AbstractA promising new approach for the production of biocatalysts comprises the use of surface‐layer (S‐layer) lattices that present functional multimeric enzymes on their surface, thereby guaranteeing most accurate spatial distribution and orientation, as well as maximal effectiveness and stability of these enzymes. For proof of concept, a tetrameric and a trimeric extremozyme are chosen for the construction of S‐layer/extremozyme fusion proteins. By using a flexible peptide linker, either one monomer of the tetrameric xylose isomerase XylA from the thermophilic Thermoanaerobacterium strain JW/SL‐YS 489 or, in another approach, one monomer of the trimeric carbonic anhydrase from the methanogenic archaeon Methanosarcina thermophila are genetically linked to one monomer of the S‐layer protein SbpA of Lysinibacillus sphaericus CCM 2177. After isolation and purification, the self‐assembly properties of both S‐layer fusion proteins as well as the specific activity of the fused enzymes are confirmed, thus indicating that the S‐layer protein moiety does not influence the nature of the multimeric enzymes and vice versa. By recrystallization of the S‐layer/extremozyme fusion proteins on solid supports, the active enzyme multimers are exposed on the surface of the square S‐layer lattice with 13.1 nm spacing.