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

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 center dot g(- 1) DCW of both fusion proteins were produced, with a specific activity of > 65 AU center dot 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.

Automated summary

In this study, two different Carbohydrate-Binding Modules (CBM3 and CBM9) were successfully fused with alcohol dehydrogenase from Saccharomyces cerevisiae. The fusion proteins were immobilized to a low-cost and highly selective cellulosic support through a one-step immobilization/purification process, retaining almost total activity. CBM9 tag proved to be useful in protein purification.