Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization

Abstract

Mannooligosaccharides are released by mannan-degrading endo-β-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze–thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.

Enzyme engineering and its industrial applications

“…Gao et al. (54) made two modifications in a mesophilic mannanase extracted from Bacillus subtilis using the isopeptide‐mediated molecular cyclization technique. With this technique, an isopeptide bond formed between Lys and Asn/Asp and caused the cyclization of the linear enzyme.…”

“…This mannanase was engineered to produce functional oligosaccharides and food additives through the degradation of nonstarch polysaccharides, which constitute a significant component of plant lignocellulosic biomass. The use of engineered chimeric enzymes, also called fusion enzymes, such as mannanase–xylanase, with synergistic catalytic activities, have great potential for industrial use since lignocellulosic biomass degradation involves several nonstarch polysaccharide degrading enzymes (54).…”

Protein Macrocyclization for Tertiary Structure Stabilization

“…Several proteins have been cyclized with the SpyTag fused to their N-terminus and SpyCatcher to the C-terminus of the protein resulting in proteins with increased thermostability. [59][60][61][62][63][64][65] In some examples, SpyTag has been fused to the C-terminus and SpyCatcher to the N-terminus, also resulting in cyclic proteins with increased thermostability. [66,67] One study compares the performance of the three pairs Pilin-C/Isopeptag, SpyTag/Catcher and SnoopTag/Catcher for the cyclization of β-lactamase.…”

“…Several proteins have been cyclized with the SpyTag fused to their N‐terminus and SpyCatcher to the C‐terminus of the protein resulting in proteins with increased thermostability. [ 59 , 60 , 61 , 62 , 63 , 64 , 65 ] In some examples, SpyTag has been fused to the C‐terminus and SpyCatcher to the N‐terminus, also resulting in cyclic proteins with increased thermostability. [ 66 , 67 ]…”

“…Other studies comparing protein cyclization via SpyTag/Catcher and SnoopTag/Catcher also observed increased tolerance towards thermal stress for the SpyTag/Catcher pair. [ 64 , 68 ] More recently, the RrgA domain of Streptococcus pneumoniae has been split into three domains providing SnoopTagJr (12 amino acids), DogTag (23 amino acids) and SnoopLigase (104 amino acids). SnoopTagJr and DogTag were fused to the N‐ and C‐terminus of a protein, respectively (Figure 4 ).…”