Enzymatic characterization of D-lactate dehydrogenase and application in alanine aminotransferase activity assay kit
| العنوان: | Enzymatic characterization of D-lactate dehydrogenase and application in alanine aminotransferase activity assay kit |
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| المؤلفون: | Guosheng Gao, Ting Cai, Yi Sun |
| المصدر: | Bioengineered, Vol 12, Iss 1, Pp 6459-6471 (2021) Bioengineered article-version (VoR) Version of Record |
| بيانات النشر: | Informa UK Limited, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | Bioengineering, Dehydrogenase, stabilizer, medicine.disease_cause, Applied Microbiology and Biotechnology, Bacterial Proteins, Enzyme Stability, medicine, Humans, Asparagine, Lactate Dehydrogenases, Escherichia coli, chemistry.chemical_classification, Lactobacillus delbrueckii, biology, Alanine Transaminase, enzymatic characterization, General Medicine, Recombinant Proteins, Enzyme assay, Amino acid, d-lactate dehydrogenase, Enzyme, chemistry, Biochemistry, D-lactate dehydrogenase, biology.protein, Specific activity, alt kit, Blood Chemical Analysis, TP248.13-248.65, Research Article, Research Paper, Biotechnology |
| الوصف: | D-lactate dehydrogenase (D-LDH) is widely used for the clinical detection of alanine aminotransferase (ALT) activity. It is a key enzyme in ALT detection kits, and its enzymatic properties directly determine sensitivity and accuracy of such kits. In this study, D-lactate dehydrogenase (WP_011543503, ldLDH) coding sequence derived from Lactobacillus delbrueckii was obtained from the NCBI database by gene mining. LdLDH was expressed and purified in Escherichia coli, and its enzyme activity, kinetic parameters, optimum temperature, and pH were characterized. Furthermore, stabilizers, including sugars, polyols, amino acids, certain salts, proteins, and polymers, were screened to improve stability of ldLDH during freeze-drying and storage. Finally, a kit based on ldLDH was tested to determine whether the enzyme had potential clinical applications. The results showed that ldLDH had a specific activity of 1,864 U/mg, Km value of 1.34 mM, optimal reaction temperature of 55°C, and an optimal pH between 7.0 and 7.5. When sucrose or asparagine was used as a stabilizer, freeze-dried ldLDH remained stable at 37°C for > 2 months without significant loss of enzymatic activity. These results indicated that ldLDH possesses high activity and stability. Test results using the ALT assay kit prepared with ldLDH were consistent with those of commercial kits, with a relative deviation |
| تدمد: | 2165-5987 2165-5979 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ddcd2393b47e1b9415d146692bd87afaTest https://doi.org/10.1080/21655979.2021.1972781Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....ddcd2393b47e1b9415d146692bd87afa |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 21655987 21655979 |
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