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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.

4α-Glucanotransferase (4α-GTase) is unique in its ability to form cyclic oligosaccharides, some of which are of industrial importance. Generally, low amount of enzymes is produced by or isolated from their natural sources: animals, plants, and microorganisms. Heterologous expressions of these enzymes, in an attempt to increase their production for applicable uses, have been widely studied since 1980s; however, the expressions are mostly performed in the prokaryotic bacteria, mostly Escherichia coli. Site-directed mutagenesis has added more value to these expressed enzymes to display the desired properties beneficial for their applications. The search for further suitable properties for food application leads to an extended research in expression by another group of host organism, the generally-recognized as safe host including the Bacillus and the eukaryotic yeast systems. Herein, our review focuses on two types of 4α-GTase: the cyclodextrin glycosyltransferase and amylomaltase. The updated studies on the general structure and properties of the two enzymes with emphasis on heterologous expression, mutagenesis for property improvement, and their industrial applications are provided.

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.

Alternansucrase catalyses the sequential transfer of glucose residues from sucrose onto another sucrose molecule to form a long chain polymer, known as “alternan”. The alternansucrase-encoding gene from Leuconostoc citreum ABK-1 (Lcalt) was successfully cloned and expressed in Escherichia coli. Lcalt encoded LcALT of 2,057 amino acid residues; the enzyme possessed an optimum temperature and pH of 40 °C and 5.0, respectively, and its’ activity was stimulated up to 2.4-fold by the presence of Mn2+. Kinetic studies of LcALT showed a high transglycosylation activity, with Km 32.2 ± 3.2 mM and kcat 290 ± 12 s−1. Alternan generated by LcALT (Lc-alternan) harbours partially alternating α-1,6 and α- 1,3 glycosidic linkages confirmed by NMR spectroscopy, methylation analysis, and partial hydrolysis of Lc-alternan products. In contrast to previously reported alternans, Lc-alternan can undergo self-assembly, forming nanoparticles with an average size of 90 nm in solution. At concentrations above 15% (w/v), Lc-alternan nanoparticles disassemble and form a high viscosity solution, while this polymer forms a transparent film once dried.