Cloning, Expression, and Characterization of L-Asparaginase from a Newly Isolated Bacillus subtilis B11-06
Cloning, Expression, and Characterization of L-Asparaginase from a Newly Isolated Bacillus subtilis B11-06
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DOI：
10.1021/jf402636w
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发表时间：
2013-10-02
影响因子：
6.1
通讯作者：
Rao, Zhiming
中科院分区：
文献类型：
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作者：
Jia, Mingmei;Xu, Meijuan;Rao, Zhiming
本研究的重点是从枯草芽孢杆菌 B11-06 非致病菌株中编码 L-天冬酰胺酶 (ansZ) 的基因的克隆、过表达和表征。重组酶表现出高热稳定性和对L-谷氨酰胺的低亲和力。 ansZ 基因编码假定的 L-天冬酰胺酶 II，通过 PCR 扩增并使用穿梭载体 pMA5 在 R subtilis 168 中表达。重组酶活力为9.98 U/mL，显着高于R subtilis B11-06。通过包括硫酸铵分级分离和疏水相互作用层析的两步程序纯化重组酶。重组酶的最适pH和温度分别为7.5和40℃。该酶在 6.0-9.0 的 pH 范围内非常稳定，在 50 或 60 摄氏度孵育 2 小时后分别表现出约 14.7 和 9.0% 的活性保留。 L-天冬酰胺的 Km 为 0.43 mM，V-max 为 0.43 mM。为77.51μM/分钟。这项研究的结果还揭示了这种酶在减少马铃薯油炸过程中乙酰胺形成方面的潜在工业应用。
This study focused on the cloning, overexpression, and characterization of the gene encoding L-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11-06. The recombinant enzyme showed high thermostability and low affinity to L-glutamine. The ansZ gene, encoding a putative L-asparaginase II, was amplified by PCR and expressed in R subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of R subtilis B11-06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 degrees C, respectively. The enzyme was quite stable at a pH range of 6.0-9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 degrees C, respectively. The Km for L-asparagine was 0.43 mM, and the V-max. was 77.51 mu M/min. Results of this study also revealed the potential industrial application of this enzyme in reducing actylamide formation during the potato frying process.