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α-Glucosidase (EC 3.2.1.20) is a carbohydrate-hydrolyzing enzyme which generally cleaves α-1,4-glycosidic bonds of oligosaccharides and starch from the nonreducing ends. In this study, the novel α-glucosidase from Weissella cibaria BBK-1 (WcAG) was biochemically and structurally characterized. WcAG belongs to glycoside hydrolase family 13 (GH13) and to the neopullanase subfamily. It exhibits distinct hydrolytic activity towards the α-1,4 linkages of short-chain oligosaccharides from the reducing end. The enzyme prefers to hydrolyse maltotriose and acarbose, while it cannot hydrolyse cyclic oligosaccharides and polysaccharides. In addition, WcAG can cleave pullulan hydrolysates and strongly exhibits transglycosylation activity in the presence of maltose. Size-exclusion chromatography and X-ray crystal structures revealed that WcAG forms a homodimer in which the N-terminal domain of one monomer is orientated in proximity to the catalytic domain of another, creating the substrate-binding groove. Crystal structures of WcAG in complexes with maltose, maltotriose and acarbose revealed a remarkable enzyme active site with accessible +2, +1 and −1 subsites, along with an Arg–Glu gate (Arg176–Glu296) in front of the active site. The −2 and −3 subsites were blocked by Met119 and Asn120 from the N-terminal domain of a different subunit, resulting in an extremely restricted substrate preference.

Aim To express amylomaltase from Thermus filiformis (TfAM) in a generally recognized as safe (GRAS) organism and to use the enzyme in starch modification. Methods and results TfAM was expressed in Saccharomyces cerevisiae, using 2% (w/v) galactose inducer under GAL1 promoter. The enzyme was thermostable with high disproportionation and cyclization activities. The main large-ring cyclodextrin (CD) products were CD24-CD29, with CD26 as maximum at all incubation times. TfAM was used to modify cassava and pea starches, the amylose content decreased 18% and 30%, respectively, when 5% (w/v) starch was treated with 0·5 U TfAM g-1 starch. The increase in short branched chain (DP, degree of polymerization, 1-5) and the broader chain length distribution pattern which extended to the longer chain (DP40) after TfAM treatment were observed. The thermal property was changed, with an increase in retrogradation of starch as suggested by a lower enthalpy. Conclusions TfAM was successfully expressed in S. cerevisiae and was used to make starches with new functionality. Significance and impact of the study This is the first report on the expression of AM in the GRAS yeast and the production of a modified starch gel from pea starch to improve the versatility of starch for food use.

Levan-typed fructooligosaccharide (LFOS), a &beta, 2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.

LsRN from Bacillus licheniformis was cloned and expressed in Escherichia coli. From a 1793 bp genomic sequence, the lsRN gene was found to be composed of a single 1446 bp ORF with a putative promoter consensus boxes and a ribosome-binding site. This ORF was predicted to encode for 482 amino acid residues. The LsRN was constitutively expressed at a relatively high level without sucrose induction. The enzyme was highly purified and an apparent size of 52 kDa with an optimum temperature and pH of 50 °C and 6.0 were determined. The wide range of M(w) of levan (1-600 kDa) was synthesized in a controlled reaction with two variable parameters: temperature and ionic strength. At high temperature (50 °C), LsRN synthesized high M(w) levan (612 kDa) as a major product while at low temperature (30 °C), low M(w) levan (11 kDa) was mainly synthesized. When 0.5M NaCl was added into the reaction, the major products at both temperatures were of the size 11 kDa. Moreover we report for the first time, an enzymatic synthesis of levan nanoparticles (NPs) by a single step reaction. The LsRN synthesized levan NPs as agglomerate with average particle size of 50 nm. The encapsulation of O-acetyl-α-tocopherol was carried out to demonstrate the applicable use of levan NPs.