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Article
Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability
Protein Engineering
  • Hsuan-Liang Liu, Iowa State University
  • Yann Doleyres, Iowa State University
  • Pedro M. Coutinho, Iowa State University
  • Clark Ford, Iowa State University
  • Peter J. Reilly, Iowa State University
Document Type
Article
Publication Date
1-1-2000
DOI
10.1093/protein/13.9.655
Abstract

Three single-residue mutations, Asp71→Asn, Gln409→Pro and Gly447→Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28-Val29-Ser30→Asn-Pro-Pro (23–30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301→Ala-Gly-Ala (297–301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Δ439–441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O-glycosylation in the already highly O-glycosylated belt region that extends around the globular catalytic domain. The 23–30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297–301 replacement mutant GA was made to understand the function of the random coil region between α-helices 9 and 10. Δ439–441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23–30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4°C increase in operating temperature at constant thermostability.

Comments

This is a post-print of an article from Protein Engineering, 13, no. 9 (2000): 655–659, doi: 10.1093/protein/13.9.655.

Copyright Owner
Oxford University Press
Language
en
Date Available
November 19, 2012
File Format
application/pdf
Citation Information
Hsuan-Liang Liu, Yann Doleyres, Pedro M. Coutinho, Clark Ford, et al.. "Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability" Protein Engineering Vol. 13 Iss. 9 (2000) p. 655 - 659
Available at: http://works.bepress.com/peter_reilly/24/