The gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD. METHODS: The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D Lombard et al. (2014) H/ Machius et al. (1995) C NMR spectroscopy and enzymatic degradation studies. RESULTS: The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose. CONCLUSIONS: In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes. GENERAL SIGNIFICANCE: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient

Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract

The glycemic carbohydrates we consume are currently viewed in an unfavorable light in both the consumer and medical research worlds. In significant part, these carbohydrates, mainly starch and sucrose, are looked upon negatively due to their rapid and abrupt glucose delivery to the body which causes a high glycemic response. However, dietary carbohydrates which are digested and release glucose in a slow manner are recognized as providing health benefits. Slow digestion of glycemic carbohydrates can be caused by several factors, including food matrix effect which impedes α-amylase access to substrate, or partial inhibition by plant secondary metabolites such as phenolic compounds. Differences in digestion rate of these carbohydrates may also be due to their specific structures (e.g. variations in degree of branching and/or glycosidic linkages present). In recent years, much has been learned about the synthesis and digestion kinetics of novel α-glucans (i.e. small oligosaccharides or larger polysaccharides based on glucose units linked in different positions by α-bonds). It is the synthesis and digestion of such structures that is the subject of this review

Discovery and development of novel glucanotransferases for healthier foods

Providing nutritious, healthy and sustainably produced food is one of the main objectives of food companies such as Nestlé. Slowly digestible carbohydrates and dietary fibers are considered beneficial for human health. The aim of this work was to expand the enzymatic toolbox and to develop enzyme processes that reduce the glycaemic index of starch-containing foods. By mining the gene pool of the Nestlé culture collection (NCC) with more than 3000 food grade strains, we identified sequences for novel glucanotransferases (Gtf). Two enzymes belonging to the sub family GtfB of the glycosyl hydrolase family 70 (GH70) originating from Lactobacillus reuteri (NCC 2613) and L. fermentum (NCC 2970) as well as the GtfD enzymes from Paenibacillus beijingenis and Azobacter chroococcum (of non-NCC origin) were expressed and biochemically characterized. All four Gtf enzymes produce unique α-glucans with alternating α(1,4) and α(1,6) or α(1,4) and α(1,3) linkages of different molecular size. In vitro digestion and process development studies were performed using raw materials (e.g. wheat flour) to evaluate the impact on starch digestibility as well as the in-process modification of cereal products

Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes

Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures

Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database:The Lactobacillus reuteri NCC 2613 GtfB

The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving (α1 → 4)-linkages in maltodextrins/starch and synthesizing new (α1 → 6)-linkages. In this work, 106 putative GtfBs were identified in the Nestlé Culture Collection genome database with ~2700 genomes, and the L. reuteri NCC 2613 one was selected for further characterization based on variations in its conserved motifs. Using amylose the L. reuteri NCC 2613 GtfB synthesizes a low-molecular-mass reuteran-like polymer consisting of linear (α1 → 4) sequences interspersed with (α1 → 6) linkages, and (α1 → 4,6) branching points. This product specificity is novel within the GtfB subfamily, mostly comprising 4,6-α-glucanotransferases synthesizing consecutive (α1 → 6)-linkages. Instead, its activity resembles that of the GtfD 4,6-α-glucanotransferases identified in non-LAB strains. This study demonstrates the potential of large-scale genome sequence data for the discovery of enzymes of interest for the food industry. The L. reuteri NCC 2613 GtfB is a valuable addition to the starch-converting GH70 enzyme toolbox. It represents a new evolutionary intermediate between families GH13 and GH70, and provides further insights into the structure-function relationships of the GtfB subfamily enzymes

4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as substrates, have been established (e.g. GtfB of Lactobacillus reuteri 121). Compared to the broad linkage specificity found in GSs, all GH70 starch-acting enzymes characterized so far possess 4,6-α-glucanotransferase activity, cleaving (α1 → 4)-linkages and synthesizing new (α1 → 6)-linkages. In this work a gene encoding a putative GH70 family enzyme was identified in the genome of Lactobacillus fermentum NCC 2970, displaying high sequence identity with L. reuteri 121 GtfB 4,6-α-glucanotransferase, but also with unique variations in some substrate-binding residues of GSs. Characterization of this L. fermentum GtfB and its products revealed that it acts as a 4,3-α-glucanotransferase, converting amylose into a new type of α-glucan with alternating (α1 → 3)/(α 1 → 4)-linkages and with (α1 → 3,4) branching points. The discovery of this novel reaction specificity in GH70 family and clan GH-H expands the range of α-glucans that can be synthesized and allows the identification of key positions governing the linkage specificity within the active site of the GtfB-like GH70 subfamily of enzymes

Structural Characterisation by ESI-MS of Feruloylated Arabino-oligosaccharides Synthesised by Chemoenzymatic Esterification

The chemoenzymatic synthesis of feruloylated arabino-oligosaccharides has been achieved, using a feruloyl esterase type C from Sporotrichum thermophile (StFaeC).The structure of the feruloylated products was confirmed by ESI-MSn

Biotechnological evaluation of feruloyl esterases as tools for the synthesis of biologically active compounds

The present PhD thesis focuses on the biotechnological evaluation of feruloyl esterases (E.C. 3.1.1.73), a class of hydrolytic enzymes, and particularly on their synthetic ability to modify hydroxy-cinnamic acids. Hydroxy-cinnamic acids belong to the family of phenolic acids and are biologically active compounds with antioxidant, antimicrobial, photoprotective, antiviral, anti-inflammatory and anticancer activity. They were modified via esterification and transesterification reactions in non conventional media (mixtures of organic solvents, ionic liquids), which are used in biocatalytic processes in order to enhance the substrates’ solubility and reverse the thermodynamic equilibrium from the hydrolysis towards the synthesis of an ester bond. The enzymatic modification of hydroxy-cinnamic acids was performed via esterification with hydrophilic compounds (mono- and oligo-saccharides, glycerol) and lipopholic compounds (aliphatic alcohols). At first, the synthetic activity of two type C feruloyl esterases was investigated (StFaeC from the fungus Sporotrichum thermophile and TsFaeC from the fungus Talaromyces stipitatus), using as reaction systems detergentless microemulsions. In these media, transesterification reactions between methyl esters of hydroxyl-cinnamic acids and mono- and oligo-saccharides were studied. The produced esterified sugars were proved to possess antimicrobial activity against the in vitro growth of mycobacterium, responsible for the tuberculosis desease. Subsequently, the detergentless microemulsions were employed as reaction media for the enzymatic lipophilisation of hydroxy-cinnamic acids and particularly for the synthesis of their butyl esters. In that case, a type A feruloyl esterase from the fungus Aspergillus niger (AnFaeA) and three commercial enzyme preparations, containing feruloyl esterase activity, were used as biocatalysts. Both free and immobilised enzymes were used. In order to immobilise them, the preparation of cross-linked enzyme aggregates (CLEAs) methodology was employed. This is the first example of immobilisation of feruloyl esterases until present. The synthesised butyl esters of hydroxy-cinnamic acids presented antioxidant activity against the inhibition of the oxidation of low-density lipoprotein (LDL) in vitro. In order to expand the synthetic activity of feruloyl esterases in other non conventional media, their activity in ionic liquids was investigated. Ionic liquids, which are salts usually in liquid form in ambient temperatures, are a new and very promising category of solvents with many advantages against conventional organic solvents and with potentiality to design their properties (designer solvents). In these media, the enzymatic esterification of glycerol with sinapic acid was succeeded using the type A feruloyl esterase AnFaeA in its free and immobilised form. The modification of glycerol, which is a biodiesel industry by-product, shows increasing interest in a wide spectrum of applications (food, pharmaceutical and cosmetic industry). This reaction is the first example reported for the activity of feruloyl esterases in ionic liquids, expanding their use in these new reaction systemsΗ παρούσα διδακτορική διατριβή έχει ως αντικείμενο τη βιοτεχνολογική αξιολόγηση μίας κατηγορίας ενζύμων, των εστερασών του φερουλικού οξέος (E.C. 3.1.1.73), ως προς τη συνθετική τους ικανότητα με στόχο την τροποποίηση των υδροξυ-κινναμικών οξέων. Τα υδροξυ-κινναμικά οξέα ανήκουν στην οικογένεια των φαινολικών οξέων, που αποτελούν βιολογικά ενεργές φυσικές ουσίες Η τροποποίηση τους πραγματοποιήθηκε μέσω ενζυμικών αντιδράσεων εστεροποίησης σε μη συμβατικά συστήματα (μίγματα οργανικών διαλυτών, ιοντικά υγρά), μέσα που αυξάνουν τη διαλυτότητα των υποστρωμάτων και μετατοπίζουν τη θερμοδυναμική ισορροπία της αντίδρασης από την υδρόλυση προς τη σύνθεση εστερικού δεσμού. Tα υδροξυ-κινναμικών οξέων τροποποιήθηκαν μέσω εστεροποίησης με υδρόφιλα μόρια (μονο- και ολιγο-σακχαρίτες, γλυκερόλη) και με λιπόφιλα μόρια (αλειφατικές αλκοόλες). Αρχικά, μελετήθηκε η συνθετική δράση δύο εστερασών του φερουλικού οξέος τύπου C (StFaeC του μύκητα Sporotrichum thermophile και η TsFaeC του μύκητα Talaromyces stipitatus), χρησιμοποιώντας ως μέσα αντιδράσεων μικρογαλακτώματα απουσία επιφανειοενεργών ουσιών. Στα συστήματα αυτά, μελετήθηκαν αντιδράσεις μετεστεροποίησης μεταξύ μεθυλεστέρων των υδροξυ-κινναμικών οξέων με μονο- και ολιγο-σακχαρίτες. Τα προκύπτοντα εστεροποιημένα σάκχαρα αποδείχθηκαν ουσίες με αντιμικροβιακή δράση έναντι της in vitro ανάπτυξης μυκοβακτηρίων που ευθύνονται για τη νόσο της φυματίωσης. Στη συνέχεια, τα προαναφερθέντα μικρογαλακτώματα χρησιμοποιήθηκαν ως μέσα αντίδρασης για την ενζυμική λιποφιλίωση των υδροξυ-κινναμικών οξέων και συγκεκριμένα, τη σύνθεση βουτυλεστέρων τους. Ως βιοκαταλύτες χρησιμοποιήθηκαν μία τύπου Α εστεράση του φερουλικού οξέος, η AnFaeA του μύκητα Aspergillus niger, καθώς και τρία εμπορικά ενζυμικά σκευάσματα με ενεργότητα εστεράσης του φερουλικού οξέος. Οι βιοκαταλύτες αυτοί χρησιμοποιήθηκαν τόσο στην ελεύθερη όσο και στην ακινητοποιημένη τους μορφή. Για την ακινητοποίησή τους, χρησιμοποιήθηκε η μέθοδος σχηματισμού ενζυμικών συσσωματωμάτων διασταυρούμενων δεσμών (Cross-linked enzyme aggregates, CLEAs). Αυτό αποτελεί και το πρώτο παράδειγμα ακινητοποίησης εστερασών του φερουλικού οξέος στη βιβλιογραφία. Οι βουτυλεστέρες των υδροξυ-κινναμικών οξέων που παρασκευάστηκαν παρουσίασαν αντιοξειδωτική δράση ως προς την παρεμπόδιση της οξείδωσης της χαμηλής πυκνότητας λιποπρωτεΐνης (LDL) in vitro. Mε στόχο τη διεύρυνση της συνθετικής ικανότητας των εστερασών του φερουλικού οξέος σε άλλα μη συμβατικά συστήματα, ερευνήθηκε η δράση τους σε ιοντικά υγρά, τα οποία είναι άλατα σε υγρή ως επί το πλείστον μορφή σε θερμοκρασίες περιβάλλοντος. Αποτελούν μία νέα και πολλά υποσχόμενη κατηγορία διαλυτών λόγω των πλεονεκτημάτων τους έναντι των κλασσικών οργανικών διαλυτών, αλλά και της δυνατότητας σχεδιασμού των ιδιοτήτων τους ανάλογα με την εκάστοτε διεργασία (designer solvents). Στα συστήματα αυτά πραγματοποιήθηκε επιτυχώς η ενζυμική εστεροποίηση της γλυκερόλης με σιναπικό οξύ με τη δράση της εστεράσης του φερουλικού οξέος AnFaeA, στην ελεύθερη και ακινητοποιημένη της μορφή. Η τροποποίηση της γλυκερόλης, που είναι παραπροϊόν της βιομηχανίας biodiesel, παρουσιάζει αυξανόμενο ενδιαφέρον σε ένα ευρύ πεδίο εφαρμογών (βιομηχανία τροφίμων, καλλυντικών, φαρμάκων κ.ά). Η αντίδραση αυτή αποτελεί το πρώτο παράδειγμα δράσης εστερασών του φερουλικού οξέος σε ιοντικά υγρά, επεκτείνοντας τις χρήσεις των ενζύμων αυτών σε νέα συστήματα αντιδράσεων

Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation

The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid's antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid

Structural Characterisation by ESI-MS of Feruloylated Arabino-oligosaccharides Synthesised by Chemoenzymatic

Abstract: The chemoenzymatic synthesis of feruloylated arabino-oligosaccharides has been achieved, using a feruloyl esterase type C from Sporotrichum thermophile (StFaeC). The structure of the feruloylated products was confirmed by ESI-MS n