Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

Sanni P. Voutilainen, Susanna Nurmi-Rantala, Merja Penttilä, Anu Koivula (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

25 Citations (Scopus)

Abstract

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S-S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal- scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.

Original languageEnglish
Pages (from-to)2991-3001
JournalApplied Microbiology and Biotechnology
Volume98
Issue number7
DOIs
Publication statusPublished - 1 Jan 2014
MoE publication typeA1 Journal article-refereed

Keywords

  • Carbohydrate-binding module
  • CBM1
  • CBM2
  • CBM3
  • Cellulase
  • Disulphide bridge
  • GH7
  • Protein engineering
  • Saccharomyces cerevisiae
  • Talaromyces emersonii

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