Construction and immobilization of recombinant Bacillus subtilis with D-allulose 3-epimerase.

Yuxia Wei; Xian Zhang; Mengkai HU; Yu Shao; Shan Pan; Morihisa Fujita; Zhiming Rao

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Construction and immobilization of recombinant Bacillus subtilis with D-allulose 3-epimerase.

Author: Yuxia WEI ¹ ; Xian ZHANG ¹ ; Mengkai HU ¹ ; Yu SHAO ¹ ; Shan PAN ¹ ; Morihisa FUJITA ¹ ; Zhiming RAO ¹
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1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: Bacillus subtilis; D-allulose-3-epimerase; immobilized cells; short amphipathic peptides; titanium dioxide
MeSH: Bacillus subtilis/metabolism*; Carbohydrate Epimerases/genetics*; Enzyme Stability; Enzymes, Immobilized/metabolism*; Fructose; Hydrogen-Ion Concentration; Racemases and Epimerases; Temperature
From: Chinese Journal of Biotechnology 2021;37(12):4303-4313
CountryChina
Language:Chinese
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 1:4 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.