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首页> 外文期刊>African Journal of Biotechnology >Expression optimisation of recombinant -L-arabinofuranosidase from Aspergillus niger ATCC 120120 in Pichia pastoris and its biochemical characterisation
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Expression optimisation of recombinant -L-arabinofuranosidase from Aspergillus niger ATCC 120120 in Pichia pastoris and its biochemical characterisation

机译：黑曲霉ATCC 120120重组-L-阿拉伯呋喃糖苷酶在毕赤酵母中的表达优化及其生化特性

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摘要

A gene encoding α-L-arabinofuranosidase (AnabfA) from?Aspergillus niger?ATCC 120120 was successfully cloned and expressed in?Pichia pastoris?under the control of the?AOX1?promoter. The effect of cultural conditions on recombinant AnabfAproduction was studied and the enzyme was expressed as a soluble protein. Recombinant AnabfA?was expressed as an active enzyme at 28°C when cultured in BMMY medium (pH 6.0) and induced with 2% methanol every 24 h. Maximum activity was observed 5 days after induction. The purified recombinant AnabfAbefore and after treatment with PNGase F migrated by SDS-PAGE had relative molecular masses of about 83 and 66 kDa, respectively, suggesting that the AnabfA?contains?N-linked oligosaccharides. Characterisation of the purified recombinant AnabfA?showed an optimum temperature and pH of?50°C and 4, respectively. The enzyme was stable at a pH of 3 to 6 and retained more than 80% of its activity after pre-incubation at 40°C for 30 min.?The recombinant AnabfAactivity was stimulated by K+, Mn2+, Na2+?and triton X-100 and was strongly inhibited by Cu2+?and Fe2+?and the enzyme activity was relatively unaffected by Ca2+, CO2+, Mg2+?and EDTA. The?Km?and?Vmax?of the purified recombinant AnabfAactivity towards ρNPA were 0.93 mM and 17.86 μmol/ml/min, respectively.
机译：在“ AOX1”启动子的控制下，成功地从黑曲霉ATCC 120120克隆了编码α-L-阿拉伯呋喃糖苷酶（AnabfA）的基因并在巴斯德毕赤酵母中表达。研究了培养条件对重组AnabfA产生的影响，并将该酶表示为可溶性蛋白。当在BMMY培养基（pH 6.0）中培养并在每24小时用2％甲醇诱导时，重组的AnabfAα在28℃时表达为一种活性酶。诱导后5天观察到最大活性。经SDS-PAGE迁移的PNGase F处理前后的纯化的重组AnabfA的相对分子量分别为约83和66kDa，表明该AnabfAβ包含βN-连接的寡糖。纯化的重组AnabfAβ的表征分别显示出最佳温度和pH分别为约50℃和4。该酶在3至6的pH值下是稳定的，并在40°C下预孵育30分钟后保留了80％以上的活性。重组AnabfA活性被K +，Mn2 +，Na2 +和triton X-100刺激。并被Cu2 +和Fe2 +强烈抑制，而Ca2 +，CO2 +，Mg2 +α和EDTA对酶活性的影响相对较小。纯化的重组人AnabfA对ρNPA的ΔKm和ΔVmax分别为0.93mM和17.86μmol/ ml / min。

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