Amplification of Thermostable Neutral Protease Open Reading Frame from Geobacillus sp. DS3 Isolated from Sikidang Crater, Dieng Plateau, Central Java, Indonesia

DOI

10.2991/978-94-6463-086-2_101How to use a DOI?

Keywords

Amplification; Geobacillus sp. DS3; Thermostable neutral protease (NPr)

ABSTRACT

Neutral protease is a protease that are widely applied in the food industry due to its stability at neutral pH. However, the neutral protease is generally poor in thermal stability which limits its application in some industries that require high temperatures. The thermostable neutral protease-producing bacterium, Geobacillus sp. DS3, was isolated from Sikidang Dieng Crater, Central Java, Indonesia. Preliminary research showed the existence of thermostable protease activity from Geobacillus sp. DS3 that was grown optimally at 70°C on a minimal synthetic medium agar + 1% skim milk. Geobacillus sp. DS3 produces thermostable protease to hydrolyze protein into amino acid. Production of thermozymes requires high temperatures, making the process less efficient. In this work, the open reading frame of NPr gene was amplified and characterized as a starting point to express the gene of interest into mesophilic bacteria. Amplification was carried out by PCR using degenerate primers Pro_F and Pro_R resulted in ~1.6 kbp of NPr sequence. A GenBank accession number of this sequence was ON882245. Analysis using BLASTN confirmed that the amplified ORF had a high similarity with the neutral protease from Alicyclobacillus acidocaldarius. Translation of the ORF resulted 546 amino acid residues and theoretical pI/Mw of 5.60 / 59.81 kDa. The NPr had a structure similar to that of PDB 5A3Y or SAD thermolysin with 86.35% sequence identity. The domain and function of NPr were determined using SignalIP and InterPro. SignalIP results showed that the amino acid sequence of NPr consisted of signaling peptides. InterPro results showed that the NPr domain consisted of four major domains, namely, fungalysin/thermolysin/propeptide domain from residues 80–124, PepSY regulatory peptide domain from residues 140–21, and peptidase domain consisted of peptidase M4 from residues 235–380 and peptidase M4 C-terminal from residues 383–258. Further study related molecular docking must be performed to further understand the NPr structure.

Copyright

© 2023 The Author(s)

Open Access

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TY  - CONF
AU  - Lucia Dhiantika Witasari
AU  - Joshua Halim
AU  - Caroline Jilbert
AU  - Muhammad Saifur Rohman
PY  - 2022
DA  - 2022/12/28
TI  - Amplification of Thermostable Neutral Protease Open Reading Frame from Geobacillus sp. DS3 Isolated from Sikidang Crater, Dieng Plateau, Central Java, Indonesia
BT  - Proceedings of the International Conference on Sustainable Environment, Agriculture and Tourism (ICOSEAT 2022)
PB  - Atlantis Press
SP  - 770
EP  - 777
SN  - 2468-5747
UR  - https://doi.org/10.2991/978-94-6463-086-2_101
DO  - 10.2991/978-94-6463-086-2_101
ID  - Witasari2022
ER  -