Cloning, expression and purification/immobilisation of CBM–tagged enzymes involved in multienzymatic production of lactic acid

Abstract

The present thesis responds to the need to reduce the operational costs associated with the production, purification and immobilisation of biocatalysts used in bioprocesses. The process intensification strategies described have been developed and validated for three different enzymes -an alcohol dehydrogenase (ADH), a pyruvate decarboxylase (PDC) and a lactate dehydrogenase (LDH)- which are involved in a biocatalytic system proposed by the European BIOCON-CO2 project, which aims to synthesize lactic acid. In this context, in order to obtain the biocatalysts of interest in the most efficient way, the integration of several procedures from different fields is attempted, including gene cloning methods and processes for the production of recombinant proteins with the Gram-negative bacterium Escherichia coli, and also with the development of a one-step biocatalyst purification/immobilisation process, based on the affinity of carbohydrate-active protein domains towards low-cost cellulosic supports. In the first part of this thesis, the E. coli strain NEB 10-ß was used for the expression of the three enzymes involved in the multienzyme system proposed to obtain lactic acid synthesis with a histidine tag fused to their N-terminal end. The production process, based on the PBAD expression system, proved to be successful, not only by using complex culture mediums but also with chemically defined medium supplemented with amino acids, used in a two-step fed batch strategy. Nevertheless, aiming to assess if higher production yields could be achieved, the three target enzymes were cloned into an in-house developed expression vector, based on the T5 lac promoter, and were subsequently produced by using one auxotrophic M15-derived strain at bench-scale reactor through fed-batch cultures and antibiotic-free defined culture medium. Thus, M15[Delta]glyA strain proved to be a more suitable and more robust E. coli host strain to produce the required enzymes at high titres, although the NEB 10-ß strain also resulted to be a feasible microbial host. The second part of this thesis was focused on the generation of new enzyme variants, containing different Carbohydrate-Binding Modules (CBM) fused to their N-terminal end, aiming to develop a one-step purification/immobilisation method which enables to recover the enzymes produced with high specificity and efficiency, by using cheap immobilisation cellulosic supports while avoiding the presence of metal ions, usually present in samples processed with immobilized-metal affinity resins. The three target enzymes were successfully co-expressed with two different CBMs -one from T. maritima Xylanase 10A (CBM9) and one from C. Thermocellum cellulosomal-scafolding protein A (CBM3)- in E. coli M15[Delta]glyA strain. The resulting fusion proteins were successfully produced at high titres in a bench-scale reactor by following the same strategy applied to produce the histidine-tagged variants. Results showed that M15[Delta]glyA strain was able to produce CBM-fused enzymes as efficiently as histidine-tagged ones, and it was also proved that CBM domains do not have a significant negative impact in enzyme performance. Finally, it was developed and characterised the one-step purification/immobilisation process of the different CBM-tagged variants onto low-cost and highly selective cellulosic supports, including the study of storage stability of the immobilised derivatives and the determination of maximum loading capacity of the supports. CBM9-fused enzymes proved to bind effectively to regenerated amorphous cellulose (RAC) at high loads, whereas CBM3-tagged enzymes bound with higher affinity onto microcrystalline cellulose (MC). Moreover, RAC support was also used for protein purification by fast liquid protein chromatography (FPLC), aiming to establish an alternative to metal affinity chromatography, by which only CBM9 proved to be a suitable purification tag for the three enzymes tested.