Efficient biotransformation of naringenin to naringenin α-glucoside, a novel α-glucosidase inhibitor, by amylosucrase from Deinococcus wulumuquiensis.

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1-16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The kcat/Km value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin.

Characterization of a unique pH-dependent amylosucrase from Deinococcus cellulosilyticus.

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health.

Enzymatic Synthesis of α-Glucan Microparticles Using Amylosucrases from Bifidobacterium Species and Its Physicochemical Properties.

α-Glucan microparticles (GMPs) have significant potential as high-value biomaterials in various industries. This study proposes a bottom-up approach for producing GMPs using four amylosucrases from Bifidobacterium sp. (BASs). The physicochemical characteristics of these GMPs were analyzed, and the results showed that the properties of the GMPs varied depending on the type of enzymes used in their synthesis. As common properties, all GMPs exhibited typical B-type crystal patterns and poor colloidal dispersion stability. Interestingly, differences in the physicochemical properties of GMPs were generated depending on the synthesis rate of linear α-glucan by the enzymes and the degree of polymerization (DP) distribution. Consequently, we found differences in the properties of GMPs depending on the DP distribution of linear glucans prepared with four BASs. Furthermore, we suggest that precise control of the type and characteristics of the enzymes provides the possibility of producing GMPs with tailored physicochemical properties for various industrial applications.

Correction to: Versatile biotechnological applications of amylosucrase, a novel glucosyltransferase.

[This corrects the article DOI: 10.1007/s10068-019-00686-6.].

Whole-cell bioconversion using non-Leloir transglycosylation reactions: a review.

Microbial biocatalysts are evolving technological tools for glycosylation research in food, feed and pharmaceuticals. Advances in bioengineered Leloir and non-Leloir carbohydrate-active enzymes allow for whole-cell biocatalysts to curtail production costs of purified enzymes while enhancing glucan synthesis through continued enzyme expression. Unlike sugar nucleotide-dependent Leloir glycosyltransferases, non-Leloir enzymes require inexpensive sugar donors and can be designed to match the high value, yield and selectivity of the former. This review addresses the current state of bacterial cell-based production of glucans and glycoconjugates via transglycosylation, and describes how alterations made to microbial hosts to surpass purified enzymes as the preferred mode of catalysis are steadily being acquired through genetic engineering, rational design and process optimization. A comprehensive exploration of relevant literature has been summarized to describe whole-cell biocatalysis in non-Leloir glycosylation reactions with various donors and acceptors, and the characterization, application and latest developments in the optimization of their use.

Modulation of gut microbiota by rice starch enzymatically modified using amylosucrase from Deinococcus geothermalis.

Amylosucrase can increase the amount of resistant starch (RS) in starch by transferring glucose from sucrose to amylopectin. Here, rice starch was modified using amylosucrase from Deinococcus geothermalis (DgAS). DgAS-modified rice starch (DMRS) increased the side-chain length of amylopectin and appeared in the form of B-type crystals. In vitro digestion analyses revealed that DMRS had a higher RS contents and lower digestion rate than native rice starch. When high-fat diet (HFD)-induced C57BL/6 mice were orally administered DMRS, body weight and white fat tissues of DMRS-fed HFD mice were not significantly different. However, serum leptin and glucose levels were significantly decreased and serum glucagon like peptide-1was increased in these mice. The cecal microbiome in DMRS-fed HFD mice was identified to investigate the role of DMRS in gut microbiota regulation. DMRS supplementation increased the relative abundance of Bacteroides, Faecalibaculum, and Ruminococcus in mouse gut microbiota. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01238-1.

Acceptor dependent catalytic properties of GH57 4-α-glucanotransferase from Pyrococcus sp. ST04.

The 4-α-glucanotransferase (4-α-GTase or amylomaltase) is an essential enzyme in maltodextrin metabolism. Generally, most bacterial 4-α-GTase is classified into glycoside hydrolase (GH) family 77. However, hyperthermophiles have unique 4-α-GTases belonging to GH family 57. These enzymes are the main amylolytic protein in hyperthermophiles, but their mode of action in maltooligosaccharide utilization is poorly understood. In the present study, we investigated the catalytic properties of 4-α-GTase from the hyperthermophile Pyrococcus sp. ST04 (PSGT) in the presence of maltooligosaccharides of various lengths. Unlike 4-α-GTases in GH family 77, GH family 57 PSGT produced maltotriose in the early stage of reaction and preferred maltose and maltotriose over glucose as the acceptor. The kinetic analysis showed that maltotriose had the lowest KM value, which increased amylose degradation activity by 18.3-fold. Structural models of PSGT based on molecular dynamic simulation revealed two aromatic amino acids interacting with the substrate at the +2 and +3 binding sites, and the mutational study demonstrated they play a critical role in maltotriose binding. These results clarify the mode of action in carbohydrate utilization and explain acceptor binding mechanism of GH57 family 4-α-GTases in hyperthermophilic archaea.

Different physicochemical properties of entirely α-glucan-coated starch from various botanical sources.

Amylosucrase from Neisseria polysaccharea (NpAS) synthesizes α-1,4 glucan polymer from sucrose. In this study, we coated various botanical sources of raw starch with an α-glucan layer generated by NpAS to improve physicochemical properties. Field-emission scanning electron microscopy demonstrated that all surfaces of the starch granules were successfully coated via the NpAS reaction. X-ray diffraction analysis revealed that the crystallinity decreased and the crystal pattern changed to C-type as an amylose layer formed around the surface of the starch granules. Based on rapid viscosity and differential scanning calorimetry analyses, the gelatinization resistance of the α-glucan-coated starch increased owing to decreased viscosity and increased melting temperature. Therefore, the α-glucans coated the starches by enzymatic reactions of various botanical sources; these have applicability in the food and starch industries owing to various physicochemical properties such as enhanced thermostability. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01113-z.

Human gut commensal bacterium Ruminococcus species FMB-CY1 completely degrades the granules of resistant starch.

Resistant starch (RS) in the diet reaches the large intestine and is fermented by the gut microbiota, providing beneficial effects on human health. The human gut bacterium FMB-CY1 was isolated and identified as a new species closest to Ruminococcus bromii. Ruminococcus sp. FMB-CY1 completely degraded RS including commercial RS types 2, 3, and 4, and generated glucose and maltose; however, it did not assimilate glucose. Genome analysis revealed 15 amylolytic enzymes (Amy) present in FMB-CY1. The evolutionary trees revealed that the Amys were well divided each other. All Amys (4, 9, 10, 12, and 16) containing cohesin and/or dockerin and scaffolding proteins known to be involved in constituting the amylosome, were identified. A new species of Ruminococcus, strain FMB-CY1, was considered to have the ability to form amylosomes for the degradation of RS. This new RS-degrading Ruminococcus species provides insights into the mechanism(s) underlying RS degradation in the human gut. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-01027-2.

Enzymatic analysis of truncation mutants of a type II pullulanase from Bifidobacterium adolescentis P2P3, a resistant starch-degrading gut bacterium.

A putative type II pullulanase gene, pulP, was identified in Bifidobacterium adolescentis P2P3. PulP possesses an α-amylase domain at the N-terminus and a pullulanase type I domain at the C-terminus, as well as three carbohydrate-binding modules (one CBM25 and two CBM41s) between them. The native PulP and four truncated mutant recombinant proteins (PulPΔCΔP, PulPΔP, PulPΔAΔC, and PulPΔA), in which each of the two catalytic domains and/or the CBMs were deleted, were produced in Escherichia coli and their specific properties were characterized. The removal of either catalytic domain abolished the corresponding catalytic activity of the wild-type enzyme. Deletion of the C-terminal domain resulted in a drastic decrease in the optimal temperature and thermostability, indicating that the pullulanase domain might be related to the temperature dependency of the enzyme. In addition, the elimination of the CBMs in the mutant proteins led to a loss of binding affinity toward raw substrates as well as the loss of their hydrolysis activities compared to the wild-type enzyme. HPAEC and TLC analyses proved that PulP and its mutants could hydrolyze α-glucans into maltotriose as their main product. These results suggest that PulP may play an important role in α-glucan metabolism in B. adolescentis P2P3.

Biosynthesis of glyceride glycoside (nonionic surfactant) by amylosucrase, a powerful glycosyltransferase.

Amylosucrase (ASase, E.C. 2.4.1.4) is a powerful transglycosylation enzyme that can transfer glucose from sucrose to the hydroxyl (-OH) group of various compounds. In this study, recombinant ASases from Deinococcus geothermalis (DgAS) and Bifidobacterium thermophilum (BtAS) were used to synthesize biosurfactants based on the computational analysis of predicted docking simulations. Successful predictions of the binding affinities, conformations, and three-dimensional structures of three surfactants were computed from receptor-ligand binding modes. DgAS and BtAS were effective in the synthesis of biosurfactants from glyceryl caprylate, glyceryl caprate, and polyglyceryl-2 caprate. The results of the transglycosylation reaction were consistent for both ASases, with glyceryl caprylate acceptor showing the highest concentration, as confirmed by thin layer chromatography. Furthermore, the transglycosylation reactions of DgAS were more effective than those of BtAS. Among the three substrates, glyceryl caprylate glycoside and glyceryl caprate glycoside were successfully purified by liquid chromatography-mass spectrometry (LC-MS) with the corresponding molecular weights.

Genome analysis of 1-deoxynojirimycin (1-DNJ)-producing Bacillus velezensis K26 and distribution of Bacillus sp. harboring a 1-DNJ biosynthetic gene cluster.

1-Deoxynojirumycin (1-DNJ) is a representative iminosugar with α-glucosidase inhibition (AGI) activity. In this study, the full genome sequencing of 1-DNJ-producing Bacillus velezensis K26 was performed. The genome consists of a circular chromosome (4,047,350 bps) with two types of putative virulence factors, five antibiotic resistance genes, and seven secondary metabolite biosynthetic gene clusters. Genomic analysis of a wide range of Bacillus species revealed that a 1-DNJ biosynthetic gene cluster was commonly present in four Bacillus species (B. velezensis, B. pseudomycoides, B. amyloliquefaciens, and B. atrophaeus). In vitro experiments revealed that the increased mRNA expression levels of the three 1-DNJ biosynthetic genes were closely related to increased AGI activity. Genomic comparison and alignment of multiple gene sequences indicated the conservation of the 1-DNJ biosynthetic gene cluster in each Bacillus species. This genomic analysis of Bacillus species having a 1-DNJ biosynthetic gene cluster could provide a basis for further research on 1-DNJ-producing bacteria.

4,4'-Diaponeurosporene from Lactobacillus plantarum subsp. plantarum KCCP11226: Low Temperature Stress-Induced Production Enhancement and In Vitro Antioxidant Activity.

Carotenoids, which have biologically beneficial effects and occur naturally in microorganisms and plants, are pigments widely applied in the food, cosmetics and pharmaceutical industries. The compound 4,4'-diaponeurosporene is a C30 carotenoid produced by some Lactobacillus species, and Lactobacillus plantarum is the main species producing it. In this study, the antioxidant activity of 4,4'-diaponeurosporene extracted from L. plantarum subsp. plantarum KCCP11226 was examined. Maximum carotenoid content (0.74 ± 0.2 at A470) was obtained at a relatively low temperature (20°C). The DPPH radical scavenging ability of 4,4'-diaponeurosporene (1 mM) was approximately 1.7-fold higher than that of butylated hydroxytoluene (BHT), a well-known antioxidant food additive. In addition, the ABTS radical scavenging ability was shown to be 2.3- to 7.5-fold higher than that of BHT at the range of concentration from 0.25 mM to 1 mM. The FRAP analysis confirmed that 4,4'- diaponeurosporene (0.25 mM) was able to reduce Fe3+ by 8.0-fold higher than that of BHT. Meanwhile, 4,4'-diaponeurosporene has been confirmed to be highly resistant to various external stresses (acid/bile, high temperature, and lysozyme conditions). In conclusion, L. plantarum subsp. plantarum KCCP11226, which produces 4,4'-diaponeurosporene as a functional antioxidant, may be a potentially useful strain for the development of functional probiotic industries.

Molecular Docking and Kinetic Studies of the A226N Mutant of Deinococcus geothermalis Amylosucrase with Enhanced Transglucosylation Activity.

Amylosucrase (ASase, E.C. 2.4.1.4) is capable of efficient glucose transfer from sucrose, acting as the sole donor molecule, to various functional acceptor compounds, such as polyphenols and flavonoids. An ASase variant from Deinococcus geothermalis, in which the 226th alanine is replaced with asparagine (DgAS-A226N), shows increased polymerization activity due to changes in the flexibility of the loop near the active site. In this study, we further investigated how the mutation modulates the enzymatic activity of DgAS using molecular dynamics and docking simulations to evaluate interactions between the enzyme and phenolic compounds. The computational analysis revealed that the A226N mutation could induce and stabilize structural changes near the substratebinding site to increase glucose transfer efficiency to phenolic compounds. Kinetic parameters of DgAS-A226N and WT DgAS were determined with sucrose and 4-methylumbelliferone (MU) as donor and acceptor molecules, respectively. The kcat/Km value of DgAS-A226N with MU (6.352 mM-1min-1) was significantly higher than that of DgAS (5.296 mM-1min-1). The enzymatic activity was tested with a small phenolic compound, hydroquinone, and there was a 1.4-fold increase in α-arbutin production. From the results of the study, it was concluded that DgAS-A226N has improved acceptor specificity toward small phenolic compounds by way of stabilizing the active conformation of these compounds.

The presence of resistant starch-degrading amylases in Bifidobacterium adolescentis of the human gut.

Resistant starch (RS) is a complex prebiotic carbohydrate beneficial to the human gut. In the present study, four genes encoding for putative amylolytic enzymes, likely to be responsible for RS-degradation, were identified in the genome of Bifidobacterium adolescentis P2P3 by comparative genomic analysis. Our results showed that only three enzymes (RSD1, RSD2, and RSD3) exhibited non-gelatinized high amylose corn starch (HACS)-degrading activity in addition to typical α-amylase activity. These three RS-degrading enzymes (RSD) were composed of multiple domains, including signal peptide, catalytic domain, carbohydrate binding domains, and putative cell wall-anchoring domains. Typical catalytic domains were conserved by exhibiting seven typical conserved regions (I-VII) found mostly in α-amylases. Analysis of enzymatic activity revealed that RSD2 displayed stronger activity toward HACS-granules than RSD1 and RSD3. Comparative genomics in combination with enzymatic experiments confirmed that RSDs might be the key enzymes used by RS-degrading bifidobacteria to degrade RS in a particular ecological niche, such as the human gut.

Starch nanoparticles prepared by enzymatic hydrolysis and self-assembly of short-chain glucans.

Enzymatic hydrolysis and self-assembly are considered promising methods for preparation of starch nanoparticles (SNPs) because they are environmentally friendly, and time- and cost-effective. These methods are based on the self-assembly of short-chain glucans released from the α-1,6 bonds in amylopectin. Since their discovery, many studies have described the structural and physicochemical properties of self-assembled SNPs. Self-assembled SNPs can be prepared by two methods: using only the soluble portion containing the short-chain glucans, or using the whole hydrolyzate including both insoluble and soluble fractions. Although the structural and physical properties of self-assembled SNPs can be attributed to the composition of the hydrolyzates that participate in self-assembly, this aspect has not yet been discussed. This review focuses on SNPs self-assembled with only soluble short-chain glucans and addresses their characteristics, including formation mechanisms as well as structural and physicochemical properties, compared with SNPs prepared with total hydrolyzates.

Genome analysis of Lactobacillus plantarum subsp. plantarum KCCP11226 reveals a well-conserved C30 carotenoid biosynthetic pathway.

Carotenoids are group of colored terpenoids with antioxidant properties and widespread in nature including in microorganisms. Lactobacillus plantarum subsp. plantarum KCCP11226 was previously isolated from kimchi, while exhibiting the production of 4,4'-diaponeurosporene as a C30 carotenoid. In this study, full genome sequencing of the strain KCCP11226 was performed. Genome analysis revealed that the dehydrosqualene synthase (crtM) and dehydrosqualene desaturase (crtN) genes, which are major genes for biosynthesis of 4,4'-diaponeurosporene, were shown to act as an operon in most L. plantarum strains, but they were uncommon in other Lactobacillus species. In vitro experiments revealed that the production of 4,4'-diaponeurosporene was greatly increased by oxidative stress. In this situation, mRNA expressions of crtN and crtM were also significantly increased. In conclusion, genome analysis of L. plantarum subsp. plantarum KCCP11226 suggested the presence of a well-conserved C30 carotenoid biosynthetic pathway that includes the crtM-crtN operon. The genomic information on L. plantarum subsp. plantarum KCCP11226 could further elucidate the functions of genes involved in isoprenoid biosynthetic pathway, especially in C30 carotenoid biosynthesis.

Genome Analysis of Enterococcus mundtii Pe103, a Human Gut-Originated Pectinolytic Bacterium.

Pectin exists in significant amounts in vegetables and fruits as a component of the plant cell wall. In human diet, pectin is not degraded by the human digestive enzymes due to its complex structure; only gut bacteria degrade pectin in the large intestine. To date, although pectin is one of the most important sources of dietary fiber in human diet, there have been only few reports on human gut-originated pectinolytic bacteria. In this study, the strain Enterococcus mundtii Pe103, a bacterium with pectin-degrading activity, was isolated from the feces of a healthy Korean adult female. Culture experiments revealed that it could grow on pectin as the sole carbon source by degrading pectin to approximately 35% within 13 h. We report the complete genome data of human gut E. mundtii Pe103. It consists of a circular chromosome (3,084,146 bps) and two plasmids (63,713 and 56,223 bps). Genomic analysis suggested that at least nine putative enzymes related to pectin degradation are present in E. mundtii Pe103. These enzymes may be involved in the degradation of pectin. The whole genome information of E. mundtii Pe103 could improve the understanding of the mechanism underlying the degradation of pectin by human gut microbiota.

Comparative study on four amylosucrases from Bifidobacterium species.

Amylosucrase (ASase) is α-glucan-producing enzyme. Four putative ASase genes (bdas, blas, bpas, and btas) were cloned from Bifidobacterium sp. and expressed in Escherichia coli. All ASases from Bifidobacterium sp. (BAS) displayed typical ASase properties with slightly different characteristics. Among the BASs studied, BdAS and BpAS showed maximal enzyme activities at 35 and 30 °C, respectively, whereas BlAS and BtAS were maximally active at higher temperatures, i.e., 45 and 50 °C, respectively. BpAS exhibited optimum pH under slightly basic conditions (pH 8.0), while BdAS, BlAS, and BtAS preferred weakly acidic conditions (pH 5.0-6.0). All BASs showed higher isomerization activities. Particularly, BlAS produced more trehalulose than turanose. Although polymerization was the highest for BtAS, BtAS synthesized α-1, 4-glucans with a lower degree of polymerization than that of the other BASs. The versatile properties of the BASs described could contribute to the efficient production of highly valuable biomaterials for the agriculture, food, and pharmaceutical industries.

Heterologous expression of Deinococcus geothermalis amylosucrase in Corynebacterium glutamicum for luteolin glucoside production.

Amylosucrase (ASase) has great industrial potential owing to its multifunctional activities, including transglucosylation, polymerization, and isomerization. In the present study, the properties of Deinococcus geothermalis ASase (DGAS) expressed in Corynebacterium glutamicum (cDGAS) and purified via Ni-NTA affinity chromatography were compared to those of DGAS expressed in Escherichia coli (eDGAS). The pH profile of cDGAS was similar to that of eDGAS, whereas the temperature profile of cDGAS was lower than that of eDGAS. The melting temperature of both enzymes did not differ significantly. Interestingly, polymerization activity was slightly lower in cDGAS than in eDGAS, whereas luteolin (an acceptor molecule) transglucosylation activity in cDGAS was 10 % higher than that in eDGAS. Analysis of protein secondary structure via circular dichroism spectroscopy revealed that cDGAS had a lower strand/helix ratio than eDGAS. The present results indicate that cDGAS is of greater industrial significance than eDGAS.