Heterologous expression of 4α-glucanotransferase: overproduction and properties for industrial applications.

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.

A GH13 α-glucosidase from Weissella cibaria uncommonly acts on short-chain maltooligosaccharides.

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.

Levansucrase from Bacillus amyloliquefaciens KK9 and Its Y237S Variant Producing the High Bioactive Levan-Type Fructooligosaccharides.

Levan-typed fructooligosaccharide (LFOS), a ß-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.

An α-1,6-and α-1,3-linked glucan produced by Leuconostoc citreum ABK-1 alternansucrase with nanoparticle and film-forming properties.

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.

Identification of essential tryptophan in amylomaltase from Corynebacterium glutamicum.

This work aims to identify essential tryptophan residue(s) of amylomaltase from Corynebacterium glutamicum (CgAM) through chemical modification and site-directed mutagenesis techniques. The recombinant enzyme expressed by Escherichia coli was purified and treated with N-bromosuccinimide (NBS), a modifying agent for tryptophan. A significant decrease in enzyme activity was observed indicating that tryptophan is important for catalysis. Inactivation kinetics with NBS resulted in pseudo first-order rate constant (kinact) of 2.31 min(-1). Substrate protection experiment confirmed the active site localization of the NBS-modified tryptophan residue(s) in CgAM. Site-directed mutagenesis was performed on W330, W425 and W673 to localize essential tryptophan residues. Substitution by alanine resulted in the loss of intra- and intermolecular transglucosylation activities for all mutated CgAMs. Analysis of circular dichroism spectra showed no change in the secondary structure of W425A but a significant change for W330A and W673A from that of the WT. From these results in combination with X-ray structural data and interpretation from the binding interactions in the active site region, W425 was confirmed to be essential for catalytic activity of CgAM. The hydrophobicity of this tryptophan was thought to be critical for substrate binding and supporting catalytic action of the three carboxylate residues at the active site.

High expression level of levansucrase from Bacillus licheniformis RN-01 and synthesis of levan nanoparticles.

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.