木瓜乳汁乾燥製品之幾丁質酶及幾丁聚醣酶研究

Studies on Chitinase and Chitosanase from Dried Papaya Latex

廖倍瑜(Bei-Yu Liao) ； 吳珮甄(Pei-Chen Wu) ； 江翠蓮(Chui-Liang Chiang) ； 張珍田(Chen-Tien Chang)

《臺灣農業化學與食品科學》 44卷4期 (2006/08) Pp. 217-227

https://doi.org/10.6578/TJACFS.2006.025

摘要

木瓜乳汁乾燥製品所含幾丁聚合物水解酵素經由緩衝液（含1.66 mM p-hydroxymercuribenzoic acid）抽取、硫酸銨沉澱（70%飽和度）及Sephacryl S-100膠體過濾層析等連續步驟純化，可獲得幾丁三糖酶及幾丁聚醣酶兩種幾丁聚合物水解酵素。其中幾丁三糖酶為一種可水解4-methylumbelliferyl N，N'，N”-triacetyl-β-chitotrioside(4-MU-GlcNAc3)之幾丁質酶，其水解4-MU-GlcNAc3之最適pH為4，最適溫度為40~50℃，而Km值為9.33μM。此外，幾丁三糖酶亦可水解p-nitrophenyl-N-acety-β-D-glucosaminide及p-nitrophenyl-N-acetylchitooligosaccharides（聚合度3~5；pNP-β-GlcNAc(下標 3~5)），顯示純化之幾丁三糖酶尚含有β-N-乙醯胺基葡萄糖苷酶。另一純化之幾丁聚醣酶水解幾丁聚醣之最適pH為4.6，最適溫度為70℃，而Km值為1.5mg/mL。以膠體過濾層析測得酵素原態分子量為12.4kDa，熱穩定性分析顯示酵素對熱頗穩定（70℃保溫30分鐘尚有75%左右活性），基質專一性分析顯示酵素對22~94%去乙醯度幾丁聚醣均具活性，其中以對50~60%去乙醯度幾丁聚醣具有最高活性。幾丁聚醣醇水解幾丁聚醣之主要產物為低分子量幾丁聚醣及幾丁寡醣(26.3~2.4kDa)。

關鍵字

木瓜乳汁 ； 幾丁三糖酶 ； 幾丁聚醣酶 ； 純化 ； 性質

並列摘要

Two chitinolytic enzymes, chitotriosidase and chitosanase, were purified sequentially from a commercial dried papaya latex by buffer (containing 0.166 M p-hydroxymercuribenzoic acid) extraction, (NH4)2SO4 (70% saturation) precipitation and Sephacryl S-100 gel filtration. Chitotriosidase is a chitinase that can hydrolyze chitotrioside derivative 4-methylumbelliferyl N, N', N”-triacetyl-β-chitotrioside (4-MU-GlcNAc3). The optimal pH for 4-MU-GlcNAc3 hydrolysis of the purified chitotriosidase was 4, the optimal temperature was 40~50℃ and the Km was 9.33μM. The enzyme also showed activity toward p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-β-GlcNAc) and p-nitrophenyl-N-acetylchitooligosaccharides with chain length from 3 to 5 (pNP-β-GlcNAc(subscript 3-5)) for releasing p-nitrophenol. These results indicate that the purified chitotriosidase contains β-N-acetylglucosaminidase. The optimal pH for chitosan hydrolysis of the purified chitosanase was 4.6, the optimal temperature was 70℃ and Km was 1.5 mg/mL. The molecular mass of the enzyme was 12.4 kDa, as estimated by gel filtration. This enzyme was thermostable, as it retained~75% of its activity after incubation at 70℃ for 30 mim. Chitosan polymers exhibiting various degrees of deacetylation (22~94%) were all susceptible to the enzyme, with the most susceptible being 50~60% deacetylated. The end products of the hydrolysis catalyzed by the enzyme were low molecular weight chitosan polymers and oligomers (26.3~2.4 kDa).

並列關鍵字

Papaya latex ； Chitotriosidase ； Chitosanase ； Purification and characterization