Purification and characterization of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia

Sotharith Phon; Andriati Ningrum; Lucia Dhiantika Witasari

doi:10.22146/ijbiotech.65822

Purification and characterization of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia

https://doi.org/10.22146/ijbiotech.65822

Sotharith Phon⁽¹⁾, Andriati Ningrum⁽²⁾, Lucia Dhiantika Witasari^(3*)

(1) Department of Food and Agricultural Products Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada, Jl. Flora No. 1, Yogyakarta 55281, Indonesia
(2) Department of Food and Agricultural Products Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada, Jl. Flora No. 1, Yogyakarta 55281, Indonesia
(3) Department of Food and Agricultural Products Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada, Jl. Flora No. 1, Yogyakarta 55281, Indonesia
(*) Corresponding Author

Abstract

Thermostable proteases that optimally withstand the high‐temperature conditions of thermophilic bacteria could be produced and purified, which would be highly beneficial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditions. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytic index was measured in a solid medium. The expression of protease was optimized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purified using ammonium sulfate (40‐60%) and DEAE Sephadex A‐25 resin. Using SDS‐PAGE, the molecular weight of the enzyme was predicted to be around 32 kDa. Purified thermostable protease was highly activated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classified as a thermostable alkaline serine protease. Its kinetic study revealed specific activity of 0.41 U/mg (Vmax) and 0.25 mg/mL (KM). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high activity at high temperatures and alkaline pH, which is vital for application in industries such as leather processing and detergent formulation.

Keywords

Thermostable serine alkaline protease; Geobacillus sp. DS3; enzyme purification, DEAE Sephadex A‐25

Full Text:

PDF

References

Baykara SG, Sürmeli Y, Şanlı Mohamed G. 2021. Purification and Biochemical Characterization of a Novel Thermostable Serine Protease from Geobacillus sp. GS53. Appl. Biochem. Biotechnol. 193(5):1574– 1584. doi:10.1007/s12010021035120.

Bhatt HB, Singh SP. 2020. Cloning, expression, and structural elucidation of a biotechnologically potential alkaline serine protease from a newly isolated haloalkaliphilic Bacillus lehensis JO26. Front. Microbiol. 11:1–16. doi:10.3389/fmicb.2020.00941.

Chang C, Gong S, Liu Z, Yan Q, Jiang Z. 2021. High level expression and biochemical characterization of an alkaline serine protease from Geobacillus stearothermophilus to prepare antihypertensive whey protein hydrolysate. BMC Biotechnol. 21(1):1–13. doi:10.1186/s12896021006787.

Hussein AH, Lisowska BK, Leak DJ. 2015. The genus geobacillus and their biotechnological potential. Adv. Appl. Microbiol. 92:1–48. doi:10.1016/bs.aambs.2015.03.001.

Iqbal I, Aftab MN, Afzal M, UrRehman A, Aftab S, Zafar A, UdDin Z, Khuharo AR, Iqbal J, UlHaq I. 2015. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus. J. Basic Microbiol. 55(2):160– 171. doi:10.1002/jobm.201400190.

Lakshmi B, Muni Kumar D, Hemalatha K. 2018. Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8. J. Genet. Eng. Biotechnol. 16(2):295–304. doi:10.1016/j.jgeb.2018.05.009.

Pambudi NA. 2018. Geothermal Power Generation in Indonesia, a country within the Ring of Fire: Current Status, future development and policy. Renewable Sustainable Energy Rev. 81:2893–2901. doi:10.1016/j.rser.2017.06.096.

Rai SK, Mukherjee AK. 2009. Ecological significance and some biotechnological application of an organic solvent stable alkaline serine protease from Bacillus subtilis strain DM04. Bioresour. Technol. 100(9):2642– 2645. doi:10.1016/j.biortech.2008.11.042.

Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD. 2018. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the panther database. Nucleic Acids Res. 46(D1):D624–D632. doi:10.1093/nar/gkx1134.

Sharma KM, Kumar R, Panwar S, Kumar A. 2017. Microbial alkaline proteases: Optimization of production parameters and their properties. J. Genet. Eng. Biotechnol. 15(1):115–126. doi:10.1016/j.jgeb.2017.02.001.

Shrinivas D, Naik G. 2011. Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity. Int. Biodeterior. Biodegradation 65(1):29–35. doi:10.1016/j.ibiod.2010.04.013.

Singh R, Kumar M, Mittal A, Mehta PK. 2016. Microbial Enzymes: Industrial progress in 21st Century. 3 Biotech 6(2):1–15. doi:10.1007/s1320501604858.

Souza PM, Bittencourt ML, Caprara CC, Freitas Md, Almeida RP, Silveira D, Fonseca YM, Ferreira Filho EX, Pessoa Junior A, Magalhães PO, et al. 2015. A biotechnology perspective of fungal proteases. Braz. J. Microbiol. 46(2):337–346. doi:10.1590/s1517 838246220140359.

Stetter KO. 2006. Hyperthermophiles in the history of life. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 361(1474):1837–1843. doi:10.1098/rstb.2006.1907.

Suberu Y, Akande I, Samuel T, Lawal A, Olaniran A. 2019a. Cloning, expression, purification and characterisation of serine alkaline protease from Bacillus subtilis RD7. Biocatal. Agric. Biotechnol. 20:101264. doi:10.1016/j.bcab.2019.101264.

Suberu Y, I A, Samuel T, Lawal A, Olaniran A. 2019b. Optimization of protease production in indigenous Bacillus species isolated from soil samples in Lagos, Nigeria using response surface methodology. Biocatal. Agric. Biotechnol. 18:101011. doi:10.1016/j.bcab.2019.01.049.

Tavano OL, BerenguerMurcia A, Secundo F, FernandezLafuente R. 2018. Biotechnological applications of proteases in Food Technology. Compr. Rev. Food Sci. Food Saf. 17(2):412–436. doi:10.1111/1541 4337.12326.

Witasari LD, Prijambada ID, Widada J, Andang D,Wibawa A. 2010. Cloning of thermostable DNA polymerase gene from a thermophilic Brevibacillus sp. isolated from Sikidang Crater, Dieng Plateu, Central Java. Indones. J. Biotechnol. 15(2):72–78. doi:10.22146/ijbiotech.7825.

Zhou C, Qin H, Chen X, Zhang Y, Xue Y, Ma Y. 2018. A novel alkaline protease from alkaliphilic Idiomarina sp. C91 with potential application for ecofriendly enzymatic dehairing in the leather industry. Scientific Rep. 8(1):1–18. doi:10.1038/s41598018344165.

Zilda DS, Harmayani E, Widada J, Asmara W, Irianto HE, Patantis G, Fawzya YN. 2013. Screening of thermostable protease producing microorganisms isolated from Indonesian hotspring. Squalen Bull. Mar. Fish. Postharvest Biotechnol. 7(3):105. doi:10.15578/squalen.v7i3.5.

DOI: https://doi.org/10.22146/ijbiotech.65822