Development of an efficient bioprocess for turanose production by sucrose isomerisation reaction of amylosucrase

Turanose, a structural isomer of sucrose, is a non-cariogenic and low-calorigenic disaccharide. It has a promising potential to be a high-quality functional sweetener, but there has been no competitive way to produce turanose until now. In this study, a recombinant amylosucrase, originating from Neisseria polysaccharea (NpAS), was utilised to efficiently convert sucrose to turanose. With 2.5 M sucrose and 400 U/l of NpAS, the production yield of turanose maximally reached to 56.2% at 35 °C after a 120 h reaction. Main transglycosylating reaction pathway of NpAS clearly shifted from α-(1,4)-glucan synthesis to turanose production by simply increasing sucrose substrate concentration. Purification, by a preparative recycling HPLC, effectively separated turanose from the reaction mixture with a high purity of 94.7% and a high recovery yield of 97.5%. Chemical structure of the purified turanose was confirmed by NMR analysis, and three tautomers, namely 3-O-α-d-glucopyranosyl-α-d-fructofuranose, 3-O-α-d-glucopyranosyl-β-d-fructofuranose, and 3-O-α-d-glucopyranosyl-β-d-fructopyranose, were identified with a ratio of approximately 1:2:2.     

Characterisation of low‐digestible starch fractions isolated from amylosucrase‐modified waxy corn starch

Amylosucrase (AS) modification of starch increases the slowly digestible (SDS) and resistant starch (RS) fractions. However, the characteristics and formation mechanism of each fraction of AS‐modified starch have not been determined yet. Therefore, this study isolated SDS and/or RS from AS‐modified waxy corn starches and investigated their structural characteristics. The amount of SDS+RS and RS had a positive correlation with the proportion of the medium length (13–24 of degree of polymerisation) branched chains of amylopectin. The relative crystallinity increased in the order of AS‐modified starch < SDS+RS < RS, while maintaining the B‐type crystalline structure. The thermal transition temperature ranges of the isolated fractions were also higher than those of undigested starches. The medium branched chains of amylopectin were presumably the clincher for the SDS and/or RS formation in AS‐modified starches. The principal causes of SDS and RS formation were the chain length elongation and the subsequent retrogradation‐like process, respectively.     

Biosynthesis of glyceride glycoside (nonionic surfactant) by amylosucrase,a powerful glycosyltransferase

Amylosucrase (ASase, E.C. 2.4.1.4) is a powerful transglycosylation enzyme that can transfer glucose from sucrose to the hydroxyl (-OH) group of various compounds. In this study, recombinant ASases from Deinococcus geothermalis (DgAS) and Bifidobacterium thermophilum (BtAS) were used to synthesize biosurfactants based on the computational analysis of predicted docking simulations. Successful predictions of the binding affinities, conformations, and three-dimensional structures of three surfactants were computed from receptor-ligand binding modes. DgAS and BtAS were effective in the synthesis of biosurfactants from glyceryl caprylate, glyceryl caprate, and polyglyceryl-2 caprate. The results of the transglycosylation reaction were consistent for both ASases, with glyceryl caprylate acceptor showing the highest concentration, as confirmed by thin layer chromatography. Furthermore, the transglycosylation reactions of DgAS were more effective than those of BtAS. Among the three substrates, glyceryl caprylate glycoside and glyceryl caprate glycoside were successfully purified by liquid chromatography–mass spectrometry (LC–MS) with the corresponding molecular weights.     

Production and characterization of digestion‐resistant starch by the reaction of Neisseria polysaccharea amylosucrase

Recombinant amylosucrase (200 U/mL) from Neisseria polysaccharea was used to produce digestion‐resistant starch (RS) using 1–3% (w/v) corn starches and 0.1–0.5 M sucrose incubated at 35°C for 24 h. Characterization of the obtained enzyme‐modified starches was investigated. Results show that the yields of the enzyme‐modified starches were inversely proportional to the original amylose contents of corn starches. After enzymatic reaction, insoluble RS contents increased by 22.3 and 20.7% from 6.9% of waxy and 7.7% of normal corn starches, respectively, using 3.0% starch as acceptor and 0.3 M sucrose as donor, while amylomaize VII showed the lowest increase (8.5%) in RS content. The crystalline polymorph of these enzyme‐modified starches resulted in the B‐type immediately after enzymatic reaction. The enzyme‐modified starches displayed higher melting peak temperatures (85.6–100.6°C) compared to their native starch counterparts (70.1–78.4°C). After enzymatic reaction, pasting temperature increased in waxy (71.9 → 77.6°C) and normal corn starches (75.3 → 80.6°C), and the peak viscosity of waxy corn starches increased from 264 to 349 RVU, whereas that of normal corn starches decreased from 235 to 66 RVU.