Serratia marcescens NTU-17所產生幾丁質分解酵素 之研究

Study on Chitinolytic Enzymes from Serratia marcescens NTU-17

林芫如(Yuan-Ju Lin)

指導教授 ： 陳俊任

共同指導教授 ： 盧福明

國立臺灣大學/生物資源暨農學院/生物產業機電工程學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.00801

摘要

目前已知N-乙醯幾丁寡糖 (聚合度4-7)具有特殊生理活性如能提高免疫力及抑制腫瘤生長，故本研究目的擬由富含幾丁質環境中篩選出可以分解幾丁質產生N-乙醯幾丁寡醣之微生物。在初期篩選中，以粗酵素水解膠態幾丁質並以高效能液相層析 (HPLC)確認其產物，挑選出一株產物中有較長鏈N-乙醯幾丁寡糖之微生物，經鑑定命名為Serratia marcescens NTU-17。以反應區面法 (response surface methodology, RSM)之中心混成設計 (central composite design, CCD)得到最佳酵素生產的培養基條件為0.4 g/l colloidal chitin、1.6 g/l casein於30。C及pH 7.5下培養；以此條件下震盪培養18小時後可生產出最高的酵素活性。粗酵素液經35-70%硫酸銨沉澱後，以Sephacryl 200進行膠體過濾，接著以DEAE-Sephacel離子交換層析進行純化，最後分別得到二種純化之幾丁質酶，經SDS-PAGE分析其分子量，分別為53 kDa (chitinase 1)及39 kDa (chitinase 2)，並以活性染色方式確認此2種蛋白質皆具有幾丁質酶活性，純化之幾丁質酶活性大約為0.3 U/ml且比活性增加2.5倍，而酵素回收率則約為12%。在酵素特性部分，chitinase 1在pH 3反應溫度為50℃時有最佳的活性，而chitinase 2在pH 3-12的範圍中皆具有幾丁質酶活性。

關鍵字

幾丁質

並列摘要

Abstract
N-acetylchitooligosaccharides (degree of polymerization 4-6) have specific biological activities such as antitumor activity and immuno-enhancing effects. In this study, we aimed to isolate environmental microorganisms which could produce enzymes to hydrolyze chitin into N-acetylchitooligosaccharides. At the initial stage, we hydrolyzed colloidal chitin with crude microbial enzymes and analyzed the products by HPLC. From this screening, we found that the crude enzyme from one bacterial isolate could hydrolyze chitin and produce N-acetylchitooligosaccharides. The bacterial strain was identified by 16S rRNA sequencing and phylogenetic analysis to belong Serratia marcescens and was named S. marcescens NTU-17. We used central composite design (CCD) of response surface methodology (RSM) to obtain the optimal culture condition for chitinase production: 0.4 g/l colloidal chitin, 1.6 g/l casein, 30。C and pH 7.5; the highest chitinase activity was produced at 18 hours after inoculation. The crude enzyme from culture broth of S. marcescens NTU-17 was subjected to successive steps of purification. After ammonium sulfate fractionation (35-70%), gel filtration-Sephacryl 200 chromatography, and DEAE-Sephacel column chromatography, two species of chitinase were purified and the molecular weights were determined by SDS-PAGE to be 53 kDa (chitinase 1) and 39 kDa (chitinase 2). The chitinase activity of chitinase 1 and 2 were also verified by an in-gel chitinase activity assay. After purification, the specific activity of chitinase was increased by 2.5 fold and the yield was 12%. Chitinase 1 exhibited the optimal activity at pH 3 and 50℃, and chitinase 2 showed the optimal activity at 30℃ and similar activities at pH 3-12.

並列關鍵字

chitin

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