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. 2023 Feb 24;11:1156953. doi: 10.3389/fbioe.2023.1156953

FIGURE 2.

FIGURE 2

Optimization of the whole-cell catalyst. (A) Expression of the fusion enzymes. The calculated molecular weights were as follows: GP 81.49 kDa, GS1P 81.80 kDa, GS2P 82.11 kDa, GS3P 82.42 kDa, GE1P 81.96 kDa, GE2P 82.42 kDa, GE3P 82.89 kDa. M refers to marker. C refers to the control, which contain the empty pET-28a (PB) plasmid. (B) HPIC analysis of the enzymatic cascades (C) Efficiency of the enzymatic cascades in converting D-glucose to D-allulose. GP refers to the fusion protein linked AcceGI and CcDPEase directly. Other fusion protein are those linked AcceGI and CcDPEase through corresponding flexible or rigid linkers. G-P refers to the cascade composed of free AcceGI and CcDPEase. Standards refer to the D-glucose, D-fructose, and D-allulose mixture. (D) Analysis of rate limiting step of the enzymatic cascade by stepwise increasing gene copy number (E) Improving the activity of the whole-cell catalyst by optimizing the enzyme expression levels. (F) Expression level analysis of AcceGI and CcDPEase. M refers to marker. C refers to the control, which contain the empty pET-28a (PB) plasmid. G indicates the bands of AcceGI; P indicates the bands of CcDPEase.