Cyclodextrin Glycosyltransferase Research Articles

Efficient glycosylation of polyphenols via dynamic complexation of cyclodextrin and synchronous coupling reaction with cyclodextrin glycosyltransferase in water

Glycosylation is an effective way to promote the total intake of polyphenols in humans by increasing the solubility of polyphenols. In this study, an efficient glycosylation system was built via the dynamic complexation of CD with polyphenols and synchronous coupling reaction with cyclodextrin glycosyltransferase (CGTase) in water. The glycosylation efficiencies of quercetin, naringenin, rutin, resveratrol and caffeic acid were 20.9, 3.6, 2.7, 3.4 and 1.5 times higher than the non-complexed system. To quantify conversion rate and determine the rate-limiting step, the mixed product was treated with amyloglucosidase to obtain α-glucosyl rutin, which was identified as rutin 4”-O-α-D-glucopyranoside with purity of 93.6 % and yield of 34.8 % from NMR, MS and HPLC analysis. The results of half-reaction kinetics showed that the catalytic efficiencies of ring-opening of γ-CD (k1) and glycosylation reaction of rutin (k2) were 621.92 and 9.43 mM−1·s−1. The rate-limiting step was clarified for the first time, showing that the ring-opening ability of CGTase to CD was much higher than its glycosylation ability to polyphenols. It is speculated that the rapid ring-opening reaction of CD affected its dynamic complexation, releasing many polyphenols which were not utilized by CGTase in time. Therefore, adjusting the ratio and concentration of CD resulted in an optimal glycosylation molar yield of 84.1 % for rutin, which was the highest yield reported so far in water. This study established a universal system and clarified the rate-limiting step in the enzymatic glycosylation, providing theoretical guidance for efficient production of polyphenol glycosylation.

Enhanced antimicrobial activity against oral bacteria Actinomyces viscous by cinnamaldehyde emulsion microencapsulated with cyclodextrin glycosyltransferase-catalyzed products

Actinomyces viscous (A. viscous) is well documented as a major cariogenic bacterium in the oral cavity and needs to be inhibited and removed timely. Essential oils (EOs) are recognized as secure antibacterial agents for treating oral diseases, but their volatility and insolubility limit their application. In this study, cinnamaldehyde was screened as the optimum EO for inhibiting the A. viscous growth by a micro-agar dilution method and microencapsulated by cyclodextrin glycosyltransferase (CGTase)-catalyzed products. The antibacterial effects against A. viscous were investigated and compared with the free cinnamaldehyde. Antibacterial diameter, antibacterial efficiency and stability, and time-kill curve results revealed that the cinnamaldehyde emulsion had better antibacterial properties. 1 MIC of the cinnamaldehyde emulsion had an inhibitory zone of 9.92 nm, a 100 % inhibition rate when acting for 2 min or 5 min, and still maintained the same inhibitory effect for 2 years. The extracellular environment showed more pH decrease, conductivity increase, and protein leakage, suggesting damage to the cell membrane. Microstructure and flow cytometric analysis further revealed that the CGTase-catalyzed products induced more changes in the A. viscous membrane integrity. Based on the results, CGTase-catalyzed products can be used as a potential substance for encapsulating EOs for treating oral bacteria.

Reactivities of 4-amino salicylic acid and its ‘Green’ inhibition into nano sized β-cyclodextrin for Anti-Tuberculosis drug delivery

In the present work, 4-amino salicylic acid (PAS) which is an oral antibiotic against tuberculosis is being investigated for its potential “Green” effectivity in trapped conditions within nano sized good solubilizing drug receptor. The process is totally “green” as no toxic materials, chemicals and solvent other than water has been used throughout the encapsulation /complexation process. Cyclodextrin glycosyltransferase (3CGT) and its by-product beta (β)-cyclodextrin (β-CD) usually act as nano sized good solubilizing agents for many drugs. In aqueous medium both 3CGT and β-CD can create drug: receptor complex by encapsulating the drug into its nano sized cavity and can increase the aqueous solubility of probe drug depending on the ionic strength. PAS was initially screened by its drug likeness properties by ADMET analysis. Structural analysis and chemical reactivity of PAS have been checked by density functional theory with basis set B3LYP/6-311G++*(d,p) in aqueous environment. The electronic properties (atomic charges, electrostatic potential, and uv–visible absorption & fluorescence analysis) have been studied to check the possibility of 1:1 complex formation between PAS: β-CD. To understand the type and strength of interactions between PAS and receptor, molecular docking and molecular dynamics simulations have been performed between PAS and cyclodextrin glycosyltransferase (3GGT). PAS has shown strong interaction possibilities both as 1:1 inclusion complex (PAS: β-CD) and as PAS: 3CGT complex. Our in-silico and experimental outcomes have recommended 4-amino salicylic acid’s better efficacy as a promising inhibitor inside cyclodextrin nano-cavity for targeting tuberculosis.

Effects of cyclodextrin glycosyltransferase on physicochemical, digestion, and gel properties of corn and potato starches

The application of natural starches in the food industry is sometimes limited because of their high viscosity, strong susceptibility to retrogradation, and poor water retention capacity. In this study, cyclodextrin glycosyltransferase (CGTase) was used to modify corn starch (CS) and potato starch (PS) to investigate their physicochemical, digestion and gel properties. Rapid Visco-Analyzer (RVA) was used to determine the impact of this enzyme treatment on the gelatinization and retrogradation behavior of these two starches. The CGTase treatment reduced the viscosity and inhibited the short-term retrogradation of both starches. Enzymatic modification weakened the gel structure of corn starch but enhanced the gel structure of potato starch. The microstructure confirmed the above conclusion. The gel wall of CS became thinner, but more and smaller grids were generated, and the water holding capacity was enhanced after enzyme treatment, while the wall thickness of PS was not significantly changed, and the water holding capacity was relatively good on the whole. Compared with native starch, the enzyme modified starch exhibited a similar digestibility but the content of rapidly digestible starch was slightly higher. The crystal structure of starches was disrupted by the enzyme treatment, but that the covalent bonds within starches were not affected. Starch gels formed by CGTase treatment could be successfully used as edible inks in 3D food printing applications, which may be useful for the creation of personalized foods.

Use of ultrasound to improve the activity of cyclodextrin glycosyltransferase in the producing of β-cyclodextrins: Impact on enzyme activity, stability and insights into changes on enzyme macrostructure

This work explored the impact of ultrasound (US) on the activity, stability, and macrostructural conformation of cyclodextrin glycosyltransferase (CGTase) and how these changes could maximize the production of β-cyclodextrins (β-CDs). The results showed that ultrasonic pretreatment (20 kHz and 38 W/L) at pH 6.0 promoted increased enzymatic activity. Specifically, after sonication at 25 °C/30 min, there was a maximum activity increase of 93 % and 68 % when biocatalysis was carried out at 25 and 55 °C, respectively. For activity measured at 80 °C, maximum increase (31 %) was observed after sonication at 25 °C/60 min. Comparatively, US pretreatment at low pH (pH = 4.0) resulted in a lower activity increase (max. 28 %). These activation levels were maintained after 24 h of storage at 8 °C, suggesting that changes on CGTase after ultrasonic pretreatment were not transitory. These pretreatments altered the conformational structure of CGTase, revealed by an up to 11 % increase in intrinsic fluorescence intensity, and resulted in macrostructural modifications, such as a decrease in particle size and polydispersion index (up to 85 % and 45.8 %, respectively). Therefore, the sonication of CGTase under specific conditions of pH, time, and temperature (especially at pH 6.0/ 30 min/ 25 °C) promotes macrostructural changes in CGTase that induce enzyme activation and, consequently, higher production of β-CDs.

Kinetics guided engineering of cyclodextrin glycosyltransferase with enhanced intermolecular transglycosylation activity

AbstractCyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization‐coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization‐coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the −3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild‐type. Kinetic analysis reveals a fourfold improvement of kcat/KmA for disproportionation and an 85.5% reduction in kcat/Km for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin.

Isolation of β-Cyclodextrin Gycosyl Transferase (β-CGTase) Producing Bacteria from Potato Plantation Soil

Cyclodextrin glycosyltransferase (CGTase) is an extracellular enzyme produced by several microorganisms, particularly bacteria, that converts starch into cyclodextrin (CD). CD has various applications in fields such as food, textiles, pharmaceuticals, chemistry, and biotechnology. The objective of this study was to isolate β-CGTase-producing bacteria from soil in a potato plantation located in Kerinci, and to conduct their characterization. Screening was conducted using Horikoshi agar media with phenolphthalein and methyl orange color indicators. The results indicated that one isolate, coded CK-2, produced the optimum β-CGTase enzyme when incubated for 30 hours at 37 ºC and pH 10. The CGTase enzyme converted starch to CD when incubated for 60 minutes at 37 ºC and pH 7. The CK-2 isolate was identified as monobacilli Gram-positive bacteria, having spores, positive in the catalase test, and motile. The study concluded that the bacteria producing β-CGTase enzyme are found in the soil of potato plantations in Kerinci, which is believed to originate from the genus Bacillus sp.

Improving cyclodextrin yield via retarding retrogradation of debranched starches during the preparation of cyclodextrin with cyclodextrin glycosyltransferase and pullulanase

Improving substrate utilization is crucial for industrial conversion of starch. The introduction of pullulanase in cyclodextrin (CD) production has improved substrate utilization. However, complete utilization of debranched starches has not been achieved, mainly due to the retrogradation of debranched starches and the inhibitory effect of CDs on pullulanase during the catalytic reaction. This study aimed to establish an efficient CD preparation system by regulating the substrate concentration and reaction temperature. A Bacillus thermoleovorans pullulanase (BtPul) was selected for investigation of its interaction with CDs through inhibition pattern assays, fluorescence spectroscopy, and molecular docking. Results clarified that CDs inhibited BtPul, with α-CD showing the highest inhibition, followed by β-CD and γ-CD. γ-CD inhibited BtPul through a mixed mode of competitive and noncompetitive inhibition, while the other CDs inhibited BtPul competitively. When pullulan was used as the substrate, increasing the substrate concentration weakened the inhibitory effect of CDs to some degree, while changes in reaction temperature only had a minor contribution. When BtPul and β-CGTase collaboratively converted 30% (w/v) starch substrate, an increase in reaction temperature improved the conversion rate of β-CD from 67.6% to 86.8%. Optimization of substrate concentration showed that high concentration starch substrate had an adverse effect on CDs yield due to retrogradation. The optimal yields of α-CD and β-CD produced by BtPul and α-/β-CGTases were 55.7% and 90.3%, respectively. This study provides a reliable method and reference for the efficient conversion of starch into CDs.

Enhancement of β-Cyclodextrin Production Using a Glycogen Debranching Enzyme from Saccharolobus solfataricus STB09.

Efficient production of cyclodextrins (CDs) has always been challenging. CDs are primarily produced from starch via cyclodextrin glycosyltransferase (CGTase), which acts on α-1,4 glucosidic bonds; however, α-1,6 glucosidic bonds in starch suppress the enzymatic production of CDs. In this study, a glycogen debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE) was utilized to promote the production of β-CD by hydrolyzing α-1,6 glucosidic bonds. The addition of SsGDE (750 U/g of starch) at the liquefaction stage remarkably improved the β-CD yield, with a 43.9% increase. Further mechanism exploration revealed that SsGDE addition could hydrolyze specific branches with less generation of byproducts, thereby promoting CD production. The chain segments of a degree of polymerization ≥13 produced by SsGDE debranching could also be utilized by β-CGTase to convert into CDs. Overall, these findings proposed a new approach of combining SsGDE with β-CGTase to enhance the CD yield.

Immobilization of Cyclodextrin glycosyltransferase onto three dimensional- hydrophobic and two dimensional- hydrophilic supports: A comparative study.

Cyclodextrin glycosyltransferase (CGTase) degrades starch into cyclodextrin via enzymatic activity. In this study, we immobilize CGTase from Thermoanaerobacter sp. on two supports, namely graphene nanoplatelets (GNP) consisting of short stacks of graphene nanoparticles and a calcium-based two-dimensional metal organic framework (Ca-TMA). The uptakes of CGTase on GNP and Ca-TMA reached 40 and 21mg g-1 respectively, but immobilized CGTase on Ca-TMA showed a higher specific activity (38 U mg-1 ) than that on GNP (28 U mg-1 ). Analysis of secondary structures of CGTase, shows that immobilization reduces the proportion of β-sheets in CGTase from 56% in the free to 49% and 51.3% for GNP and Ca-TMA respectively, α-helix from 38.5% to 18.1 and 37.5%, but led to increased β-turns from 5.5 to 40% and 11.2% for GNP and Ca-TMA, respectively. Lower levels of conformational changes were observed over the more hydrophilic Ca-TMA compared to hydrophobic GNP, resulting in its better activity. Increased β-turns were found to correlate with lower β-CD production, while more β-sheets and α-helix favored more β-CD. Reusability studies revealed that GNP retains up to 74% of initial CGTase activity, while Ca-TMA dropped to 33% after eight consecutive uses. The results obtained in this work provide insight on the effect of support's surface properties on CGTase performance and can assist in developing robust CGTase-based biocatalysts for industrial application.

Surface engineering of cyclodextrin glycosyltransferase reveals structural compactness and rigidity responsible for enhanced organic solvents resistance

Cyclodextrin glycosyltransferase (CGTase) is a preferable biocatalyst for production of cyclodextrin and phenols glycosylated derivatives due to its cost-effective donors and broad range of acceptors. Organic solvents (OSs) are often beneficial for solubilizing hydrophobic substrates and product specificity, but often harmful towards CGTases. Herein, using Paenibacillus macerans CGTase (PmCGTase) as a template and DMSO as a screening solvent, semi-rational engineering was performed at 18 surface residues by a developed high throughput screening method. Using site saturation mutagenesis and iterative saturation mutagenesis, 5 OSs-resistant variants (G539I, G539V, G539I/R146F, G539V/R146A, G539I/R146F/D147N) were obtained. Compared with WT, the best variant G539I/R146F/D147N showed a 79.3 % enhanced residual activity at 30 % DMSO and a 60 % increased t1/2 value at both 40 and 50 °C. In addition, a decreased Km value and an increased kcat value led to 41.9 % higher catalytic efficiency of G539I/R146F/D147N than WT in 15 % DMSO. This variant was also applied to glycosylation of sophoricoside in 20 % DMSO, leading to 26.2 % increased conversion. Molecular dynamics demonstrates that its enhanced OSs resistance may be attributed to the strengthened structural compactness and rigidity. Our results provide guidance for engineering OSs resistance of PmCGTase, which would further improve its application potential.

Bioconversion of starch to Cyclodextrin using Cyclodextrin glycosyltransferase immobilized on metal organic framework

The superiority of a metal organic framework, namely, MIL-101 (Cr) over conventional support, zeolite Y, as immobilization support for Cyclodextrin glycosyltransferase (CGTase) was investigated. The adsorption capacity, stability, and secondary structures of CGTase upon immobilization were investigated. CGTase adsorption on MIL-101 and zeolite Y was best represented by the Langmuir isotherm with optimum adsorption capacities of 37.5 and 6.1 mg/g on two supports, respectively. The deconvolution of the amide I band of the FTIR spectrum indicated that free CGTase molecules predominantly contain β-sheets, which increased with the immobilization on MIL-101 and zeolite Y from 56% to 84.1% and 69.7%, respectively, suggesting the existence of CGTase agglomerates on the supports. The CGTase immobilized on MIL-101 showed relative activity of about 50% at a substrate concentration of up to 4 g/L (compared with free enzymes) and above 3 times higher selectivity towards the more valuable α-CD. A diffusion–reaction model was used to predict the behavior of the immobilized system, and reusability studies revealed that immobilized CGTase may be utilized for numerous reaction cycles, with possible improvements via covalent attachment using glutaraldehyde. Overall, the obtained results in this study provide insights into the usefulness of MIL-101 as a suitable support for CGTase.

Economical synthesis of γ-cyclodextrin catalyzed by oriented cyclodextrin glycosyltransferase displayed on bacterial polyhydroxyalkanoate nanogranules

BackgroundThe advantages of γ-cyclodextrin (γ-CD) include its high solubility, ability to form inclusion complexes with various poorly water-soluble molecules, and favorable toxicological profile; thus, γ-CD is an attractive functional excipient widely used in many industrial settings. Unfortunately, the high cost of γ-CD caused by the low activity and stability of γ-cyclodextrin glycosyltransferase (γ-CGTase) has hampered large-scale production and application.ResultsThis study reports the in vivo one-step production of immobilized γ-CGTase decorated on the surface of polyhydroxyalkanoate (PHA) nanogranules by the N-terminal fusion of γ-CGTase to PHA synthase via a designed linker. The immobilized γ-CGTase-PHA nanogranules showed outstanding cyclization activity of 61.25 ± 3.94 U/mg (γ-CGTase protein) and hydrolysis activity of 36,273.99 ± 1892.49 U/mg, 44.74% and 18.83% higher than that of free γ-CGTase, respectively. The nanogranules also exhibited wider optimal pH (cyclization activity 7.0–9.0, hydrolysis activity 10.0–11.0) and temperature (55–60 °C) ranges and remarkable thermo- and pH-stability, expanding its utility to adapt to wider and more severe reaction conditions than the free enzyme. A high yield of CDs (22.73%) converted from starch and a high ratio (90.86%) of γ-CD in the catalysate were achieved at pH 9.0 and 50 °C for 10 h with 1 mmol/L K+, Ca2+, and Mg2+ added to the reaction system. Moreover, γ-CGTase-PHA beads can be used at least eight times, retaining 82.04% of its initial hydrolysis activity and 75.73% of its initial cyclization activity.ConclusionsThis study provides a promising nanobiocatalyst for the cost-efficient production of γ-CD, which could greatly facilitate process control and economize the production cost.

Engineering of cyclodextrin glycosyltransferase improves the conversion efficiency of rebaudioside A to glucosylated steviol glycosides and increases the content of short-chain glycosylated steviol glycoside

BackgroundCompared 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.ResultsHere, 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.ConclusionsThis 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.

Rational Design of Cyclodextrin Glycosyltransferase with Improved Hesperidin Glycosylation Activity

Cyclodextrin glycosyltransferase (CGTase) can catalyze the glycosylation of hesperidin, resulting in α-glycosyl hesperidin with significantly improved water solubility. In this study, a rational design of CGTase to improve its hesperidin glycosylation activity was investigated. The strategy we employed involved docking hesperidin in its near-attack conformation and virtually mutating the surrounding residues, followed by calculating the changes in binding energy using Rosetta flex-ddG. The mutations with a stabilization effect were then subjected to an activity assay. Starting from CGTase-Y217F, we obtained three double-point mutants, Y217F/M351F, Y217F/M351L, and Y217F/D393H, with improved hesperidin glycosylation activities after screening twenty variants. The best variant, Y217F/D393H, exhibited a catalytic activity of 1305 U/g, and its kcat/KmA is 2.36 times higher compared to CGTase-Y217F and 15.14 times higher compared to the wild-type CGTase. Molecular dynamic simulations indicated that hesperidin was repulsed by CGTase-Y217F when bound in a near-attack conformation. However, by introducing a second-point mutation with a stabilization effect, the repulsion effect is weakened, resulting in a reduction in the distances between the bond-forming atoms and, thus, favoring the reaction.

Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

Heterologous expression of cyclodextrin glycosyltransferase from Bacillus stearothermophilus in Bacillus subtilis and its application in glycosyl rutin production.

The online version contains supplementary material available at 10.1007/s13205-023-03510-5.

Aided-efflux and high production of β-amyrin realized by β-cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae.

As a plant-derived pentacyclic triterpenoid, β-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β-cyclodextrin (β-CD) has been previously employed to aid the efflux of β-amyrin out of the cells. Nevertheless, the supplemented β-CD in the medium is not consistent with β-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of β-CD was developed in this study to enhance the biosynthesis of β-amyrin and its efflux. The in situ synthesis of β-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β-CD could capture 16% of the intracellular β-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of β-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.

Glycosyltransferases improve breadmaking quality by altering multiscale structure in gluten-free bread

In gluten-free bread (GFB), starch and its interplay with structural agents were vital for product quality due to the absence of gluten. During breadmaking, amylolytic enzymes modified starch in situ , and their reaction modes determined starch structure and hence how interactions occurred. Herein, based on the matrices of rice protein and hydroxypropyl methylcellulose (HPMC), α-amylase and multifunctional enzymes of glucan branching enzyme (GBE) and cyclodextrin glycosyltransferases (CGTase) were adopted, aiming to understand the mechanism of these enzymes on breadmaking quality of fresh bread from the multiscale structure of GFB. Results demonstrated that all enzymes, especially CGTase and GBE, markedly increased GFB specific volume to 107%–134% of Control. Compared with α-amylase, CGTase and GBE more depended on the structural changes rather than fermentation to improve GFB volume. Considering the texture, CGTase and GBE reduced crumb stickiness and still maintained a relatively softness and springiness crumb, while α-amylase induced a stick and less elastic crumb. Starch molecular changes enhanced the entanglement of starch and HPMC to form extended and bold fibril bundles and finally improved strength and pore size of gel network structure. Moreover, the starch structure modifications and specific enzymatic products of CGTase and GBE altered the microstructure and thus facilitated the even distribution of rice protein, which further improved GFB texture properties. Amylolytic enzymes, that acted both hydrolysis and glycosyl transfer, moderately hydrolyzed starch and generated specific enzymatic products, were more suitable to enhance GFB structure and eliminate the weak crumb texture. • α-amylase (α-Amy) and glycosyltransferases (GTases) were included. • Breadmaking quality of gluten-free bread (GFB) with α-Amy and GTases was compared. • GFB with GTases performed better in crumb structure and texture. • GTases depended more on structure rather than gas to improve volume. • Interactions of enzymatic starch and structural agents was crucial for GFB quality.

Novel α-glycosyl compounds from glycosylation of rubusoside

In this work we investigated mixtures from α-glycosylation of rubusoside with cyclodextrin glycosyltransferases. In addition to the previously known α-1,4 glycosylated derivatives, nine new compounds with rare α-1,3-glycosidic bonds were identified based on nuclear magnetic resonance spectroscopy and mass spectrometric analysis. Furthermore, sensory properties of monoglycosylated rubusoside derivatives were investigated and compared to previously described monoglycosylated compounds. Additionally, digestion with α-amylase from human saliva was investigated for different glycosylated rubusoside derivatives.