Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases.
Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases.
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用于热稳定性 L-天冬酰胺酶高通量筛选的同步细胞破碎和半定量活性测定
DOI：
10.1038/s41598-018-26241-7
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发表时间：
2018-05-21
影响因子：
4.6
通讯作者：
Yang S
中科院分区：
文献类型：
--
作者：
Li X;Zhang X;Xu S;Zhang H;Xu M;Yang T;Wang L;Qian H;Zhang H;Fang H;Osire T;Rao Z;Yang S
L-天冬酰胺酶催化L-天冬酰胺水解生成L-天冬氨酸，由于其在医药和食品工业中的广泛应用而引起了研究人员的关注。本研究克隆并在枯草芽孢杆菌168中高效表达了一种新型的耐热L天冬酰胺酶。为了获得耐热的L-天冬酰胺酶高活性突变体，建立了一种针对嗜热酶的高通量筛选方法。在这一过程中，细胞破坏和酶活性分析在96深孔板中同时进行。结合容易出错的聚合酶链式反应和筛选，鉴定出6个亮丽的阳性变异和4个关键的氨基酸残基突变。四个残基的联合突变显示了较高的比活力(3108U/mg)，是野生型酶的2.1倍。用该突变株在5-L发酵罐中发酵，L天冬酰胺酶活力为2168 U/mL。
L-asparaginase, which catalyses the hydrolysis of L-asparagine to L-aspartate, has attracted the attention of researchers due to its expanded applications in medicine and the food industry. In this study, a novel thermostable L-asparaginase fromPyrococcus yayanosiiCH1 was cloned and over-expressed inBacillus subtilis168. To obtain thermostable L-asparaginase mutants with higher activity, a robust high-throughput screening process was developed specifically for thermophilic enzymes. In this process, cell disruption and enzyme activity assays are simultaneously performed in 96-deep well plates. By combining error-prone PCR and screening, six brilliant positive variants and four key amino acid residue mutations were identified. Combined mutation of the four residues showed relatively high specific activity (3108 U/mg) that was 2.1 times greater than that of the wild-type enzyme. Fermentation with the mutant strain in a 5-L fermenter yielded L-asparaginase activity of 2168 U/mL.
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影响因子：
6.1
作者：
Jia, Mingmei;Xu, Meijuan;Rao, Zhiming
通讯作者：
Rao, Zhiming
影响因子：
2.8
作者：
Bansal, S.;Gnaneswari, D.;Kundu, B.
通讯作者：
Kundu, B.
DOI：
10.1016/s0167-4838(00)00179-5
发表时间：
2000-09-29
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
影响因子：
--
作者：
Aung, HP;Bocola, M;Röhm, KH
通讯作者：
Röhm, KH
影响因子：
5
作者：
Khushoo, A;Pal, Y;Mukherjee, KJ
通讯作者：
Mukherjee, KJ
影响因子：
5.4
作者：
Binter, Alexandra;Staunig, Nicole;Macheroux, Peter
通讯作者：
Macheroux, Peter