Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases.

ABSTRACT: 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

SUBMITTER: Li X 

PROVIDER: S-EPMC5962637 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature