Cyclodextrin glycosyltransferase (CGTase) is a significant extracellular enzyme with diverse functions. CGTase is widely used in production of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch via transglycosylation reaction. Recent discoveries of novel CGTases from different microorganisms have expanded its applications but natural CGTase have lower yield, leading to heterologous expression for increased production to meet various needs. Moreover, significant advancements in directed evolution approach have been explored to alter the molecular structure of CGTase to enhance its performance. This review comprehensively summarizes the strategies employed in heterologous expression to boost CGTase production and secretion in various host. It also outlines molecular engineering approaches aimed to improving CGTase properties, including product and substrate specificity, catalytic efficiency, and thermal stability. Additionally, a considerable stability against changes in temperature and organic solvents can be obtained by immobilization.

Isomaltomegalosaccharides with α-(1 → 4) and α-(1 → 6)-segments solubilize water-insoluble ligands since the former complexes with the ligand and the latter solubilizes the complex. Previously, we enzymatically synthesized isomaltomegalosaccharide with a single α-(1 → 4)-segment at the reducing end (S-IMS) by dextran dextrinase (DDase), but the chain length [average degree of polymerization (DP) ≤ 9] was insufficient for strong encapsulation. We hypothesized that the conjugation of longer α-(1 → 4)-segment afforded the promising function although DDase is incapable to do so. In this study, the cyclodextrin glucanotransferase-catalyzed coupling reaction of α-cyclodextrin to S-IMS synthesized a new α-(1 → 4)-segment at the nonreducing end (N-4S) of S-IMS to form D-IMS [IMS harboring double α-(1 → 4)-segments]. The length of N-4S was modulated by the ratio between α-cyclodextrin and S-IMS, generating N-4Ss with DPs of 7-50. Based on phase-solubility analysis, D-IMS-28.3/13/3 bearing amylose-like helical N-4S with DP of 28.3 displayed a water-soluble complex with aromatic drugs and curcumin. Small-angle X-ray scattering revealed the chain adapted to rigid in solution in which the radius of gyration was estimated to 2.4 nm. Furthermore, D-IMS with short N-4S solubilized flavonoids of less-soluble multifunctional substances. In our research, enzyme-generated functional biomaterials from DDase were developed to maximize the hydrophobic binding efficacy towards water-insoluble bioactive compounds.

Amylopectin clusters (APCs) are produced by cyclodextrin glucanotransferase (EC 2.4.1.19). Their solubility rate in aqueous solution was found to be 16.7 %. The weight-average molecular weight of APCs is ∼105 Da, as determined by multiangle laser light scattering analysis. Side chain length analysis indicated that the relative proportions of side chains with a degree of polymerization in the ranges of 2-8 and 25-50 decreased and increased, respectively, during preparation of APCs. In the exercise experiment, the blood glucose level of rats was higher in the APC-treated group than in the groups treated with commercial carbohydrate supplement (CCD) and glucose. In the forced swimming test, the swimming time in the APC and CCD groups increased by 22.6 % and 31.1 %, respectively, compared with the glucose administration group. The insulin levels were also similar between the APC and CCD groups. However, the glycogen levels in the liver and muscles of mice were significantly higher in the APC group than control group. These results suggest that APCs could potentially enhance endurance when added to sports drinks.

α-Monoglucosyl hesperidin is a promising food additive with various activities. However, there are a few reports about the production of α-monoglucosyl hesperidin. Here, to develop a practical and safe process for α-monoglucosyl hesperidin synthesis, we used nonpathogenic Bacillus subtilis as a host to express cyclodextrin glucanotransferase (CGTase) from Bacillus sp. A2-5a. The promoters and signal peptides were screened to optimize the transcription and secretion of CGTase in B. subtilis. The results of optimization showed that the best signal peptide and promoter were YdjM and PaprE, respectively. Finally, the enzyme activity increased to 46.5 U mL-1, 8.7 times that of the enzyme expressed from the strain containing pPHpaII-LipA, and the highest yield of α-monoglucosyl hesperidin was 2.70 g L-1 by enzymatic synthesis using the supernatant of the recombinant B. subtilis WB800 harboring the plasmid pPaprE-YdjM. This is the highest α-monoglucosyl hesperidin production level using recombinant CGTase to date. This work provides a generally applicable method for the scaled-up production of α-monoglucosyl hesperidin. KEY POINTS: • A three-step procedure was created for high throughput signal peptide screening. • YdjM and PaprE were screened from 173 signal peptides and 13 promoters. • α-Monoglucosyl hesperidin was synthesized by CGTase with a yield of 2.70 g L-1.

BACKGROUND: Compared with steviol glycosides, the taste of glucosylated steviol glycosides is better and more similar to that of sucrose. At present, cyclodextrin glucanotransferase (CGTase) is primarily used to catalyze the conversion of steviol glycosides to glucosylated steviol glycosides, with soluble starch serving as a glycosyl donor. The main disadvantages of enzymatic transglycosylation are the limited number of enzymes available, the low conversion rates that result in low yields, and the lack of selectivity in the degree of glycosylation of the products. In order to fill these gaps, the proteome of Alkalihalobacillus oshimensis (also named Bacillus oshimensis) was used for mining novel CGTases.
RESULTS: Here, CGTase-15, a novel β-CGTase with a wide pH adaptation range, was identified and characterized. The catalyzed product of CGTase-15 tasted better than that of the commercial enzyme (Toruzyme® 3.0 L). In addition, two amino acid sites, Y199 and G265, which play important roles in the conversion of steviol glycosides to glucosylated steviol glycosides were identified by site-directed mutagenesis. Compared with CGTase-15, CGTase-15-Y199F mutant significantly increased the conversion rate of rebaudioside A (RA) to glucosylated steviol glycosides. Compared with CGTase-15, the content of short-chain glycosylated steviol glycosides catalyzed by CGTase-15-G265A mutant was significantly increased. Moreover, the function of Y199 and G265 was verified in other CGTases. The above mutation pattern has also been applied to CGTase-13 (a CGTase discovered by our laboratory with great potential in the production of glycosylated steviol glycosides), confirming that the catalytic product of CGTase-13-Y189F/G255A mutant has a better taste than that of CGTase-13.
CONCLUSIONS: This is the first report on the improvement of the sensory profiles of glycosylated steviol glycosides through site-directed mutagenesis of CGTase, which is significant for the production of glycosylated steviol glycosides.

Baicalin is a biologically active flavone glucuronide with poor water solubility that can be enhanced via glucosylation. In this study, the transglucosylation of baicalin was successfully achieved with CGTases from Thermoanaerobacter sp. and Bacillus macerans using α-cyclodextrin as a glucosyl donor. The synthesis of baicalin glucosides was optimized with CGTase from Thermoanaerobacter sp. Enzymatically modified baicalin derivatives were α-glucosylated with 1 to 17 glucose moieties. The two main glucosides were identified as Baicalein-7-O-α-D-Glucuronidyl-(1→4\')-O-α-D-Glucopyranoside (BG1) and Baicalein-7-O-α-D-Glucuronidyl-(1→4\')-O-α-D-Maltoside (BG2), thereby confirming recent findings reporting that glucuronyl groups are acceptors of this CGTase. Optimized conditions allowed for the attainment of yields above 85% (with a total glucoside content higher than 30 mM). BG1 and BG2 were purified via centrifugal partition chromatography after an enrichment through deglucosylation with amyloglucosidase. Transglucosylation increased the water solubility of BG1 by a factor of 188 in comparison to that of baicalin (molar concentrations), while the same value for BG2 was increased by a factor of 320. Finally, BG1 and BG2 were evaluated using antioxidant and anti-glycation assays. Both glucosides presented antioxidant and anti-glycation properties in the same order of magnitude as that of baicalin, thereby indicating their potential biological activity.

To improve the applicability of quercetin (QCT), we produced a QCT and cycloamylose (CA-QCT) inclusion complex based on the cyclization activity of cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19). The encapsulated QCT was purified using recycling preparative high-performance liquid chromatography, and its formation was analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The water solubility of CA-QCT was 55,000-fold higher than that of QCT. CA-QCT had 97 % stability for one week at pH 8 in a 4 °C water bath. According to a 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay, CA-QCT activity in aqueous solution was 24 times higher than that of an equal amount of QCT in aqueous solution. In an anti-inflammatory assay using lipopolysaccharide-induced RAW264.7 macrophages, CA-QCT in aqueous solution decreased nitric oxide production in a similar manner to QCT in dimethyl sulfoxide (DMSO). Additionally, even under aqueous conditions, CA-QCT more effectively inhibited the production of inflammatory mediators, such as interleukin-1β, interleukin-6, and cyclooxygenase, compared with QCT dissolved in DMSO.

Understanding the mechanisms of enzyme specificity is increasingly important from a fundamental viewpoint and for practical applications. Transglycosylation has attracted many attentions due to its importance in improving the functional properties of acceptor substrates both in vivo and in vitro. Cyclodextrin glucanotransferase (CGTase) is one of the key enzymes in transglycosylation, it has a broad substrate spectrum and utilizes sugar as the donor. However, little is known about the acceptor selectivity of CGTase, which greatly hampers efforts toward the rational design of desirable transglycosylated derivatives. In this study, we found that the CGTase from Bacillus circulans, BcCGTase, was able to form glycosylated products with diverse ginsenosides. In particular, it not only carries out diverse mono-, di-, and even higher-order glycosylations via the transfer of glucose moieties to the COGlc positions, but also can glycosylate the C3-OH position of ginsenosides. In contrast, another CGTase from Bacillus licheniformis (BlCGTase) showed relatively specific acceptor preference with only several ginsenosides. Structural comparison between BcCGTase and BlCGTase revealed that the Arg74/K81 position within the acceptor-binding sites of BcCGTase/BlCGTase was responsible for the differences in catalytic specificity for ginsenoside F1. Further mutagenesis confirmed their roles in the acceptor selection. In conclusion, our study not only demonstrates the acceptor selectivity of CGTases, but also provides insight into the catalytic mechanism of CGTases, which will potentially increase the utility of CGTase for biosynthesis of new, rationally designed transglycosylated derivatives.

Maltoheptaose (G7) is one of the mixtures of maltodextrin widely used in the food, pharmaceutical, and cosmetics industries. A genetically engineered strain, which simultaneously expressed cyclodextrin glucanotransferase (CGTase) from Gracilibacillus alcaliphilus SK51.001 and cyclomaltodextrinase (CDase) from Bacillus sphaericus E-244, two enzymes, was constructed by cloning the above two genes into a plasmid and transformed into the host Escherichia coli BL21(DE3) (E. coli) strain, resulted in recombinant cells harboring the vector pETDuet-GaCGT/BsCD (pGaBs). These cells were used as whole-cell catalysts for the biotransformation of G7 from the inexpensive substrate (starch). Due to the high molecular weight of starch, the cell membrane prevents the entry of starch into the cellular system. Therefore, the pGaBs cell wall was permeabilized by lysozyme, EDTA, and heat treatment. After reaching the optimized conditions of permeabilized pGaBs cell amount, lysozyme amount, reaction temperature, and metal ion concentration, approximately 4.1 g/L of G7 was produced from 30 g/L starch in 1 h with the addition of Ca2+. This co-expression system offers a one-pot synthesis approach to the production of G7 using an inexpensive substrate, avoiding enzyme purification steps.

The use of unmodified starch in frozen foods can cause extremely undesirable textural changes after the freeze-thaw process. In this study, using cyclodextrin glucanotransferase (CGTase) and branching enzymes, an amylopectin cluster with high freeze-thaw stability was produced, and was named CBAC. It was found to have a water solubility seven times higher, and a molecular weight 77 times lower, than corn starch. According to the results of a differential scanning calorimetry (DSC) analysis, dough containing 5% CBAC lost 19% less water than a control dough after three freeze-thaw cycles. During storage for 7 days at 4 °C, bread produced using CBAC-treated dough exhibited a 14% smaller retrogradation peak and 37% less hardness than a control dough, suggesting that CBAC could be a potential candidate for clean label starch, providing high-level food stability under repeated freeze-thaw conditions.