Efficient Soluble Expression and Fermentation Conditions of D-Allulose 3-Epimerase in <i>Escherichia coli</i>

LI Qiufeng; CHEN Jing; ZHAO Jingyi; WEI Xin; WANG Zhiqi; LIU Jidong

doi:10.13386/j.issn1002-0306.2021120208

Volume 43 Issue 22

Nov. 2022

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Science and Technology of Food Industry > 2022 > 43(22): 136-143. > DOI: 10.13386/j.issn1002-0306.2021120208

LI Qiufeng, CHEN Jing, ZHAO Jingyi, et al. Efficient Soluble Expression and Fermentation Conditions of D-Allulose 3-Epimerase in Escherichia coli[J]. Science and Technology of Food Industry, 2022, 43(22): 136−143. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021120208.

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Efficient Soluble Expression and Fermentation Conditions of D-Allulose 3-Epimerase in Escherichia coli

1.
College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China
2.
Guangxi Academy of Agricultural Sciences/Guangxi South Subtropical Agricultural Science Research Institute, Chongzuo 532415, China

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Received Date: December 19, 2021
Available Online: September 07, 2022

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Abstract

Abstract

The DAEase gene sequence derived from Clostridium bolteae ATCCBAA-613 was synthesized by codon optimization. Using pCold TF as the expression vector, the cold-shock promoter CspA induced the expression of the DAEase gene in Escherichia coli BL21(DE3) at low temperature. Then, the highly soluble recombinant Cb-DAEase was obtained and purified by Ni-chelating affinity chromatography. Results showed that, the Cb-DAEase exhibited maximum activity at pH7.0 and 55 ℃. Additionally, the Cb-DAEase showed different sensitivities to the various metal ions when Co²⁺ was able to significantly (P<0.05) enhance its enzyme activity. The optimum fermentation conditions were determined as follows, 7 g/L glycerol, 10 g/L yeast extract, 1% inoculation volume, 0.25 mmol/L IPTG inducer, and incubation 5 h before the induction. Eventually, the secretion level of Cb-DAEase reached (10.11±0.02) U/g, which was 7.33-fold higher than that control (1.38±0.01) U/g. Through optimizing conditions, D-allulose was produced effectively by the whole-cell biotransformation system when 120 g/L D-fructose was used as the substrate for 0.5 h, the yield reached (11.47±0.04) g/L, which was 11.14-fold higher than that control (1.03±0.02) g/L. Overall, the recombinant strain constructed based on cold-shock expression increased significantly (P<0.05) Cb-DAEase activity after fermentation optimization, which would provide a theoretical basis for the efficient preparation of D-allulose.
- D-allulose 3-epimerase,
- Escherichia coli,
- cold-shock,
- soluble expression,
- enzymatic properties,
- fermentation optimization

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