Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5

  • M. Yusuf IPB University
  • C. Budiman Department of Animal Production and Technology, Faculty of Animal Science, IPB University
  • I. I. Arief Department of Animal Production and Technology, Faculty of Animal Science, IPB University
  • C. Sumantri Department of Animal Production and Technology, Faculty of Animal Science, IPB University
Keywords: Clp-Protease, heterologous expression, Lactobacillus plantarum IIA-1A5, structural homology modelling, synthetic gene

Abstract

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.

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Published
2021-11-12
How to Cite
YusufM., BudimanC., AriefI. I., & SumantriC. (2021). Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5. Tropical Animal Science Journal, 44(4), 520-530. https://doi.org/10.5398/tasj.2021.44.4.520