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 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.

Design of a high‐efficiency synthetic system for l‐asparaginase production in Bacillus subtilis

“…A series of strategies have been used to improve L-asparaginase production, such as optimization of fermentation medium and process [9][10][11], signal peptide screening, promoter mutation, and N-terminal deletion [12]. To date, the highest yield of L-asparaginase obtained was 2168 U/mL, which was reported in our previous work [13].…”

“…To further improve the production of L-asparaginase, 300 RBS sequences were designed, and they were divided into 10 groups on average with a gradient of translation initiation rates for statistical convenience. After assaying by the high-throughput screening method developed in our previous study [13], we found most of RBS in the group 1 × 10 3 -2 × 10 3 au, 2 × 10 3 -5 × 10 3 au, 5 × 10 3 -1 × 10 4 au, 1 × 10 4 -5 × 10 4 au, 5 × 10 4 -1 × 10 5 au, 1 × 10 5 -2 × 10 5 au, 2 × 10 5 -5 × 10 5 au, 5 × 10 5 -1 × 10 6 au, 1 × 10 6 -2 × 10 6 au, and 2 × 10 6 -max (5 687 190 au) increased the enzyme activity in the range of 1-1.2%, 2-4%, 4.1-5.2%, 8.8-29.0%, 29.2-51.2%, 51.7-62.1%, 62.5-74.5%, 74.9-81.7%, and 82-96%, respectively (Figure 2). The predicted translation initiation rates and the enzyme activities of most of synthetic RBS were directly proportional.…”

“…The pMA5‐S17G/A90S/R156S/K272A plasmid, harboring the mutant Pyrococcus yayanosii CH1 l ‐asparaginase gene that was constructed in our previous study , was used as the template to clone the l ‐asparaginase gene (pyasnaseMut) with the primer pair F1/R1. The plasmid pMA5 was used as the template to clone the gene with the PHpaII promoter accompanied by the RBS sequence from pMA5 with the primer pair F2/R2.…”

“…According to the evaluation of the translation initiation rate, 300 synthetic RBS sequences were automatically designed by the RBS Calculator (https://www.denovodna.com/software/) . Screening for strains with high l ‐asparaginase activity was conducted as described previously , and B. subtilis 168 / pMA5‐P 43 ‐pyasnase Mut was used as a control. The screening method for the strains with high l ‐asparaginase activity involved simultaneous cell disruption and semiquantitative activity assays.…”

“…The expression, purification, and activity assays were conducted as described in our previous study . The sum of the intracellular and extracellular enzymatic activities was regarded as the recombinant strain productivity.…”