Aeromonas hydrophila Too12生產N-乙醯幾丁寡醣之發酵培養條件探討

Study of the Cultivation Conditions of Aeromonas hydrophila Too12 for Producing N-acetylchitooligosaccharides

顧振旗(ZHEN-QI GU) ； 吳淑姿(SHWU-TZY WU) ； 余世宗(SHIH-TSUNG YU)

《科學與工程技術期刊》 18卷1期 (2022/03) Pp. 1-13

摘要

探討不同培養條件（不同碳源、氮源、α-幾丁質濃度、β-幾丁質濃度和通氣量）對菌株Aeromonas hydrophila Too12 生產N-乙醯幾丁寡醣的影響。菌株培養於2% α-幾丁質與膠態幾丁質72 h，水解產物以N-乙醯幾丁三醣為主，分別為0.48與0.47 g/L。菌株培養於5% α-幾丁質144 h，水解產物N-乙醯葡萄糖胺2.85 g/L與N-乙醯幾丁三醣2.42 g/L。培養於4% β-幾丁質，生成水解產物含有N-乙醯幾丁四醣0.68 g/L。以蛋白腖、酵母抽出物及胰化蛋白為氮源之培養，其幾丁質分解酶活性、還原醣生成量及幾丁質水解產物相近。於高溶氧的批次發酵培養96 h，N-乙醯幾丁三醣產量為0.64 g/L。於低溶氧的批次發酵培養72 h，N-乙醯葡萄糖胺與N-乙醯幾丁三醣，分別有1.26與1.1 g/L。不通氣的批次發酵培養96 h，N-乙醯葡萄糖胺為2.55 g/L。培養菌株之粗酵素液，經硫酸銨沉澱、透析、離子交換層析（DEAE-Sepharose CL-6B）和膠體過濾層析（Sephacryl S-100）純化步驟，收集具活性之波峰，利用活性染色分析，33 kDa蛋白質於膠片上有明顯活性。酵素最適反應溫度與pH值分別為40℃與6.0。Hg^(2+)、Zn^(2+)及Mn^(2+)金屬離子對幾丁質分解酶活性具抑制作用，EDTA對幾丁分解酶之活性具促進作用。

關鍵字

N-乙醯幾丁寡醣 ； N-乙醯幾丁三醣 ； 幾丁質分解酶

並列摘要

This study explored the effect of different cultivation conditions (different carbon sources, nitrogen sources, α-chitin concentration, β-chitin concentrations, and ventilation) on the production of N-acetylchitooligosaccharides using the strain Aeromonas hydrophila Too12. The strains were cultivated in 2% α-chitin and colloidal chitin for 72 h using N-acetyl chitosan triose hydrolysate at 0.48 and 0.47 g/L, respectively. The strain that was cultivated in 5% α-chitin for 144 h and N-2.85 g/L acetylglucosamine and 2.42 g/L N-acetylchitotriose. Under cultivation in 4% β-chitin, the hydrolysate contained 0.68 g/L N-acetylchitotetraose. With the cultivation of peptone using yeast extract and pancreatic protein as nitrogen sources, the activity of chitin-decomposing enzyme, amount of reducing sugar, and amount of chitin hydrolysate were similar. After 96 h of fermentation in batches with high dissolved oxygen, the yield of N-acetylchitotriose was 0.64 g/L. After fermentation and cultivation in batches with low dissolved oxygen for 72 h, the yield of N-acetylglucosamine and N-acetylchitotriose was 1.26 and 1.1 g/L, respectively. The nonaerated batch was fermented for 96 h, producing 2.55 g/L N-acetylglucosamine. The crude enzyme solution of the cultured strain was purified through ammonium sulfate precipitation, dialysis, ion exchange chromatography, and colloidal filtration chromatography. The active peaks were collected and analyzed using active staining; 3-kDa protein exhibited obvious activity on the film. The optimal reaction temperature and pH of the enzyme were 40°C and 6, respectively. The results revealed that Hg^(2+), Zn^(2+), and Mn^(2+) metal ions have an inhibitory effect on chitin-degrading enzyme activity, whereas ethylenediaminetetraacetic acid can promote such activity.

並列關鍵字

N-acetylchitooligosaccharides ； N-acetylchitotriose ； chitinase

參考文獻

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