嗜碱芽孢杆菌C-125木糖苷酶基因的表达与酶特征鉴定

Expression and characterization of a xylosidase (Bxyl) from Bacillus halodurans C-125

摘要:

嗜碱芽孢杆菌(Bacillus halodurans)C-125菌株的基因组中, 一个编码木糖苷酶的基因(BH1068)被克隆并在大肠杆菌中获得高效表达。通过全面分析纯化蛋白, 确证了它的木糖苷酶功能。该酶在pH 4~9的范围内保持稳定, 最适pH值为中性, 有较宽的最适温度(35°C~45°C), 且能在45°C范围内保持稳定。这些特性使得该酶可在较为宽广的条件下对木聚糖进行酶促降解。该酶对人工合成底物对硝基苯-b-木糖苷(p-nitrophenyl-b-xylose, pNPX)的比活力为174 mU/mg蛋白质, 且木糖对其反馈抑制较弱(抑制常数Ki为300 mmol/L)。结果显示该酶是活性较高且较耐木糖抑制的细菌源木糖苷酶。该酶与商品化的木聚糖酶一起水解山毛举木聚糖(Beechwood xylan)时显示了增效作用, 且水解率可获40%。该酶最适pH为中性, 对木糖耐受等特性与大多数来源于真菌、最适pH为酸性、对木糖敏感的木糖苷酶将有较好的互补。结果表明该酶在木聚糖或含木聚糖多糖的单糖化过程可能发挥重要作用。

Abstract:

A xylosidase gene, labeled as BH1068 in genome of Bacillus halodurans C-125, was successfully cloned and overexpressed in Escherichia coli JM109. The purified enzyme was thoroughly characterized and its xylosidase function was unambiguously confirmed. It has maximum activities in neutral condition and is stable over a wide range of pH (4.5–9.0). The enzyme has a broad temperature optimal (35°C–45°C) and is quite stable at temperature up to 45°C. The unique pH and temperature profiles of the enzyme should allow a wide range of xylanolytic operational conditions. With high specific activity of 174 mU/mg protein for its artificial substrate (p-nitrophenyl-b-xylose) and low xylose inhibition (inhibitor constant Ki = 300 mmol/L), this enzyme is among the most active and high tolerant bacterial xylosidase to xylose inhibition. Its high synergy with commercial xylanase has been demonstrated with beechwood xylan hydrolysis, achieving a hydrolysis yield of 40%. Its neutral pH optimal and high tolerance to product inhibition complements well with its fungal counterparts that are only optimal at acidic pH and susceptible to xylose inhibition. In conclusion, this enzyme has high potential in the saccharification of xylan and xylan-containing polysaccharides.