Fructooligosaccharides production by immobilized Pichia pastoris cells expressing Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase

Abstract

Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Keywords: Calcium alginate; Cell immobilization; Fructooligosaccharides; Fructosyltransferase; Pichia pastoris.

Fig. 1.

Performance of Pichia pastoris PGFT6x-308 in fed-batch fermentation using molasses as a carbon source. Periplasmic (black bars) and extracellular (white bars) Sa1-SSTrec activities were measured in intact cells and the culture supernatant, respectively. Biomass yield (line) is expressed as dry weight.

Fig. 2.

Effect of biomass loading on cell entrapment efficiency (line) and Sa1-SSTrec activity (bars). Immobilization yield expresses the percentage of initial Sa1-SSTrec activity which was not recovered in the entrapped cells but remained in the CaCl₂ solution. Data are averages of three experiments with standard deviation. Different letters show significant differences between enzyme activities per bead for each biomass loading (Student–Newman–Keuls test, α = 0.05).

Fig. 3.

Permeabilization of immobilized Pichia pastoris PGFT6x-308 cells. (a) Effect of toluene concentration on the Sa1-SSTrec activity of the calcium-alginate beads. (b) Effect of toluene on the stability of free Sa1-SSTrec. (c) Reduction of CO₂ formation by toluene-permeabilized cells during transformation of sucrose (1.75 M) at 30°C. Cut glasses contain beads previously incubated with 0, 3, or 5% (vol/vol) toluene. Data are the means of triplicate measurements ± standard deviation. Different letters are significant differences between relative activity at the three concentrations with respect to the control (Student–Newman–Keuls test, α = 0.05).

Fig. 4.

Effect of pH (a) and temperature (b) on the initial rates of Sa1-SSTrec activity in free (□) and immobilized (◆) cells. Values are means of three experiments with standard deviation.

Fig. 5.

Fructooligosaccharides production by the immobilized whole-cell biocatalyst reacting with different sucrose sources in a stirred tank reactor. Fructose (○), glucose (◊), sucrose (∆), 1-kestotriose (■), and 1,1-kestotetraose (◆). Raw data are included as a supplementary table.

Fig. 6.

Chromatographic analysis of fructooligosaccharides synthesized by immobilized cells from different sucrose sources in repetitive cycles. (a) TLC chromatograms of samples collected after batches 1, 3, 5, 10, and 15. S means mixture of standard sugars. (b–d) HPLC chromatograms. (b) Refined and raw sugar. (c) Refined sugar after the first batch. (d) Raw sugar after the first batch. GF₃, 1,1-kestotetraose; GF₂, 1-kestotriose; GF, sucrose; G, glucose; F, fructose.

Fig. 7.

Sa1-SSTrec stability in immobilized cells after 15 cycles of batchwise operation. Different letters show significant differences between residual activities in each sucrose source (Student–Newman–Keuls test, α = 0.05).