Characterization of cysteine sulfinic acid decarboxylase from Tribolium castaneum and its application in the production of β-alanine
β-alanine is a precursor for the production of pharmaceuticals and food additives that is produced by chemical methods in industry. As concerns about the environment and energy are increasing, biocatalysis using L-aspartate-α-decarboxylase (ADC) to convert L-aspartate to β-alanine has great potential. Many studies have focused on the catalytic activity of ADC, but these researches were limited to the prokaryotic enzymes. In this study, the gene encoding cysteine sulfinic acid decarboxylase from Tribolium castaneum (TcCSADC) was synthesized and overexpressed in Escherichia coli, and the enzyme was purified and characterized for the first time. It could use L-aspartate as its substrate, and the specific activity was 4.83 μmol/min/mg, which was much higher than that of ADCs from prokaryotes. A homology modeling assay indicated that TcCSADC had a dimer structure. Based on the evolutionary information from thermophilic bacteria, twenty-three variants were constructed to attempt to improve its abilities that transform L-aspartate to β-alanine. One mutant, G369A, was screened that had improved thermal stability. An analysis of the suitability of the catalytic process showed that the up to 162 g/L β-alanine could be produced using cells expressing the recombinant G369A variant, which is the highest yield to date. The CSADC from T. castaneum has important value for studies of the mechanism of ADCs and CSADCs from eukaryotes, and the engineered strain containing the G369A variant has great potential for the industrial production of β-alanine.
KeywordsCysteine sulfinic acid decarboxylase L-aspartate-α-decarboxylation β-alanine Molecular engineering Whole-cell catalysis Tribolium castaneum
This study was funded by National Key R&D Program of China (2016YFE0127400), the Fundamental Research Funds for the Central Universities (JUSRP51713B), the national first-class discipline program of Light Industry Technology and Engineering (LITE2018-04), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06) and National natural science foundation of China (3140078).
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Conflict of Interest:
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.