Simultaneous enzyme production, Levan-type FOS synthesis and sugar by-products elimination using a recombinant Pichia pastoris strain expressing a levansucrase-endolevanase fusion enzyme.

BACKGROUND: Although Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products. RESULTS: The expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h. At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level. CONCLUSION: We demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.

Structure-Function Relationship Studies of Multidomain Levansucrases from Leuconostocaceae Family.

Levansucrase LevS from Leuconostoc mesenteroides B-512F is a multidomain fructansucrase (MD-FN) that contains additional domains (ADs) to the catalytic domain. However, the understanding of the effect that these ADs have on enzyme activity remains vague. To this aim, structure-function relationship studies of these LevS ADs were performed by evaluating both biochemical properties and the enzymatic capacity of truncated versions of LevS. Joint participation of the N- and C-terminal domains is essential for stability, activity, specificity, and polymerization processes. Specifically, the N-terminal region is involved in stability, while the transition region plays an essential role in the transfructosylation reaction and polymer elongation. Based on our results, we suggest that ADs interact with each other, adopting a U-shaped topology. The importance of these ADs observed in the MD-FN of the Leuconostocaceae family is not shared by the Lactobacillaceae family. Phylogenetic analysis of LevS AD suggests that MD-FN from Lactobacillaceae and Leuconostocaceae have different evolutionary origins. This is the first study on the structure-function relationship of multidomain levansucrases from the Leuconostocaceae family. Our results point towards the functional role of AD in MD-FN and its involvement in fructan synthesis.

Co-production of Fructooligosaccharides and Levan by Levansucrase from Bacillus subtilis natto with Potential Application in the Food Industry.

Fructooligosaccharides and levan have a wide range of applications in the food industry due to their physiological and functional properties. The enzymatic synthesis of these molecules exhibits great advantages when compared with microbial fermentation. In this study, the production of levansucrase from Bacillus subtilis natto and its utilization in fructooligosaccharides and levan syntheses using different reaction conditions were described. The best condition for levansucrase production was 420.7 g L-1 of sucrose at pH 7.0, which reached 23.9 U ml-1 of transfructosylation activity. In a bioreactor, the highest production of fructooligosaccharides was 41.3 g L-1 using a medium containing 350 g L-1 sucrose at 35 °C for 36 h. The enzymatic synthesis of levan resulted in 86.9 g L-1 when conditions similar to those used for fructooligosaccharides synthesis were applied. These results indicate that the levansucrase from B. subtilis natto could be applied for the co-production of fructooligosaccharides and levan, which are biomolecules that have health benefits and are used successfully in the food industry.

Levan-type fructooligosaccharides synthesis by a levansucrase-endolevanase fusion enzyme (LevB1SacB).

We describe here the enzymatic production of levan type-fructooligosaccharides (L-FOS) with a DP from 2 to 10, through simultaneous synthesis and hydrolysis reactions. This was accomplished by LevB1SacB, a new enzyme resulting from the fusion of SacB, a levansucrase from Bacillus subtilis and LevB1, an endolevanase from B. licheniformis. In the fusion enzyme, SacB retains its catalytic behavior with a decrease in kcat from 164 to 108s-1. LevB1 in LevB1SacB kinetic behavior improves considerably reaching saturation with levan and following Michaelis-Menten kinetics, quite differently from the previously reported first order kinetic behavior. We also report that LevB1SacB or both enzymes (LevB1 & SacB) at equimolar concentrations in simultaneous reactions result in an optimal, wide and diverse L-FOS profile, including 6-kestose, levanbiose and blastose among other L-FOS and 1-kestose, which accumulates as by-product of SacB levan synthesis. Yields of around 40% (w/w) were obtained from 600g/l sucrose with either LevB1SacB or LevB1 & SacB. The reaction was successfully scaled up to a stirred 2l bioreactor.

A novel two-step enzymatic synthesis of blastose, a ß-d-fructofuranosyl-(2↔6)-d-glucopyranose sucrose analogue.

Blastose, a natural disaccharide found in honey, is usually found as a byproduct of fructo-oligosaccharide synthesis from sucrose with fructosyltransferases. In this study, we describe a novel two-step biosynthetic route to obtain blastose, designed from a detailed observation of B. subtilis levansucrase (SacB) acceptor structural requirements for fructosylation. The strategy consisted first in the synthesis of the trisaccharide O-ß-d-Fruf-(2↔6)-O-α-d-Glcp-(1↔1)-α-d-Glcp, through a regioselective ß-d-transfructosylation of trehalose (Tre) which acts as acceptor in a reaction catalyzed by SacB using sucrose or levan as fructosyl donor. In this reaction, levansucrase (LS) transfers regioselectively a fructosyl residue to either C6-OH group of the glucose residues in Tre. The resulting trisaccharide obtained in 23% molar yield based on trehalose, was purified and fully characterized by extensive NMR studies. In the second step, the trisaccharide is specifically hydrolyzed by trehalase, to obtain blastose in 43.2% molar yield based on the trisaccharide. This is the first report describing the formation of blastose through a sequential transfuctosylation-hydrolysis reaction.

Size product modulation by enzyme concentration reveals two distinct levan elongation mechanisms in Bacillus subtilis levansucrase.

Two levan distributions are produced typically by Bacillus subtilis levansucrase (SacB): a high-molecular weight (HMW) levan with an average molecular weight of 2300 kDa, and a low-molecular weight (LMW) levan with 7.2 kDa. Previous results have demonstrated how reaction conditions modulate levan molecular weight distribution. Here we demonstrate that the SacB enzyme is able to perform two mechanisms: a processive mechanism for the synthesis of HMW levan and a non-processive mechanism for the synthesis of LMW levan. Furthermore, the effect of enzyme and substrate concentration on the elongation mechanism was studied. While a negligible effect of substrate concentration was observed, we found that SacB elongation mechanism is determined by enzyme concentration. A high concentration of enzyme is required to synthesize LMW levan, involving the sequential formation of a wide variety of intermediate size levan oligosaccharides with a degree of polymerization (DP) up to ∼70. In contrast, an HMW levan distribution is synthesized through a processive mechanism producing oligosaccharides with DP <20, in reactions occurring at low enzyme concentration. Additionally, reactions where levansucrase concentration was varied while the total enzyme activity was kept constant (using a combination of active SacB and an inactive SacB E342A/D86A) allowed us to demonstrate that enzyme concentration and not enzyme activity affects the final levan molecular weight distribution. The effect of enzyme concentration on the elongation mechanism is discussed in detail, finding that protein-product interactions are responsible for the mechanism shift.

Levans production by Gluconacetobacter diazotrophicus

Background: Growth of Gluconacetobacter diazotrophicus with glucose as carbon an energy source has been extensively studied. However, there are no reports in the literature describing growth of G. diazotrophicus in cultures containing sucrose as carbon source. The first step in sucrose pathway and production of levans was investigated. Biomass, levans, gluconic acid and keto gluconic acids production and levansucrase activity were determined in cultures with different sucrose concentration and nitrogen sources. Results: The biomass production was maximal in cultures containing 100 g x L-1 sucrose and inorganic nitrogen. Gluconic acid production was observed under all conditions tested, at levels up to 9 g x L-1 in cultures with sucrose excess and biological N2-fixation (BNF). Keto gluconic acids were detectable only in cultures with sucrose excess and supplemented with organic nitrogen sources. Levans production, although observed in all cultures, was maximal in batch culture with 100 g x L-1 of sucrose and BNF, concomitant with a significant expression of extracellular levansucrase. Conclusions: Ours results not only describe some unknown aspects of G. diazotrophicus physiology, but open up the possibility of developing a technology of levans production by this organism using culture media with sucrose (or some cheaper substitute, like molasses) and without the addition of any N-source because of its ability of fixing atmospheric N2.