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A New Method for Screening α-Glucosidase Inhibitors and Application to Marine Microorganisms

A New Method for Screening α-Glucosidase Inhibitors and Application to Marine Microorganisms. Marine Microorganisms 較少作為 glucosidase inhibitors 的來源是因為缺乏適合且有效率的酵素分析方法。. Spectrophotometrical method 缺點如醱酵培養基中的 protease, 植物卒取液中的菌體等都會干擾結果。

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A New Method for Screening α-Glucosidase Inhibitors and Application to Marine Microorganisms

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  1. A New Method for Screening α-Glucosidase Inhibitors and Application to Marine Microorganisms

  2. Marine Microorganisms較少作為glucosidase inhibitors的來源是因為缺乏適合且有效率的酵素分析方法。

  3. Spectrophotometrical method缺點如醱酵培養基中的protease,植物卒取液中的菌體等都會干擾結果。 • Antimicrobial assay非專一性的針對篩選glucosidase inhibitors，使用此方法沒有抗生素特性但有glucosidase inhibitors的微生物不會被篩選出來。 • Kurihara’s assay缺點為抑制環不易辨別、需使用大量酵素及受質、分析樣本少。

  4. α-glucosidase: 將多醣和寡醣水解成單分子。 • α-glucosidase inhibitors: 結構與醣類相似，所以會與醣類競爭醣類消化酵素，它與α-glucosidase的親合力比醣類強。

  5. Thin-Layer Chromatography(TLC) Plate Method • 將樣本液滴一小點在TLC Sheet上(直徑小於２㎜) • 將agar(含1.6％澱粉)倒在TLC Sheet上 • 用Enzyme(2㎎/ml,溶於0.1mol/l acetate buffer(pH4.5))將紙浸溼 • 將紙平舖在agar上 • 培養1.5小時在55℃下 • 將紙拿開，以0.05mol/l 碘液倒在表面 • 用水將TLC Sheet上過量的碘液沖洗掉

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  7. Figure 1. The inhibitory rings of different doses of acarbose. Thedoses of acarbose from left to right are 0.312, 0.624, 1.248, 2.496,and 4.992mmol, respectively. Table 1. Inhibitory activity of different doses of acarbose.

  8. 選擇glucoamylase作為enzyme的優點 • glucoamylase的受質是澱粉，使用碘液後產生紫色，與α-glucosidase相比(淡黃色)，其與marine microorganism broth的顏色有較大差異(黃色)。 • glucoamylase比α-glucosidase更有效率。(glucosidase inhibitors ：acarbose、deoxynojirimycin，對glucoamylase有較高的敏感度)

  9. TLC Plate Method的優點 • 快速、簡單且精準的篩選出α-glucosidase inhibitors • 可做semiquantitative analysis • 如將各樣本的量控制一樣，抑制環的大小、顏色可反應酵素活性

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