Cloning and expression of the sucrose phosphorylase gene from Leuconostoc mesenteroides in Escherichia coli

Abstract

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K (m) of 3 mM with sucrose as a substrate; optimum activity was at 37 degrees C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP).

Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae

“…A previously constructed S. cerevisiae strain lacking all native sucrose proton-symporters and hydrolases, which remained sucrose-negative upon strong selective pressures (Marques et al, 2017), was used as a platform to avoid interference by native sucrose metabolising enzymes. For the phosphorolytic cleavage reaction, SPase from Leuconostoc mesenteroides ATTC 12291 was chosen in view of the compatibility of its temperature and pH optima with expression in yeast (Aerts et al, 2011;Goedl et al, 2010Goedl et al, , 2007Kawasaki et al, 1996;Lee et al, 2008). Several proposed sucrose facilitators from plant origins were screened for their ability to support growth of the platform strain on sucrose: Phaseolus vulgaris SUF1 (PvSUF1), Pisum sativum SUF1 and SUF4 (PsSUF1 and PsSUF4), Arabidopsis thaliana SWEET12 (AtSWEET12) and Oryza sativa SWEET11 (OsSWEET11).…”

Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

“…Before this study, SP was commonly expressed as a cytoplasmic protein in E. coli , and systematic investigations of its production are rare . Through the above study, naturally cytoplasmic SP Lc was produced in the extracellular medium.…”

“…Before this study, SP was commonly expressed as a cytoplasmic protein in E. coli, and systematic investigations of its production are rare. [37][38][39] Through the above study, naturally cytoplasmic SP Lc was produced in the extracellular medium. Extracellular production depended on the enhanced permeability of the cell membrane induced by the phospholipid hydrolase activity of co-expressed PLC.…”

Transglycosylation Activity and Characterization of Recombinant Sucrose Phosphorylase From Leuconostoc Mesenteroides MBFWRS-3(1) Expressed in Escherichia Coli

“…Although three potential catalytic amino acid residues, namely, Asp-196, Glu-237, and Asp-295, are located in the conserved sequences of the L. mesenteroides SPases, there exists some dissent on the diversity of the transglycosylation properties of SPases with respect to the acceptors and their products [2,17,21,22]. This variability could be due to the involvement of the C-terminal region.…”

“…The activity of SPase from L. mesenteroides has been used in a variety of industrial applications, including to transfer the glucosyl moiety of G-1-P to various sugars and sugar alcohols [12], to transfer the glucosyl moiety of sucrose to phenolic or alcoholic hydroxide groups of various substances [12][13][14][15][16], and to transfer the glucosyl moiety of sucrose or of G-1-P to acceptors such as galactose, maltose, and glucose or beta linkage compounds such as cellobiose and gentiobiose using sucrose as a glucosyl donor to produce various acceptor reaction products [17]. Because LAB is considered safe for humans, the production of enzymes in some species for food-and health-related industrial applications is highly promising.…”