clues for developing new α-glycosidase inhibitors.Attentions should be paid on the study of Bacillus firmus in further.