文章基本信息

标题：Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases
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作者：Xu Li ; Xian Zhang ; Shuqin Xu 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2018
卷号：8
期号：1
页码：7915
DOI：10.1038/s41598-018-26241-7
语种：English
出版社：Springer Nature
摘要：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 from Pyrococcus yayanosii CH1 was cloned and over-expressed in Bacillus subtilis 168. 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.