ε-聚離胺酸分解酵素之生化研究（二）

Biochemical Study of ε-Polylysine Depolymerase: Part 2

吳芳禎(FANG-CHEN WU)；李建德(JEN-DER LEE)；施英隆(ING-LUNG SHIH)

ε-聚離胺酸 ； 聚離胺酸分解酵素 ； 放線菌白色鏈球菌 ； 酵素純化 ； 離子交換層析 ； 外切型酵素 ； ε-Poly lysine ； ε-PL-degrading enzyme ； Streptomyces albulus ； enzyme purification ； ion exchange chromatography ； exo-type degradation

科學與工程技術期刊

16卷2期（2020 / 09 / 01）

27 - 34

繁體中文

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，它的應用潛力廣，然而生合成作用機制還未明，調控機制則仍十分模糊。ε-PL分解酵素（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。我們曾篩選出一株具有能忍受ε-PL之菌株且具ε-PL合成與ε-PL分解能力之菌株S. albulus PLT1，此菌株之ε-PL分解酵素為膜結合酵素。S. albulus PLT1之ε-PL分解酵素經分離純化，已知其分子量約為39.5 kDa。本研究接續先前之研究，進行ε-PL分解酵素之特性鑑定及探討其生化活性；ε-PL分解酵素的特性分析發現，其對ε-PL之作用型態為外切型酵素，以L-lysyl-p-nitroanilide（Lys−PNA）為基質時，最適反應溫度為30℃，最適pH值為7.0，在20-40℃保存時酵素活性穩定，在50℃以上保存時酵素活性迅速下降。最大反應速率V_(max) = 0.068mmol/L/min，米氏常數K_m= 0.127 mmol/L。ε-PL分解酵素是一種金屬酶，EDTA處理後酵素活性完全消失，加入Ca^(2+)、Fe^(3+)、Mg^(2+)及Zn^(2+)可有效恢復酵素活性，分別為94%、92%、85%及70%。Co^(+2)可恢復酵素活性42%，Fe^(2+)、Mn^(2+)、Cu^(2+)、Ni^(2+)及Hg^(2+)則無法恢復酵素活性。

Microbial fermentation produces ε-polylysine (ε-PL), which is a naturally occurring biomaterial. It has potential applications in various fields, such as in the food, medicine, pesticides, and electronic and chemical materials. Studies have reported promising results for ε-PL biosynthesis; however, the biosynthetic mechanism has yet not been elucidated, and the biosynthetic enzymes have yet to be isolated or characterized. We previously isolated an ε-PL-tolerant strain (Streptomyces albulus PLT1) that showed both ε-PL-producing and ε-PL-degrading capacity. The ε-PL-degrading enzyme (a cell membrane-associated protein) of Streptomyces albulus PLT1 was purified and analyzed using polyacrylamide gel electrophoresis. Its molecular mass was estimated to be approximately 39.5 kDa. In the present study, the enzyme was further characterized, and its biochemical properties were explored. The results showed that the enzyme catalyzed the exo-type degradation of ε-PL. With L-lysyl-p-nitroanilide as a substrate, the enzyme was stable between pH 6.0 and 9.0 and showed maximum activity at pH 7.0. The optimal temperature was 30℃, and activity considerably decreased at temperatures exceeding 50 ℃. The enzyme is a metalloenzyme that was completely deactivated by ethylenediaminetetraacetic acid treatment, but 94%, 92%, 85%, 70%, and 40% of its activity was restored through the addition of Ca^(2+), Fe^(3+), Mg^(2+), Zn^(2+), and Co^(2+), respectively. However, the addition of Fe^(2+), Mn^(2+), Cu^(2+), and Ni^(2+) was unable to restore activity. The apparent K_m with L-lysyl-p-nitroanilide as the substrate was 0.127 mM, and the V_(max) was 0.068 mmol/L/min.

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