Fructo-oligosaccharides production by the Gluconacetobacter diazotrophicuslevansucrase expressed in the methylotrophic yeast Pichia pastoris

Levansucrase (LsdA) (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (fo rmerly Aretobacter diazotrophicus) yields high levels of fructo-oligosaccha rides (FOS) from sucrose. A DNA fragment encoding the precursor LsdA lackin g the first 57 amino acids was fused to the pho1 signal sequence under the control of the Pichia pastoris-alcohol oxidase I (AOXI) promoter. Methanol induction of a P. pastoris strain harboring a single copy of the lsdA expre ssion cassette integrated in the genome resulted in the production of activ e levansucrase. After fermentation of the recombinant yeast, LsdA activity was detected in the periplasmic fraction (81%) and in the culture supernata nt (18%) with an overall yield of 1% of total protein. The recombinant LsdA was glycosylated and displayed optimal pH and temperature for enzyme activ ity similar to those of the native enzyme, but thermal stability was increa sed. Neither fructosylpolymerase activity nor FOS production was affected. Incubation of recombinant LsdA in sucrose (500 g l(-1)) yielded 43% (w/w) o f total sugar as 1-kestose, with a conversion efficiency about 70%. Intact recombinant yeast cells also converted sucrose to FOS although for a 30% ef ficiency. (C) 2001 Elsevier Science Inc. All rights reserved.