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High-efficiency expression of Sulfolobus acidocaldarius maltooligosyl trehalose trehalohydrolase in Escherichia coli through host strain and induction strategy optimization

  • Lingqia Su
  • Shixiong Wu
  • Jinyun Feng
  • Jing Wu
Research Paper
  • 11 Downloads

Abstract

Maltooligosyl trehalose trehalohydrolase (MTHase, EC 3.2.1.141) catalyzes the release of trehalose, a novel food ingredient, by splitting the α-1,4-glucosidic linkage adjacent to the α-1,1-glucosidic linkage of maltooligosyl trehalose. However, the high-yield preparation of recombinant MTHase has not yet been reported. In this study, a codon-optimized synthetic gene encoding Sulfolobus acidocaldarius MTHase was expressed in Escherichia coli. In initial expression experiments conducted using pET-24a (+) and E. coli BL21 (DE3), the MTHase activity was 10.4 U/mL and a large amount of the expression product formed inclusion bodies. The familiar strategies, including addition of additives, co-expression with molecular chaperones, and expression with a fusion partner, failed to enhance soluble MTHase expression. Considering the intermolecular disulfide bond of MTHase, expression was investigated using a system comprising plasmid pET-32a (+) and host E. coli Origami (DE3), which is conducive to cytoplasmic disulfide bond formation. The MTHase activity increased to 55.0 U/mL, a 5.3-fold increase. Optimization of the induction conditions in a 3-L fermentor showed that when the lactose was fed at 0.2 g/L/h beginning at an OD600 of 40 and the induction temperature was maintained at 30 °C, the MTHase activity reached a maximum of 204.6 U/mL. This is the first report describing a systematic effort to obtain high-efficiency MTHase production. The high yield obtained using this process provides the basis for the industrial-scale production of trehalose. This report is also expected to be valuable in the production of other enzymes containing disulfide bonds.

Keywords

Maltooligosyl trehalose trehalohydrolase Recombinant expression Escherichia coli Fermentation optimization 

Notes

Acknowledgements

This work received financial support from the National Natural Science Foundation of China (31771916, 31501419), the Natural Science Foundation of Jiangsu Province (BK20180082), the National Science Fund for Distinguished Young Scholars (31425020), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-03), and the 111 Project (No. 111-2-06).

Supplementary material

449_2018_2039_MOESM1_ESM.tiff (4.3 mb)
Fig. S1 The codon-optimized synthetic gene (treZ) sequence (TIFF 4432 KB)
449_2018_2039_MOESM2_ESM.tiff (3.3 mb)
Fig. S2 The construction diagram of recombinant plasmid pET24a-treZ (TIFF 3362 KB)
449_2018_2039_MOESM3_ESM.tiff (4.4 mb)
Fig. S3 The construction diagram of recombinant plasmid pET24a-sumo-treZ (TIFF 4512 KB)
449_2018_2039_MOESM4_ESM.tiff (36 kb)
Fig. S4 Effects of different additives on cell growth and MTHase production. ■ OD600, □ MTHase activity (TIFF 35 KB)
449_2018_2039_MOESM5_ESM.tif (6.3 mb)
Fig. S5 SDS-PAGE analysis of recombinant MTHase in the 3-L fermentor. M, molecular mass standard proteins; 1–8, the MTHase samples corresponding with that in Table 1 (TIF 6446 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Lingqia Su
    • 1
    • 2
    • 3
  • Shixiong Wu
    • 1
    • 2
    • 3
  • Jinyun Feng
    • 1
    • 2
    • 3
  • Jing Wu
    • 1
    • 2
    • 3
  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of EducationJiangnan UniversityWuxiChina
  3. 3.International Joint Laboratory on Food SafetyJiangnan UniversityWuxiChina

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