α-萄糖苷酶抑制劑 ( α-glucosidase inhibitors, aGIs )，為2型糖尿病的藥物，最主要是因為它能直接降低人體對碳水化合物的。本實驗利用Bacillus mycoides TKU040 與 Rhizobium sp. TKU041 共同培養發酵蝦頭殼粉(SHP)，以及 Paenibacillus macerans TKU029 發酵蟹殼粉(CSP)，生產α-葡萄糖苷酶抑制劑，實驗結果顯示出，推測此三種菌皆利用幾丁質做一個生物轉換，進而生成α-葡萄糖苷酶抑制劑。其中TKU040與TKU041所生成之抑制劑推測為醣類，IC50 = 5μg/mL ，抑制型態為混和型抑制劑(non-competitive-uncompetitive)。另外TKU029 發酵蟹殼粉的部分，發酵上清液中測得最高抑制劑活性為666 U/mL，以150mL 培養可得總活性為99900U。目前已從發酵上清液中分離出七個活性區段，後續將以HPLC繼續進行分離純化，並以NMR 進行結構鑑定。 大多數的aGIs對肝臟造成負擔，並引起胃腸道困擾，因此新的aGIs的發展變得非常重要。 本實驗利用Bacillus mycoides TKU040 and Rhizobium sp. TKU041共同培養以蝦頭殼作為唯一碳氮源生產aGIs。以50毫升的培養液培養(0.1% K2HPO4、0.05% MgSO4∙7H2O, pH 9.2)，內含1% 蝦頭殼粉，於 37 ºC 培養4天發酵上清液中可得到aGIs (143 U/mL)，上清液之半抑制濃度為3 mg/mL，在熱穩定性方面，可耐熱至60 ºC，30分鐘仍保有60 %的活性，在pH安定性方面，於pH11時可提高抑制活性至140 %。後續純化方面經由NMR及MALDI-TOF鑑定後，發現抑制劑結構可能為醣類化合物，抑制型態為混合型抑制劑，純化後之辦抑制濃度為5μg/mL。 另一方面本實驗利用paenibacillus macerans TKU029以螃蟹殼作為唯一碳氮源，生產aGIs，以150毫升培養液(0.1% K2HPO4、0.05% MgSO4∙7H2O, pH 4)，內含2 %的螃蟹殼，於30 ºC 培養4天，可於發酵上清液中得到aGIs (99900 U/mL)，在熱穩定性方面，可耐熱至100 ºC，30分鐘仍保有99 % 的活性。後續純化方面經由Diaion gel及Silica gel，分離出7個活性區段，NMR進行結構鑑定。 Alpha-glucosidase inhibitors (aGIs) have potential use as antidiabetic drugs for the treatment of type II diabetes. Most aGIs place a burden on the liver and cause gastrointestinal distress, therefore the development of new aGIs has become very important. In this study, some chitinous materials were utilized for aGIs production via microbial conversion. In the first study, we investigated the production of aGIs by the co-culture of Bacillus mycoides TKU040 and Rhizobium sp. TKU041 using shrimp head powder (SHP) as the sole source of carbon and nitrogen (C/N). After fermentation in 50 mL of 1% SHP-containing medium (0.1% K2HPO4 and 0.05% MgSO4∙7H2O, pH 9.2) at 37ºC for 4 days, the maximum productivity of aGIs (143 U/mL) was reached. The IC50 of the aGIs produced in the culture supernatant was 3 mg/mL. The α-glucosidase inhibitory (aGI) activity was only 60% after treatment at pH 3 for 30 min; this increased to 140% after treatment at pH 11 for 30 min. The aGI activity remained at 60% after treatment at 60ºC for 30 min. One major active compound was isolated from fermented SHP and confirmed as a carbohydrate by NMR and MALDI-TOF analysis. This isolated inhibitor possessed a low IC50 value of 5μg/mL. In the second study of our investigation, paenibacillus macerans TKU029 was found to produce aGIs by using carb shell powder (CSP) as the sole source of C/N. After fermentation in 150 mL of 2% CSP-containing medium (0.1% K2HPO4 and 0.05% MgSO4∙7H2O, pH 9.2) at 30 ºC for 4 days, the maximum productivity of aGIs (99900 U/mL) was reached. The aGI activity remained at 71% after treatment at 100 ºC for 30 min. The application of several techniques including Diaoin, Silica gel opened columns, coupled with a biological-guided assay resulted in isolating 7 active sub–fractions. NMR was also used for prediction of chemical structure of these active components.
