Purification and characterization of extracellular lipase from a thermotolerant strain: Bacillus subtilis TTP-06

Manpreet Kaur; Rakesh Kumar; Poonam Katoch; Reena Gupta

doi:10.1007/s13205-023-03717-6

Purification and characterization of extracellular lipase from a thermotolerant strain: Bacillus subtilis TTP-06

3 Biotech. 2023 Oct;13(10):343. doi: 10.1007/s13205-023-03717-6. Epub 2023 Sep 12.

Authors

Manpreet Kaur¹, Rakesh Kumar², Poonam Katoch³, Reena Gupta¹

Affiliations

¹ Department of Biotechnology, Himachal Pradesh University, SummerHill, Shimla, HP India.
² Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, India.
³ Jaypee University of Information Technology, Waknaghat, Solan, India.

Abstract

In current study, lipase from a thermotolerant Bacillus subtilis TTP-06 was purified in a stepwise manner by using ammonium sulfate precipitation and column chromatography. Thenceforth, it was subjected to sodium dodecyl sulfate- and native-polyacrylamide gel electrophoresis to check the homogeneity of the purified enzyme. The ideal substrate concentration, pH, temperature, reaction duration and lipase specificity were identified. With a yield of 11.02%, purified lipase displayed activity of 8.51 U/mg. Thenceforward, the homogeneously purified enzyme was considered to be a homo-dimer of 30 kDa subunits. Enzyme had K_m and V_max value of 9.498 mM and 19.92 mol mg^-1 min^-1, respectively. Additionally, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method was used to investigate the purified lipase and estimate its 3-D structure, which revealed a catalytic triad of serine, aspartate and histidine.

Keywords: Bacillus subtilis TTP-06; Characterization; MALDI-TOF MS; Native-PAGE; Purification; SDS-PAGE.

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