ε-聚離胺酸分解酵素之生化研究（一）

Biochemical Study of ε-Poly-lysine Depolymerase: Part 1

ε-聚離胺酸（ε-Poly-lysine；ε-PL）是由微生物發酵生產的天然生物性材料。ε-PL之應用潛力廣，然而生合成作用機制還未明，調控機制則仍十分模糊。ε-PL合成酵素（PL synthetase；PLS）與ε-PL降解酵素（PL degrading enzyme；PLD）與ε-PL之生產有密切關係，除可影響其分子量外亦為探討ε-PL之生合成機制之一重要工具，本研究先行探討ε-PL降解酵素之分離純化。本研究已完成篩選一株具有能忍受ε-PL之菌株且具ε-PL合成與ε-PL降解能力之菌株Streptomyces albulus PLT1。 S. albulus PLT1可使用葡萄糖或甘油為碳源並能生產ε-PL，是為ε-PL之生產菌。在含葡萄糖之M3G培養基中經72h培養可得0.561g／L之ε-PL，但在含甘油之M3G培養基中經72h培養可得0.385g／L之ε-PL。S. albulus PLT1之ε-PL之合成酵素活性以附著細胞膜部份最為顯著（0.13±0.02 U／mg protein），其他去菌之培養液，細胞質部份酵素活性相當低，因此應是膜結合蛋白。S. albulus PLT1之ε-PL之分解酵素活性以附著細胞膜部份最為顯著（0.066±0.002 U／mg protein），其他去菌之培養液，細胞質部份酵素活性相當低，因此亦應是膜結合蛋白。在分解ε-P實驗，經30分鐘反應後部份ε-PL已分解，新產物為L-lys，而無其他多元之較小分子量之ε-PL，因此S. albulus PLT1之ε-PL分解酵素應是外切型酵素型態。ε-PL分解酵素（PLD）之分離純化可經過DEAE-Sepharose、Source 15Q陰離子交換層析後得到純化之酵素，最後之純化倍數為65.6，回收率為25.5％，比活性達40.0 U／mg。將純化的樣品進行SDS-PAGE電泳分析，樣品之電泳分析呈現一主帶，對照標準品分子量，計算得ε-PL分解酵素分子量約為39.5 kDa。

ε-Poly-lysine (ε-PL) is a naturally occurring biomaterial produced through microbial fermentation. ε-PL has potential applications in diverse areas, such as food, medicine, pesticides, and electronic and chemical material. Remarkable studies concerning ε-PL biosynthesis have been reported; however, the biosynthetic mechanism of ε-PL production has not yet been elucidated, and the enzymes involved in its biosynthesis are yet to be isolated and characterized. Therefore, this study involved the isolation and purification of an ε-PL-degrading enzyme (Pld). An ε-PL-tolerant strain (Streptomyces albulus PLT1) that demonstrated both ε-PL-producing and ε-PL-degrading capacities was isolated. S. albulus PLT1 synthesizes ε-PL 0.561g/L or 0.385g/L ε-PL in M3G medium containing glucose or glycerol, respectively. ε-PL synthetase (Pls) and the Pld enzyme of this strain are both membrane-bound proteins. The crude preparation of the Pld enzyme catalyzes exo-type degradation of ε-PL. The ε-PL-degrading enzyme of S. albulus PLT1 was purified. Coarse enzyme extraction was subjected to DEAE-Sepharose and Source 15Q anion exchange chromatography. The final purification fold was 65.5 with recovery yield = 25.5% and specific activity = 40.0 U/mg. The purified sample was analyzed using SDS-PAGE, and the molecular mass of the enzyme was estimated to be approximately 39.5 kDa.