Biochemical Characterization of a Novel Chitotriosidase from suspension-cultured Bamboo (Bambusa oldhamii) Cells

綠竹筍懸浮細胞於含有2,4-D激素培養過程誘發幾丁質酶並分泌於培養液中，經由硫酸銨劃分（40-80%飽和度）、Phenyl-Sepharose 疏水性層析、DEAE-Sephacel離子交換層析及製備式聚丙烯醯胺膠體電泳等連續純化步驟，可由懸浮細胞培養濾液分離一種新型幾丁質酶，稱為幾丁三醣酶。此幾丁三醣酶可水解N-乙醯幾丁寡醣，但幾乎不水解高分子幾丁質，其水解N-乙醯幾丁三醣衍生物4-MUGlcNAc3之最適pH為3，最適溫度為70°C而K(下標 m)值為4.07μM。以膠體過濾法及SDS-PAGE電泳法測得酵素分子量皆為90.5 kDa，二維電泳及膠體酵素活性染色測得酵素等電點(pI)為5。純化之幾丁三醣酶於60°C保溫30分鐘或4°C貯存一年，幾無活性損失，相當安定，汞離子(0.5 mM)顯著抑制酵素活性。以薄層層析法分析顯示酵素水解幾丁質寡醣(GlcNAc(下標 n)，n=3~6)之產物為GlcNAc(下標 1~2)或GlcNAc(下標 1~3)，而可作用之最低聚合度幾丁質寡醣則為GlcNAc(下標 1~3)。

關鍵字

綠竹筍懸浮培養細胞；幾丁質酶；幾丁三醣酶；生化檢定； 4-MU-GlcNAc3

並列摘要

Chitinase was induced in suspension-cultured bamboo cells in the presence of 2,4-dichloro phenoxyacetic acid (2,4-D) and secreted into the medium during cultivation. A novel chitinase, designated chitotriosidase, was purified from the medium of the suspension-cultured cells by sequentially applying (NH4)2SO4 (40-80% saturation) fractionation, hydrophobic chromatography, DEAE-Sephacel ion-exchange chromatography, and preparative polyacrylamide gel electrophoresis. The purified chitotriosidase was active toward chitin oligomer substrates but almost inactive toward chitin polymer. The optimal pH for 4-methylumbelliferyll-β-D-N, N', N”-triacetylchitotrioside (4-MU-GlcNAc3) hydrolysis of the enzyme was 3. The optimal temperature was 70°C, and the K(subscript m) was 4.07 μM. The molecular mass was 90.5 kDa, as estimated by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5, as estimated using two dimensional electrophoresis and gel activity staining. The chitotriosidase was thermally stable, as it retained almost all of its activity after incubation at 60°C for 30 min or storage at 4°C for a year. Mercuric ion (0.5 mM) significantly inhibited the enzyme's activity. The end products of N-acetylglucosamine oligomers (GlcNAc(subscript n), n=3~6) hydrolysis catalyzed by the enzyme were GlcNAc(subscript 1~2) or GlcNA(subscript 1~3), as analyzed using thin-layer chromatography. The smallest of the chitin oligomer substrates for the enzyme action was a chitin trimer.

並列關鍵字

Bamboo Bambusa oldhamii suspension cells ； Biochemical characterization ； Chitinase ； Chitotriosidase ； 4-MU-GlcNAc3

國際替代計量

Biochemical Characterization of a Novel Chitotriosidase from suspension-cultured Bamboo (Bambusa oldhamii) Cells

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