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

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

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。Hg2+、Zn2+及 Mn2+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。

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 Hg2+, Zn2+, and Mn2+ metal ions have an inhibitory effect on chitin-degrading enzyme activity, whereas ethylenediaminetetraacetic acid can promote such activity.