Discovering and efficiently promoting the extracellular secretory expression of Thermobacillus sp. ZCTH02-B1 sucrose phosphorylase in Escherichia coli.

Sucrose phosphorylase (SPase, EC2.4.1.7) is a promising transglycosylation biocatalyst used for producing glycosylated compounds that are widely used in the food, cosmetics, and pharmaceutical industries. In this study, a recombinant SPase from the Thermobacillus sp. ZCTH02-B1 (rTSPase), which was previously reported to have high thermostability and the catalytic ability to synthesize ascorbic acid 2-glucoside, was attempted to be extracellularly expressed in Escherichia coli BL21(DE3) by fusion of endogenous osmotically-inducible protein Y. Unexpectedly, the rTSPase itself was produced outside the cells with an underestimated performance, although no typical signal peptide was predicted. Further N- and C-terminal truncation experiments revealed that both termini of rTSPase have an important role in protein folding and enzymatic activity, while its secretion was N-terminus associated. Extracellular protein concentration and rTSPase activity achieved 1.8 mg/mL and 6.2 U/mL after induction of 36 h in a 5-L fermenter. High-level extracellular rTSPase production could also be obtained from E. coli within 24 h by inducing overexpression of D, D-carboxypeptidase for cell lysis.