摘要
草菇(Volvariellavolvacea)在20L发酵罐中以稻草粉为基质进行培养,所产生的β 木糖苷酶(β 1,4 xylosidase,EC3.2.1.37)经硫酸铵沉淀、苯基 琼脂糖(Phenyl agarose)疏水性柱层析、DEAE Sephacel阴离子柱层析、TOSOHASS HW 5S分子筛过滤分离等提纯步骤,达到了电泳纯,提纯倍数为241倍,收率为14.5%.该酶反应的最适pH值为6.41～6.76,最适温度为55℃;在pH值5.78～7.68之间酶活力相对稳定,52℃的半衰期(t1/2)为1h.由凝胶过滤和SDS PAGE测得酶由4个亚基组成,酶的相对分子质量为2825000.在所测定的底物中,β 木糖苷酶仅对对硝基苯基 β D 木糖苷有专一性水解作用,其对对硝基苯基 β D 木糖苷水解的动力学参数Km值为2.4mmol/L,vmax为42μmol/(min·mg).该酶催化对硝基苯基 β 木糖苷水解将产物β 木糖反转成α 木糖.
A β-xylosidase was purified from straw powder culture of Volvareilla volvacea through following steps: a) ammonium sulfate precipetation; b) hydrophobic interaction chromatography on Phenyl-agarose; c) ion exchange chromatography on DEAE-sephacel; and d) Gel filtration on TosoHaas-HW-55S. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 241 fold, and a yield of 14.5%. The optimal reaction conditions for the enzyme were pH 6.41 ～ 6.76 at 55 ℃. At the pH range of 5.78 ～ 7.68, the enzyme was stable and had 1 h half-life at 52℃. The molecular weight was estimated by SDS-PAGE and by gel filtration as 70,100 and 282,500,respectively. Among all tested substrates, the enzyme exhibited the specific activity to p-nitrophenyl β-xylopyranoside (PNPX). The apparent Michaelis constant of the β-xylosidase was 2.4 mmol/L for PNPX, and v(max)was 42 U/mg protein. The enzyme hydrolyzed PNPX with inversion of anomeric configuration, producing α-xylose from β-xylopyranoside.
出处
《无锡轻工大学学报（食品与生物技术）》
CSCD
北大核心
2004年第3期30-35,共6页
Journal of Wuxi University of Light Industry
关键词
Β-木糖苷酶
草菇
分离纯化
酶学性质
β-xylosidase
Volvareilla volvacea
purification and characterization
