Aeromonas caviae No. 2 所生產幾丁質分解酵素之研究

Studies on chitinolytic enzymes from Aeromonas caviae No. 2

吳立森(Li-Sen Wu)

指導教授 ： 陳俊任

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01249

摘要

過去研究指出聚合度4-7 的N-乙醯幾丁寡醣 (N-acetylchitooligosacharides) (GlcNAc) 4-7具有抗腫瘤、提高小鼠免疫能力的生物活性。本研究由富含幾丁質環境中篩選出具有可分解幾丁質產生較高聚合度的N-乙醯幾丁寡醣的菌株。初期篩選中，先以幾丁質作為培養基主要碳源，篩選可利用幾丁質之微生物，再以粗酵素液水解膠態幾丁質，以 HPLC 與 TLC鑑定其產物是否具有 (GlcNAc) 4-7。經由此篩選程序，選獲一菌株可水解幾丁質產生(GlcNAc) 4-5，水解幾丁聚醣產生(GlcN/GlcNAc) 4-6 。此菌株經16S rDNA 分析，被分類與 Aeromonas caviae 之親緣關係最為接近，本研究中命名為 Aeromonas caviae No. 2 。探討此菌株的最佳培養條件，以增加幾丁質酶之生產方面，首先以不同之培養酸鹼值與溫度測試，獲得最佳培養酸鹼值為pH 6 , 溫度為25℃。另以反應曲面法 (response surface methology, RSM) 之中心混成設計 (central composite design, CCD) 得到最佳酵素生產培養基條件之碳源與氮源濃度為colloidal chitin 1.098% , soybean flour 0.735% 和 yeast extract 0.74%。以複選培養基培養之粗酵素液以一系列純化方法包括：硫酸銨分劃 (40-70%) ，專一性吸附水解法及 Sephacryl 200 膠體過濾進行純化。純化後在SDS-PAGE於 55 至 100 kDa之間得到四個可能和分解幾丁質相關的蛋白質，其中有兩個於 55-70 kDa 間於幾丁質活性染色膠體電泳中具有活性訊號。但是若以最佳化培養基培養之粗酵素液純化過後之蛋白質在 SDS-PAGE 上於 40 至 100 kDa 之間分佈 9 個蛋白質訊號，活性染色上則可測得三種幾丁質酶，其中兩種於 55 至 70 kDa 附近，而在40 kDa 附近也有一條活性訊號。另在純化後之蛋白質水解產物方面，其水解幾丁質產生大量之 (GlcNAc)2 與少量 GlcNAc 和 (GlcNAc)3，另水解幾丁聚醣之產物亦出現 (GlcNAc)4-6 以及可能為更高聚合度之寡醣。

關鍵字

N-乙醯幾丁寡醣 ； 幾丁質酶16S rDNA ； 反應曲面法 ； 中心混成設計

並列摘要

It was reported that N-acetylchitooligosaccharides (degree of polymerization 4-7) (GlcNAc)4-7 have specific biological activities such as antitumor activity and immuno-stimulating effects. In this study, we aimed to screen microorganisms from a chitin-rich environment and isolate the ones that can produce enzymes to hydrolyze chitin into N-acetylchitooligosaccharides. At the initial stage, we used the selection medium with colloidal chitin as the sole carbon source to select for microorganisms which could utilize chitin for growth. The microbial isolates were further screened by incubating the culture broth with colloidal chitin and analyzing the products by HPLC and TLC. Using this screening strategy, we obtained one bacterial isolate that could produce enzymes to hydrolyze chitin into (GlcNAc) 4-5 and hydrolyze chitosan into (GlcN/GlcNAc) 4-6. The bacterial strain was identified by 16S rDNA sequencing and phylogenetic classification to belong to Aeromonas caviae and was named Aeromonas caviae No. 2. To optimize the culture condition for producing higher amounts of chitinase, we first tested various culture pHs and temperatures and found that the highest chitinase activity was produced at pH 6 and 25℃. Using the central composite design (CCD) of response surface methodology (RSM), we further obtained the optimal concentrations of chitin and nitrogen sources in the culture medium to be colloidal chitin: 1.098%, soybean flour: 0.735% and yeast extract: 0.74%. The crude enzyme produced in selection medium went through successive steps of purification including ammonium sulfate precipitation (40-70%), chitin affinity-hydrolysis method and gel filtration chromatography (Sephacryl 200). After purification, four major proteins were found range from 55 to 100 kDa on the SDS-PAGE, and two of them showed chitinase activity range from 55 to 70 kDa on the in-gel chitinase activity assay. When proteins from the optimized culture medium were purified using nine major bands appeared range from 40 to 100 kDa on the SDS-PAGE, and three of them showed chitinase activity on the in-gel chitinase activity assay. Moreover, the purified proteins could hydrolyze chitin primarily into (GlcNAc)2 and hydrolyze chitosan into
(GlcN/GlcNAc) 4-6.

並列關鍵字

N-acetylchitooligosaccharides ； chitin ； chitinase ； response surface methology (RSM) ； central composite design (CCD)

參考文獻

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