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Construction and immobilization of recombinant Bacillus subtilis with D-allulose 3-epimerase / 生物工程学报
Chinese Journal of Biotechnology ; (12): 4303-4313, 2021.
Article in Chinese | WPRIM | ID: wpr-921507
ABSTRACT
D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 14 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.
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Full text: Available Index: WPRIM (Western Pacific) Main subject: Temperature / Bacillus subtilis / Enzyme Stability / Carbohydrate Epimerases / Racemases and Epimerases / Enzymes, Immobilized / Fructose / Hydrogen-Ion Concentration Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2021 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Temperature / Bacillus subtilis / Enzyme Stability / Carbohydrate Epimerases / Racemases and Epimerases / Enzymes, Immobilized / Fructose / Hydrogen-Ion Concentration Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2021 Type: Article