Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability.

Jiivittha Veno, Nor Hafizah Ahmad Kamarudin, Mohd Shukuri Mohamad Ali, Malihe Masomian, Raja Noor Zaliha Raja Abd Rahman

Journal: International journal of molecular sciences 2018;18(11):2202

PMID: 29113034

Free Full Text on MDPI View on PubMed

Abstract

In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from AT2 (rT-M386) was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C) demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C) and displayed a more prolonged half-life in the range of 40-60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.

Address: Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Department of Biochemistry, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].; Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. [email protected].

DOI: 10.3390/ijms18112202

Link outs

Free resources

Full Text Sources:

Other Literature Sources:

https://scite.ai/reports/29113034

Research Materials:

https://antibodies.cancer.gov/detail/CPTC-GST+Mu1-...

MeSH Terms: Amino Acid Substitution, Bacterial Proteins, Enzyme Stability, Evolution, Molecular, Lipase, Protein Denaturation, Protein Domains, Staphylococcus epidermidis