Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase
2018
Su, Lingqia | Wang, Tian | Yu, Lingang | Wu, Jing
BACKGROUND: We previously showed that extracellular production of naturally cytoplasmic proteins could be realized through co‐expression with phospholipase C (PLC), which enhances cell membrane permeability through limited hydrolysis of membrane phospholipids. Leuconostoc mesenteroides sucrose phosphorylase (SPLc), a naturally cytoplasmic enzyme, exhibits excellent properties for synthesis of α‐arbutin, but no systemic investigation of its preparation has thus far been reported. RESULTS: Three PLCs were selected and expressed, without signal peptide, in Escherichia coli. More than 90% of the PLCs from Listeria monocytogenes and Bacillus cereus were present in the culture medium. When SPLc was co‐expressed with these PLCs, and the location of the PLC and SPLc genes in the co‐expression vector was optimized, the highest production was obtained when SPLc gene was inserted upstream of the B. cereus PLC gene, as in BL21(DE3)/pETDuet‐sp‐bcplc. SPLc production was then scaled up to a 3 L fermenter and fermentation conditions were optimized. When the initial medium contained 0.6 mmol L⁻¹ ZnCl₂ and protein expression was induced at a dry cell weight of 15 g L⁻¹ by the addition of lactose at 0.2 g L⁻¹ h⁻¹ at 30 °C, the extracellular SPLc activity reached its highest level of 1445.1 U mL⁻¹ This constituted 81.6% of total activity; the remainder of the enzyme (18.4%) remained inside the cells. CONCLUSION: This is the first report describing extracellular expression of sucrose phosphorylase, and the highest yield ever reported was obtained. This provides the basis for industrial‐scale production and application of sucrose phosphorylase. It also provides a potential method for improving the production of other proteins and fine chemicals. © 2018 Society of Chemical Industry
Afficher plus [+] Moins [-]Mots clés AGROVOC
Informations bibliographiques
Cette notice bibliographique a été fournie par National Agricultural Library
Découvrez la collection de ce fournisseur de données dans AGRIS
