Efficient biotransformation and synergetic mechanism of dual-enzyme cascade reaction in nonreducing maltoheptaose synthesis

Permeabilized whole cells containing co‐expressed cyclomaltodextrinase and maltooligosyltrehalose synthase facilitate the synthesis of nonreducing maltoheptaose (N‐G7) from β‐cyclodextrin

“…A comparison of the whole‐cell enzymatic activity under different hosts revealed that ∆ycjM ‐ ∆malS ‐ ∆lpxM is the optimal host for the whole‐cell enzyme reaction. Previous studies suggest the optimal enzymatic reaction temperature is 40 °C and pH 7.0 for recombinant CDase and MTSase 4 . The purity, conversion rate of N‐G7, and utilization rate of β ‐CD are shown in Fig.…”

“…According to the reaction mechanisms of CDase and MTSase in the synthesis of N‐G7, 4 the interaction between the two enzymes and the possibility of potential adverse responses are complex. The synthesis of N‐G7 was accompanied by hydrolysis, and the time curve of N‐G7 synthesis is shown in Fig.…”

“…Several chemical and physical approaches, including using lysozyme, ethylenediaminetetraacetic acid (EDTA), and heat treatment, have been employed to enhance membrane permeability 12,15–17 . However, the enzymatic activity of recombinant CDase and MTSase was reduced by 70% and 80% when incubated at 50 °C or above, respectively 4 . Thus, heat treatment is inapplicable to them.…”

“…These problems remain unresolved and pose challenges to the global food and other industries that use maltodextrin as a bulk ingredient. A study was conducted to synthesize nonreducing maltodextrin with a single degree of polymerization (nonreducing maltoheptaose) from β ‐cyclodextrin ( β ‐CD) using a one‐pot dual‐enzyme reaction 4 comprising two enzyme cascade reactions: β ‐CD was hydrolyzed into linear maltoheptaose (G7) by recombinant cyclomaltodextrinase (CDase); linear G7 was converted into nonreducing maltoheptaose (N‐G7) by intramolecular transglycosylation of recombinant maltooligosyltrehalose synthase (MTSase) ( α ‐1,4 glycosidic bond at the reducing end of G7 was converted to a α ‐1,1 glycosidic bond) 5 . However, recombinant CDase and MTSase are expressed intracellularly and must undergo separate fermentation and cell fragmentation to obtain soluble enzymes for enzymatic reactions, which is highly time‐ and resource‐consuming.…”

“…12,[15][16][17] However, the enzymatic activity of recombinant CDase and MTSase was reduced by 70% and 80% when incubated at 50 °C or above, respectively. 4 Thus, heat treatment is inapplicable to them. Although cell membrane permeabilization through chemical or physical treatment is efficient on a small scale, its large-scale implementation is challenging; instead, molecular engineering is recently being favored as an alternative.…”