Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1

Abstract

A new protein immobilization and purification system has been developed based on the improved plasmid vectors, designated pETChBD-X, which contained the gene coding for two novel chitin-binding domains ChBD-AB, factor Xa cleavage site and adapted for gene fusions. The ChBD-AD from Chitinolyticbacter meiyuanensis SYBC-H1 was used as a novel affinity tag to anchor fusion proteins to chitin granules. The granules carrying the ChBD-AD fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble recombination protein can be obtained after Factor Xa cleavage. The efficiency of this system has been demonstrated by reaching 95% of protein absorbed to chitin within 30 min and recycling over 75% of interest protein after Factor Xa cleavage to separate interest protein and fusion tag. Furthermore, 65% L-glutamate oxidase with this fusion tag could be purified and immobilized within only one step and to be reused in converting L-glutamate to α-ketoglutaric acid directly, the average conversion rate kept above 65% even within four batches of enzyme conversion reaction.

Fusion of Chitin-Binding Domain From Chitinolyticbacter meiyuanensis SYBC-H1 to the Leaf-Branch Compost Cutinase for Enhanced PET Hydrolysis

“…At 40°C, the maximum adsorption efficiency of 30.2% was achieved after 70 min of incubation. This is the optimal binding temperature for the chitin binding domain used in this study according to previous publications ( Zhou et al, 2020 ). With increasing incubation temperature to 65°C or 72°C, the maximum adsorption rate decreased slightly to 27% or 23.5%, which was achieved after 2 h incubation and considered beneficial for the enzymatic plastic degradation carried out at this temperature.…”

“…The gene encoding LCC ICCG cutinase, CBM, PBM and HFB4 were synthesized based on previous studies ( Ribitsch et al, 2013 ; Ribitsch et al, 2015 ; Tournier et al, 2020 ) and that for ChBD was derived from the genome of Chitinolyticbacter meiyuanensis SYBCH1 ( Zhang et al, 2018 ). LCC ICCG cutinase and ChBD expression strain E. coli BL21 (pET29a-LCC ICCG ) and E. coli BL21 (pET29a-ChBD) were constructed in previous experiments ( Zhou et al, 2020 ).…”

“…CBMs have been developed as useful affinity tags for protein immobilization due to their high adsorbing capacity to solid materials. For example, Zhou et al (2020) used chitin-binding domain as an affinity tag and achieved over 95% immobilization efficiency with their target protein. Furthermore, CBMs have a broad range of hydrophobic binding affinity with many polymer substrates including cellulose, chitin, xylan, and starch ( Oliveira et al, 2015 ).…”

Enzyme Immobilization and Co-Immobilization: Main Framework, Advances and Some Applications

“…Adsorption and ionic binding are quite simple and low-cost methods, but non-specific. Affinity binding is highly selective, enables one pot purification and immobilization and allows oriented enzyme immobilization, yet it is costly [44,[65][66][67][68][69]. Chemical attachment to the carrier involves covalent binding through amide, carbamate, ether or thio-ether bonds established between groups of suitable residues on the enzyme surface and on the carrier (Figure 1d 1 ) [36,67].…”

Recent advances in the biodegradation of polyethylene terephthalate with cutinase-like enzymes

“…Carbohydrate-binding modules (CBMs) are small noncatalytic protein domains that usually function as a part of modular enzymes (e.g., carbohydrate-active enzymes, CAZymes), which can enhance the binding affinity between enzymes and substrates ( Armenta et al, 2017 ; Weber et al, 2019 ). Consequently, CBMs have been used as efficient affinity tags for protein immobilization owing to their high adsorption capacity for solid substrates ( Oliveira et al, 2015 ; Zhou et al, 2020 ). Based on the topology of the ligand-binding site, CBMs can be classified into types A, B ( endo -type), or C ( exo -type) ( Gilbert et al, 2013 ).…”