d-amino acids metabolism reflects the evolutionary origin of higher plants and their adaptation to the environment.

d-amino acids are the d stereoisomers of the common l-amino acids found in proteins. Over the past two decades, the occurrence of d-amino acids in plants has been reported and circumstantial evidence for a role in various processes, including interaction with soil microorganisms or interference with cellular signalling, has been provided. However, examples are not numerous and d-amino acids can also be detrimental, some of them inhibiting growth and development. Thus, the persistence of d-amino acid metabolism in plants is rather surprising, and the evolutionary origins of d-amino acid metabolism are currently unclear. Systemic analysis of sequences associated with d-amino acid metabolism enzymes shows that they are not simply inherited from cyanobacterial metabolism. In fact, the history of plant d-amino acid metabolism enzymes likely involves multiple steps, cellular compartments, gene transfers and losses. Regardless of evolutionary steps, enzymes of d-amino acid metabolism, such as d-amino acid transferases or racemases, have been retained by higher plants and have not simply been eliminated, so it is likely that they fulfil important metabolic roles such as serine, folate or plastid peptidoglycan metabolism. We suggest that d-amino acid metabolism may have been critical to support metabolic functions required during the evolution of land plants.

Insights in inulin binding and inulin oligosaccharide formation by novel multi domain endo-inulinases from Botrytis cinerea.

World-wide, pathogenic fungi such as Botrytis cinerea cause tremendous yield losses in terms of food production and post-harvest food decay. Many fungi produce inulin-type oligosaccharides (IOSs) from inulin through endo-inulinases which typically show a two domain structure. B.cinerea lacks a two domain endo-inulinase but contains a three domain structure instead. Genome mining revealed three and four domain (d4) enzymes in the fungal kingdom. Here, three and two domain enzymes were compared in their capacity to produce IOSs from inulin. Hill kinetics were observed in three domain enzymes as compared to Michaelis-Menten kinetics in two domain enzymes, suggesting that the N-terminal extension functions as a carbohydrate binding module. Analysis of the IOS product profiles generated from purified GF6, GF12, GF16 and GF18 inulins and extensive sugar docking approaches led to enhanced insights in the active site functioning, revealing subtle differences between the endo-inulinases from Aspergillus niger and B. cinerea. Improved insights in structure-function relationships in fungal endo-inulinases offer opportunities to develop superior enzymes for the production of specific IOS formulations to improve plant and animal health (priming agents, prebiotics).

The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other ß-Fructofuranosidases in Bacteria.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular ß-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other ß-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other ß-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

Characterization of inulolytic enzymes from the Jerusalem artichoke-derived Glutamicibacter mishrai NJAU-1.

The rhizosphere context of inulin-accumulating plants, such as Jerusalem artichoke (Helianthus tuberosus), is an ideal starting basis for the discovery of inulolytic enzymes with potential for bio fructose production. We isolated a Glutamicibacter mishrai NJAU-1 strain from this context, showing exo-inulinase activity, releasing fructose from fructans. The growth conditions (pH 9.0; 15 °C) were adjusted, and the production of inulinase by Glutamicibacter mishrai NJAU-1 increased by 90% (0.32 U/mL). Intriguingly, both levan and inulin, but not fructose and sucrose, induced the production of exo-inulinase activity. Two exo-inulinase genes (inu1 and inu2) were cloned and heterologously expressed in Pichia pastoris. While INU2 preferentially hydrolyzed longer inulins, the smallest fructan 1-kestose appeared as the preferred substrate for INU1, also efficiently degrading nystose and sucrose. Active site docking studies with GFn- and Fn-type small inulins (G is glucose, F is fructose, and n is the number of ß (2-1) bound fructose moieties) revealed subtle substrate differences between INU1 and INU2. A possible explanation about substrate specificity and INU's protein structure is then suggested. KEY POINTS: • A Glutamicibacter mishrai strain harbored exo-inulinase activity. • Fructans induced the inulolytic activity in G. mishrai while the inulolytic activity was optimized at pH 9.0 and 15 °C. • Two exo-inulinases with differential substrate specificity were characterized.

A novel chicory fructanase can degrade common microbial fructan product profiles and displays positive cooperativity.

Fructan metabolism in bacteria and plants relies on fructosyltransferases and fructanases. Plant fructanases (fructan exohydrolase, FEH) only hydrolyse terminal fructose residues. Levan (ß-2,6 linkages) is the most abundant fructan type in bacteria. Dicot fructan accumulators, such as chicory (Cichorium intybus), accumulate inulin (ß-2,1 linkages), harbouring several 1-FEH isoforms for their degradation. Here, a novel chicory fructanase with high affinity for levan was characterized, providing evidence that such enzymes widely occur in higher plants. It is adapted to common microbial fructan profiles, but has low affinity towards chicory inulin, in line with a function in trimming of microbial fructans in the extracellular environment. Docking experiments indicate the importance of an N-glycosylation site close to the active site for substrate specificity. Optimal pH and temperature for levan hydrolysis are 5.0 and 43.7 °C, respectively. Docking experiments suggested multiple substrate binding sites and levan-mediated enzyme dimerization, explaining the observed positive cooperativity. Alignments show a single amino acid shift in the position of a conserved DXX(R/K) couple, typical for sucrose binding in cell wall invertases. A possible involvement of plant fructanases in levan trimming is discussed, in line with the emerging 'fructan detour' concepts, suggesting that levan oligosaccharides act as signalling entities during plant-microbial interactions.

Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase.

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

Implications of the mutation S164A on Bacillus subtilis levansucrase product specificity and insights into protein interactions acting upon levan synthesis.

Mutation S164A largely affects the transfructosylation properties of Bacillus subtilis levansucrase (SacB). The variant uses acceptors such as glucose and short levans with an average molecular weight of 7.6 kDa more efficiently than SacB, leading to the enhanced synthesis of medium and high molecular weight polymer and a blasto-oligosaccharide series with a polymerization degree of 2-10. A 3-fold increase in blasto-oligosaccharides yield is provoked by the modified interplay between the variant and glucose. Despite its modified product specificity, protein-carbohydrate and protein-protein interactions are still a major factor affecting size and distribution of levan molecular weight. This study highlights the importance of critical factors such as protein concentration in the analysis of wild-type and mutagenized levansucrases. Docking experiments with the crystal structures of SacB and variant S164A - the latter obtained at a 2.6 Å resolution - identified unreported potential binding subsites for fructosyl moieties on the surface of both enzymes.

A close look at the structural features and reaction conditions that modulate the synthesis of low and high molecular weight fructans by levansucrases.

The physicochemical properties and biological activity of levan, a generic term given to oligo- and polysaccharides consisting of fructose units linked predominantly by ß(2-6) bonds, are attributable to both its size and structural complexity. Branching in ß(2-1) contributes to diversify levan structures and properties. There is a broad spectrum of applications for levan and accordingly it has been the subject of several comprehensive reviews. A thorough analysis focused on the product specificity of enzymes from the Glycoside-Hydrolase family 68 that synthesize levan is however missing. We analyze here traditional and novel strategies to manipulate bacterial levansucrases in favor of the generation of low- or high-molecular weight levan, including site directed mutagenesis and chemical engineering. A comparison of highly variable structural elements of levansucrases is presented in the context of their capacity to synthesize saccharides of different sizes, employing the levansucrases from Bacillus subtilis and Bacillus megaterium as references.

Frucooligosaccharides purification: Complexing simple sugars with phenylboronic acid.

Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic reaction with fructosyltransferases (FTFs) using sucrose as both donor and acceptor. In these reactions glucose results as the most abundant by-product, arising from each fructosyl transfer event and, together with fructose, because of the inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in nutraceutical foods, the reduction or total elimination of monosaccharides is required. In this work the selective elimination of monosaccharides from a synthetic FOS mixture was achieved through the selective complexation of glucose and fructose with phenyl boronic acid (PBAc) followed by ethyl-acetate extraction. The process was applied to a complex mixture of FOS obtained in an enzymatic synthesis reaction containing 40% glucose, 15.8% fructose and 35% of FOS, elimination of the sugars was achieved through 3:1 molar reactions, resulting in a levan-type FOS product with 97% purity.

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.

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.

Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB).

Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final levan profile in reactions with sucrose as substrate.

Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight.

Levan is a fructan polymer that offers a variety of applications in the chemical, health, cosmetic and food industries. Most of the levan applications depend on levan molecular weight, which in turn depends on the source of the synthesizing enzyme and/or on reaction conditions. Here we demonstrate that in the particular case of levansucrase from Bacillus subtilis 168, enzyme concentration is also a factor defining the molecular weight levan distribution. While a bimodal distribution has been reported at the usual enzyme concentrations (1 U/ml equivalent to 0.1 µM levansucrase) we found that a low molecular weight normal distribution is solely obtained al high enzyme concentrations (>5 U/ml equivalent to 0.5 µM levansucrase) while a high normal molecular weight distribution is synthesized at low enzyme doses (0.1 U/ml equivalent to 0.01 µM of levansucrase).

Enzymatic synthesis of poly-L-lactide and poly-L-lactide-co-glycolide in an ionic liquid.

The syntheses of poly-L-lactide (PLLA) and poly-L-lactide-co-glycolide (PLLGA) is reported in the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate [HMIM][PF(6)] mediated by the enzyme lipase B from Candida antarctica (Novozyme 435). The highest PLLA yield (63%) was attained at 90 degrees C with a molecular weight (M(n)) of 37.8 x 10(3) g/mol determined by size exclusion chromatography. This procedure produced relatively high crystalline polymers (up to 85% PLLA) as determined by DSC. In experiments at 90 degrees C product synthesis also occurred without biocatalyst, however, PLLA synthesis in [HMIM][PF(6)] at 65 degrees C followed only the enzymatic mechanism as ring opening was not observed without the enzyme. In addition, the enzymatic synthesis of PLLGA is first reported here using Novozyme 435 biocatalyst with up to 19% of lactyl units in the resulting copolymer as determined by NMR. Materials were also characterized by TGA, MALDI-TOF-MS, X-ray diffraction, polarimetry and rheology.