Improvement of Thermal Stability of Ganoderma lucidum Protein LZ-8 by Site-directed Mutation of Amino Acids

doi:10.13560/J.cnki.biotech.bull.1985.2019-0529

Abstract

Abstract: This work is to improve the thermal stability of Ganoderma lucidum immunoregulatory protein LZ-8 by site-directed mutagenesis of amino acids. Molecular dynamics simulation combined with temperature factor prediction were used to reasonably design LZ-8 amino acid mutation site,LZ-8 mutant protein was constructed and expressed in Pichia pastoris X33 strain,the biological activity and thermodynamic parameters of LZ-8 before and after mutation were detected and compared by HeLa cell growth inhibition experiment and differential scanning calorimetric（DSC）study. Results showed that LZ-8 N-terminal alpha helix was the temperature sensitive region predicted by theory. F8W and R9K double site mutations occurred in this region. After mutation,the thermal stability of LZ-8 was improved,the phase transition temperature Tm increased by 0.92℃,and the phase transition enthalpy ΔH increased by 23.14 kJ/mol. However,the biological activity of LZ-8 after mutation was basically unchanged. The IC₅₀ of LZ-8 and LZ-8 mutant on HeLa cell growth inhibition was 2.238 μg/mL and 2.407 μg/mL,respectively. Ganoderma lucidum LZ-8 mutant with improved stability but unchanged biological activity is obtained by rational design of amino acid mutation sites.

Key words: LZ-8, thermal stability, molecular dynamics simulation, temperature factor, site-directed mutation

SUN Xi-lin, JIANG Zhen-yan, LIU Zhi-yi, DAI Lu, SUN Fei, HUANG Wei. Improvement of Thermal Stability of Ganoderma lucidum Protein LZ-8 by Site-directed Mutation of Amino Acids[J]. Biotechnology Bulletin, 2020, 36(1): 23-28.

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