Discovery of new levansucrase enzymes with interesting properties and improved catalytic activity to produce levan and fructooligosaccharides

Abstract

Mining for new levansucrase enzymes with high levan production, transfructosylating activity, and thermal stability and studying their kinetics and acceptor specificity.

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates

“…The inulin-type glycosidic linkage found on 1-kestose and neokestose may have made it difficult for further transfructosylation, which is supported by the low amounts of nystose and the accumulation of neokestose. High oligolevan/levan production (82% w/w at 2 h) corresponds with the results previously reported in [22]. This high-level oligolevan/levan production reveals that the LS from G. oxydans predominately performs a processive reaction, retaining the product in the active site, ready to accept more fructosyl residues.…”

“…This decrease was mainly due to the release of monosaccharides; indeed, the amount of transfructosylation products only decreased from 78 g/L to 63 g/L, while the monosaccharides content increased from 15 g/L to 100 g/L. The high production of hydrolysis products at 50 h corresponded with the kinetic parameters of the LS from V. natriegens , where both the turnover rate (k cat ) and the catalytic efficiency were higher for hydrolysis (246 s −1 , 101 s −1 ·mM −1 ) than they were for transfructosylation (152 s −1 , 0.350 s −1 ·mM −1 ) [ 22 ].…”

“…Indeed, both the turnover rate for transfructosylation (9.19 s −1 ) and hydrolysis (7.98 s −1 ) were relatively similar for LS from B. indica subsp. indica , but the catalytic efficiency was much higher for hydrolysis (0.382 s −1 ·mM −1 ) than for transfructosylation (0.020 s −1 ·mM −1 ) activities [ 22 ].…”

“…The high bioconversion of sucrose by LS from P. graminis may be attributed to its high stability and/or to the low substrate/product inhibitions. Indeed, this LS was found to have very high thermal stability, with a half-life of 291 min at 50 °C [ 22 ]. There were less peaks indicating larger oligosaccharides as compared with the other LSs in this study, with the largest identified as X 7 , while the highest quantity product was 1-kestose (74% w / w transfructosylation products), with no levan produced.…”

“…Indeed, both the turnover rate for transfructosylation (9.19 s −1 ) and hydrolysis (7.98 s −1 ) were relatively similar for LS from B. indica subsp. indica, but the catalytic efficiency was much higher for hydrolysis (0.382 s −1 •mM −1 ) than for transfructosylation (0.020 s −1 •mM −1 ) activities [22]. Figure 1 indicates that LS from V. natriegens produced levan-type oligo/polysaccharides (85 g/L; 76% w/w transfructosylation products) at 12 h (maximum oligolevan/levan production), and 18% oligosaccharide (24%, w/w total transfructosylation products) made of a mixture of trisaccharides (X 3 ), tetrasaccharides (X 4 ), and pentasaccharides (X 5 ).…”