綠竹筍β-N-乙醯胺基六碳糖苷酶及幾丁三糖酶之純化與性質研究

新鮮採收綠竹筍經幾丁聚醣溶液處理後，其可食部含高活性之幾丁質酶、幾丁聚醣酶等防禦性酵素，此外亦含有β-N-乙醯胺基六碳糖苷酶(β-NAHA)及幾丁三糖酶活性，可能是配合幾丁質酶降解入侵真菌細胞壁幾丁質，所以也具防禦性酵素功能。本研究之目的即在分離純化綠竹筍可食部所含β-NAHA及幾丁三糖酶，並探討其酵素性質。新鮮綠竹筍採收後以0.1﹪幾丁聚醣處理，置室溫 (24±2℃)貯存42小時，將可食部冷凍乾燥並磨成粉末，作為粗酵素粉末，儲存於-20℃。粗酵素粉末經由0.025 M imidazole-HCl緩衝液(pH7.4)抽取，硫酸銨沉澱(80 ﹪飽和度) ，Sephacryl S-200膠體過濾層析，DEAE-Sephacel離子交換層析，PBE-94等電焦集層析及Superose 12 HR 膠體過濾層析等連續步驟純化，可使β-NAHA酵素純度提高72.7倍，並獲得5.9％之活性回收率。膠體過濾層析測得酵素原態分子量為44.3 kDa，以人工合成之pNP-β-N-GlcNAc為基質，純化綠竹筍β-NAHA之最適pH值為5.0，最適溫度為50℃，熱穩定性分析顯示酵素於30至45℃保溫40分鐘頗穩定，但超過50℃則顯著失去活性， Km值為1.16mM而最大反應速率為4.22umol/min/mg。重金屬離子 Hg2+ (0.25 mM)完全抑制酵素活性，而Zn2+(5 mM)和Ag+(0.25 mM)則分別抑制69%及33%酵素活性。化學修飾劑Woodward’s reagent K (50 mM)完全抑制酵素活性，N-bromosuccinimide (5 mM) 抑制90%左右酵素活性，p-hydroxymercuribenzoate(0.5 mM) 抑制60%左右酵素活性。由基質特異性測定得知酵素對β-糖苷鍵具有專一性，對β-N-乙醯胺基葡萄糖苷水解之速率約為β-N-乙醯胺基半乳糖苷之四倍。粗酵素粉末經由0.025 M imidazole-HCl緩衝液(pH7.4)抽取，硫酸銨沉澱(80 ﹪飽和度) ，Sephacryl S-200膠體過濾層析，DEAE-Sephacel離子交換層析等連續步驟純化，可使幾丁三糖酶酵素純度提高10.2倍，並獲得70％之活性回收率。以人工合成之4-MU-GlcNAc3為基質，純化綠竹筍幾丁三糖酶之最適pH值為4.0，最適溫度為55℃。重金屬離子 Hg2+ (0.25 mM)抑制30％酵素活性，其餘重金屬離子對酵素活性無顯著影響。

關鍵字

綠竹筍；純化；幾丁三糖酶； β-N乙醯胺基葡萄糖苷酶

並列摘要

After the storage of fresh harvest bamboo shoots treated with chitosan, the edible portion of bamboo shoots had high activities of chitinolytic,enzymes. It also contains the enzyme of β-N-acetyl- hexosaminidase (β-NAHA) and chitotriosidase which may play an important role for degradation of fungi cell walls in defense mechanism. The purpose of this study is to investigate the purification and characterization of β-NAHA and chitotriosidase from edible portion of bamboo shoots. Fresh harvest bamboo shoots were treated with 0.1％ chitosan and stored at room temperature for 42 hours. The dried powders from edible portion of bamboo shoots by lyophilization were stored at -20℃. β-NAHA was purified by the following sequential steps: buffer extraction, ammoniumsulfate.fractionation Sephacryl S-200 gelfiltration,DEAE- Sephacel,ion-exchange,chromatography,PBE-94chromatofocusing and Superose 12 HR gel filtration. We found that the purity of β-NAHA increased 72.7 fold and the recovery of activity was 5.9％. The molecular mass estimated by gel filtration was 44.3 kDa. The optimal pH for pNP-β-N-GlcNAc hydrolysis was 5.0 and the optimal temperature was at 50℃. To assess thermostability, the enzyme almost retained all of its activity after incubation at 30℃〜45℃ for 30 min , but significantly loss activity after 50℃. The Km was 1.16 mM and Vmax was 4.22 umole/min/mg . Heavy metal ions of Hg2+ (0.25 mM), chemical modification agents N-bromosuccinimide (0.5 mM) and Woodward’s reagent K (50 mM) significantly or completely inhibited the activity of the enzyme. From the measure of substrate specificity, the enzyme had the property of specific for β-glycosidic linkage. chitotriosidase was purified by the following sequential steps: buffer extraction, ammonium sulfate fractionation, Sephacryl S-200 gel filtration, DEAE-Sephacel ion-exchange chromatography, We also found that the purity of chitotriosidase increased 10.2 fold and the recovery of activity was 70％. The optimal pH for 4-MU-GlcNAc3 hydrolysis was 4.0 and the optimal temperature was at 55℃. Heavy metal ions of Hg2+ (0.25 mM) inhibited the 30％ activity of the enzyme.

並列關鍵字

Purification ； bamboo shoots ； β-N-acetylhexosaminid

參考文獻

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余詔儒‧2004‧綠竹筍外殼β-N-乙醯胺基葡萄糖苷酶之純化及性質研究。靜宜大學食品營養研究所碩士論文。

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綠竹筍β-N-乙醯胺基六碳糖苷酶及幾丁三糖酶之純化與性質研究

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