Biotechnology Letters

, Volume 32, Issue 12, pp 1869–1875

Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum

Original Research Paper

DOI: 10.1007/s10529-010-0363-0

Cite this article as:
Yi, ZL. & Wu, ZL. Biotechnol Lett (2010) 32: 1869. doi:10.1007/s10529-010-0363-0

Abstract

We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.

Keywords

CelAClostridium thermocellumEndoglucanaseFamily shufflingThermostability

Supplementary material

10529_2010_363_MOESM1_ESM.doc (2 mb)
Supplementary material 1 (DOC 2001 kb)

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Chengdu Institute of BiologyChinese Academy of SciencesChengduChina
  2. 2.Graduate University of the Chinese Academy of SciencesBeijingChina