鳳梨莖部凝集素純化與特性分析

Characteration and purification of a lectin from pineapple stem (Ananas comsus)

林志偉(Chih-Wei Lin)

指導教授 ： 戴守谷

國立虎尾科技大學/電機資訊學院/光電與材料科技研究所/碩士(2009年)

https://doi.org/10.6827/NFU.2009.00087

摘要

凝集素廣泛地存在各種生物體內，為具有高度醣類結合特異性之蛋白質，其被稱為外源凝集素、植物凝血素、血球凝集素、親醣蛋白質、擇素或醣結合蛋白等。醣類研究是繼基因體時代及蛋白質體學研究後重要的領域之一，凝集素是醣類或醣蛋白質研究中，重要的分析工具，相關的應用也陸續發展中。不同醣類結合活性之凝集素的發現，將更有利於醣類研究工作之進展。本研究以鳳梨莖部蛋白質為研究材料，證實鳳梨莖部蛋白質粗萃取液具有紅血球凝集活性，推測鳳梨莖部應含有凝集素相關之蛋白質。鳳梨莖部蛋白質粗萃取液，先初步進行各種醣類抑制紅血球凝集活性測試，鑑定其凝集素可能之醣類結合活性。經血球凝集活性抑制測試，發現D-Glucose、D-Mannose、N-Acetyl-D-Glucosamine、Glucosamine、Methyl-α-D-Mannopyranoside 及Trehalose 等醣類具有抑制活性。鳳梨莖部蛋白質粗萃取液經離心分離，其上清液及沉降物都具有凝集素之活性，沉降物之活性更高於上清液。以0.5M Glucose溶液抑制澱粉與凝集素結合，由澱粉沉降物中競爭出凝集素，經離心分離，上清液進行血球凝集活性測試，比活性為1.379×103 HAU/mg。利用醣類配體競爭性回溶分析，確認醣類結合之特異性與粗萃取液之測試結果一致。鳳梨凝集素熱穩定測定，結果顯示熱穩定度可達60℃，1小時。經同源結構模擬，結果指出鳳梨凝集素在結構上為典型之jacalin-related lectins，為β-prism之構形。本研究開發之純化流程精確掌握鳳梨莖部內生性澱粉與凝集素間結合活性及澱粉沉降特性，進行親和力之純化，而此步驟即可使鳳梨凝集素達95%以上之純度，為本純化製程之優勢。而此純化流程之開發，亦利於後續放大製程之發展。另外，鳳梨莖部萃取液(Bromelain)開發成為機能性食品供食用已有很長的時間。由此推論，鳳梨凝集素應用於食品或生物醫藥上，將較無安全上之顧慮。

關鍵字

鳳梨凝集素 ； 鳳梨 ； 凝集素 ； 葡萄糖 ； 醣類結合專一性

並列摘要

Lectins are carbohydrate-binding proteins that can agglutinate cells and precipitate glycoconjugates. Many Plants contain the lectins that bind preferentially to mannose or glucose in the monocot and dicot species. One of the predominant proteins in the stem of pineapple (Ananas comosus) has been identified as a lectin. The lectin designated ACL was isolated and purified from the pineapple stem by a combination of affinity precipitation and resuspension with competitive carbohydrate ligan. The ACL was homogeneous by the purification and a monomeric protein with a molecular weight of 15 kDa as demonstrated by SDS-PAGE. A cDNA clone encoding the ACL from pineapple stem has been cloned and sequenced. The coding region of ACL clone was introduced into an expression vector; pET-28a and -29a with His-tag fusion and non-fusion, and then transferred into Escherichia coli BL21 (DE3). Overexpression of recombinant ACL (rACL) in the expression system accumulated as inactive inclusion bodies. Western analysis of tissue specificity suggests that ACL is preferentially to express in the pineapple stem. The ACL is thermal stability up to 60℃ and exhibited maximum activity between pH 5 and 8. D-glucose, D-mannose , N-acetyl-D-glucosamine, D-glucosamine , Methyl-α-D-mannopyranoside and trehalose inhibited ACL-induced agglutination of human erythrocytes. The inhibition assay exhibited that ACL had nearly identical glucose/mannose-binding specificity. Multiple gene alignment revealed that ACL has sequence similarity to previously described lectins of the family of jacalin-related lectins. Accordingly, a structural modeling of ACL has the same overall β-prism fold of jacalin-related lectin . In addition, the ACL is the first documented of jacalin-related lectin, which is abundantly present in the stem of pineapple. The pineapple stem extract (Bromelain) as a health food supplement for a long time indicates that the ACL for the application will not be a food safety concern.

並列關鍵字

pineapple ； lectin ； ACL ； plant leactin ； jacalin-related lectin ； carbohydrate binding ； glucose ； mannose

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