Cloning, expression, and one-step purification/immobilization of two carbohydrate-binding module-tagged alcohol dehydrogenases

Mario Benito, Ramón Román, Garazi Ortiz, Antoni Casablancas, Gregorio Álvaro, Gloria Caminal, Gloria González*, Marina Guillén

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

Abstract

Background: The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags. Results: In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g− 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg− 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade. Conclusion: CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9.

Original languageEnglish
Article number16
Number of pages14
JournalJournal of Biological Engineering
Volume16
Issue number1
DOIs
Publication statusPublished - 28 Jun 2022

Keywords

  • Alcohol dehydrogenase
  • Carbohydrate-binding module
  • Escherichia coli
  • One-step immobilization/purification
  • Regenerated amorphous cellulose

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