Haiyan Zhou1, Wenjiao Yang1, Yun Tian2, Hanhui Peng1, Yongyao Wu1. 1. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China. 2. Key Lab of Agricultural Biochemistry and Biotransformation, Hunan Agricultural University, Changsha, 410128, China.
Abstract
BACKGROUND: The disordered residues on distal loops affect the molecular structural stability and on some occasions have regulatory roles in catalytic reaction. To increase understanding of the influence of distal residue mutation, this study explored the thermostability and enzymatic activity of mannanase Man1312 deletion mutants. The focus was on residues located on the N-terminal region because they are more disordered and changeable. The effects of N-terminal truncation on enzymatic activity and thermal dynamics were investigated by spectrophotometry, circular dichroism and differential scanning calorimetry assays. RESULTS: The deletion mutants on V3, N7 and Q11 showed a marked increase in stability, while the enzymatic activity was significantly improved when triplet deletion was carried out. Triplet deletion MandVNQ showed around double the stability of its corresponding single-site and double-site deletion mutants. The Tm value of MandVNP was about 8 °C higher than that of Man1312. MandVNP had improved characteristics of Topt by 10 °C, t1/2 by 10 min and catalytic activity by 11% in comparison with Man1312. Analysis of spectra and modeling showed that MandVNQ had increased helix and strand contents. CONCLUSION: N-terminal truncation had positive effects on the thermostability and activity of mannanase.
BACKGROUND: The disordered residues on distal loops affect the molecular structural stability and on some occasions have regulatory roles in catalytic reaction. To increase understanding of the influence of distal residue mutation, this study explored the thermostability and enzymatic activity of mannanaseMan1312 deletion mutants. The focus was on residues located on the N-terminal region because they are more disordered and changeable. The effects of N-terminal truncation on enzymatic activity and thermal dynamics were investigated by spectrophotometry, circular dichroism and differential scanning calorimetry assays. RESULTS: The deletion mutants on V3, N7 and Q11 showed a marked increase in stability, while the enzymatic activity was significantly improved when triplet deletion was carried out. Triplet deletion MandVNQ showed around double the stability of its corresponding single-site and double-site deletion mutants. The Tm value of MandVNP was about 8 °C higher than that of Man1312. MandVNP had improved characteristics of Topt by 10 °C, t1/2 by 10 min and catalytic activity by 11% in comparison with Man1312. Analysis of spectra and modeling showed that MandVNQ had increased helix and strand contents. CONCLUSION: N-terminal truncation had positive effects on the thermostability and activity of mannanase.
Authors: Eduardo Santos da Silva; Lorenz Aglas; Carina Silva Pinheiro; Emília M M de Andrade Belitardo; Elisânia Fontes Silveira; Sara Huber; Rogério Tanan Torres; Michael Wallner; Peter Briza; Peter Lackner; Josef Laimer; Luis Gustavo C Pacheco; Álvaro A Cruz; Neuza Maria Alcântara-Neves; Fatima Ferreira Journal: Clin Exp Allergy Date: 2020-05-13 Impact factor: 5.018