4.5 Article

Permeabilized whole-cell biocatalyst containing co-expressed two enzymes facilitates the synthesis of maltoheptaose (G7) from starch

期刊

ENZYME AND MICROBIAL TECHNOLOGY
卷 159, 期 -, 页码 -

出版社

ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2022.110057

关键词

Cyclodextrin glucanotransferase; Cyclomaltodextrinase; Maltoheptaose; Co-expression; Escherichia coli

资金

  1. Y Major Science and Technology Innovation Project of Shandong Province [2020CXGC010601]

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In this study, a genetically engineered strain was constructed to co-express CGTase and CDase for the synthesis of G7 from inexpensive starch substrate. After permeabilization of the cell wall, G7 was successfully produced under optimized conditions, avoiding the need for enzyme purification steps.
Maltoheptaose (G7) is one of the mixtures of maltodextrin widely used in the food, pharmaceutical, and cosmetics industries. A genetically engineered strain, which simultaneously expressed cyclodextrin glucanotransferase (CGTase) from Gracilibacillus alcaliphilus SK51.001 and cyclomaltodextrinase (CDase) from Bacillus sphaericus E-244, two enzymes, was constructed by cloning the above two genes into a plasmid and transformed into the host Escherichia coli BL21(DE3) (E. coli) strain, resulted in recombinant cells harboring the vector pETDuet-GaCGT/BsCD (pGaBs). These cells were used as whole-cell catalysts for the biotransformation of G7 from the inexpensive substrate (starch). Due to the high molecular weight of starch, the cell membrane prevents the entry of starch into the cellular system. Therefore, the pGaBs cell wall was permeabilized by lysozyme, EDTA, and heat treatment. After reaching the optimized conditions of permeabilized pGaBs cell amount, lysozyme amount, reaction temperature, and metal ion concentration, approximately 4.1 g/L of G7 was produced from 30 g/L starch in 1 h with the addition of Ca2+. This co-expression system offers a one-pot synthesis approach to the production of G7 using an inexpensive substrate, avoiding enzyme purification steps.

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