High-efficiency expression of Sulfolobus acidocaldarius maltooligosyl trehalose trehalohydrolase in Escherichia coli through host strain and induction strategy optimization
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Maltooligosyl trehalose trehalohydrolase (MTHase, EC 220.127.116.11) 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.
KeywordsMaltooligosyl trehalose trehalohydrolase Recombinant expression Escherichia coli Fermentation optimization
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).
- 7.Kim YH, Kwon TK, Park S, Seo HS, Cheong JJ, Kim CH, Kim JK, Lee JS, Choi YD (2000) Trehalose synthesis by sequential reactions of recombinant maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase from Brevibacterium helvolum. Appl Environ Microbiol 66:4620–4624CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Nakada T, Ikegami S, Chaen H, Kubota M, Fukuda S, Sugimoto T, Kurimoto M, Tsujisaka Y (1996) Purification and characterization of thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biosci Biotechnol Biochem 60:267–270CrossRefPubMedGoogle Scholar
- 15.Chang M, Qiao Y, Ding HB (2011) Cloning and expression of maltooligosyltrehalose trehalohydrolase gene from Corynebacterium glutamicum. J Agri Sci Technol 13:47–52Google Scholar