Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5
| Autor: | Irma Isnafia Arief, Cahyo Budiman, Cece Sumantri, Muhammad Yusuf |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Protease
General Veterinary biology Chemistry medicine.medical_treatment heterologous expression biology.organism_classification SF1-1100 Animal culture Clp-Protease Biochemistry Synthetic gene medicine Lactobacillus plantarum IIA-1A5 Animal Science and Zoology Heterologous expression structural homology modelling synthetic gene Lactobacillus plantarum |
| Zdroj: | Tropical Animal Science Journal, Vol 44, Iss 4 (2021) |
| ISSN: | 2615-790X 2615-787X |
| Popis: | Genome sequence of Indonesian probiotic of Lactobacillus plantarum II1A5 contains a gene encoding a proteolytic subunit of caseinolytic protease, designated as ClpP_LP. This study aims to express the Clp gene heterological and apply its proteolytic activity to some livestock products. To address this, the gene encoding ClpP_LP was optimized in silico by improving its Codon Adaptation Index and GC content to 0.94 and 53.62%, respectively. The optimized gene was then inserted into pET28a, transformed into Escherichia coli BL21(DE3), and over-expressed by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside at 37°C. The result showed that ClpP_LP was successfully over-expressed in a fully soluble form with the specific activity towards milk casein was 7739.89 AU mg-1. This activity was significantly greater than that of chymotrypsin. Further, the three-dimensional model of ClpP_LP was built using SWISS MODEL, which showed that this protein formed a homo-tetradecameric (14-mer) structure with each monomer consisting of 7 α-helix and 10 β-sheets. The identification of the active side showed that the active side of ClpP_LP is Ser-97, His-122, Asp-171, and forms a substrate-binding cavity with a size of about 29.5 Ǻ. Overall, our approach can serve as an appropriate platform for the production of ClpP_LP in a large-scale production for various applications in dairy products and derivatives. |
| Databáze: | OpenAIRE |
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