無花果蛋白酶酵素製品之幾丁質酶性質研究

無花果蛋白酶酵素製品所含幾丁質酶經由pHMB-Sepharose 4B及cystatin-Sepharose 4B管柱親和層析除去蛋白酶後，再經Superdex 75 HR管柱膠體過濾層析，可使幾丁質酶純度提高5.6倍並獲得49%之幾丁質酶活性收率。此純化之幾丁質酶可水解幾丁質聚合物，但不水解Micrococcus lysodeiktics菌體細胞壁，其水解乙二醇幾丁質之最適pH為4.5，最適溫度為70℃，Km值為4.57mg/mL而Vmax為6.95μmol GlcNAc/min/mg。此外，酵素對水解p-nitrophenyl N-acetylchitooligosaccharides（聚合度3~5）釋出p-nitrophenol亦具有活性，其中以對p-nitrophenyl tetra-N-acetyl-β-chitotetraoside具有最高活性。以膠體過濾法測得酵素分子量為16.6kDa，熱穩定性分析顯示酵素對熱穩定性良好（70℃保溫30分鐘幾無活性損失），化學修飾劑N-bromosuccinimide(0.25mM)及2,4-dinitro-1-fluorobenzene(2.5mM)則會顯著抑制酵素活性。

關鍵字

無花果蛋白酶；幾丁質酶；純化；性質

並列摘要

A chitinase was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and then gel filtration on Superdex 75 HR. By these steps, the purity of chitinase was increased 5.6 fold and the recovery of chitinase activity was 49%. The purified chitinase exhibited activity toward chitin and chitosan polymer, but showed no activity toward Micrococcus lysodeiktics cell wall. The optimal pH for ethylene glycol chitin (a water-soluble chitin) hydrolysis was 4.5, the optimal temperature was 70℃, the Km was 4.57 mg/mL and Vmax was 6.95μmol GlcNAc/min/mg. In addition, the enzyme also showed activity toward p-nitrophenyl N-acetylchitooligosaccharides with chain length from 3 to 5 (pNP-β-GlcNAc(subscript 3-5)) for releasing p-nitrophenol. Most effectively hydrolyzed was p-nitrophenyl tetra N-acetyl-β-chitotetraoside (pNP-β-GlcNAc4). The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. This enzyme was thermostable, as it retained almost all of its activity after incubation at 70℃ for 30 min. Chemical modification gents N-bromosuccinimide (0.25 mM) and 2,4-dinitro-1-fluorobenzene (2.5 mM) significantly inhibited the enzyme activity.

並列關鍵字

Ficin ； Chitinase ； Purification ； Properties

國際替代計量

無花果蛋白酶酵素製品之幾丁質酶性質研究

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