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Cold-adapted microorganisms possess cold-active enzymes with potential applications in different industries and research areas. In this study, two genes encoding β-d-galactosidases belonging to Glycoside Hydrolase families 2 and 42 from the psychrotolerant Arctic bacterium Arthrobacter sp. S3* were cloned, expressed in Escherichia coli and Komagataella phaffii, purified and characterized. The GH2 β-d-galactosidase is a tetramer with a molecular weight of 450 kDa, while the GH42 β-d-galactosidase is a 233 kDa trimer. The Bgal2 was optimally active at pH 7.5 and 22 °C and maintained 57% of maximum activity at 10 °C, whereas the Bgal42 was optimally active at pH 7.0 and 40 °C and exhibited 44% of maximum activity at 10 °C. Both enzymes hydrolyzed lactose and showed transglycosylation activity. We also found that 2 U/mL of the Bgal2 hydrolyzed 85% of lactose in milk within 10 h at 10 °C. The enzyme synthesized galactooligosaccharides, heterooligosaccharides, alkyl galactopyranosides and glycosylated salicin. The Bgal42 synthesized galactooligosaccharides and 20 U/mL of the enzyme hydrolyzed 72% of milk lactose within 24 h at 10 °C. The properties of Arthrobacter sp. S3* Bgal2 make it a candidate for lactose hydrolysis in the dairy industry and a promising tool for the glycosylation of various acceptors in the biomedical sector. [ABSTRACT FROM AUTHOR]

GH29A α‐l‐fucosidases (EC 3.2.1.51) catalyze the release of α‐l‐fucosyl moieties from the nonreducing end of glycoconjugates by hydrolysis and some also catalyze transglycosylation. The latter is particularly interesting with regard to designing enzymatic synthesis of human milk oligosaccharides (HMOs). We combined the bioinformatics tool conserved unique peptide patterns (CUPP) and phylogenetic clustering to discover new microbial GH29A α‐l‐fucosidases of the underexplored CUPP group GH29:13.1. Three uncharacterized bacterial enzymes (EaGH29, SeGH29, and PmGH29) and two previously identified GH29A α‐l‐fucosidases (BF3242 and TfFuc1) were selected for reaction optimization, biochemical, and structural characterization. Kinetics, pH‐temperature optima, and substrate preference for 2‐chloro‐4‐nitrophenyl‐α‐l‐fucopyranoside (CNP‐α‐l‐Fuc) and 2′‐fucosyllactose (2′FL) were determined. Transglycosylation was favored at high neutral to alkaline pH, especially for EaGH29, SeGH29, TfFuc1, and BF3242, mainly because hydrolysis was decreased. The α‐l‐fucosidases exhibited medium regioselectivity in transglycosylation, generally forming two out of five detected lacto‐N‐fucopentaose (LNFP) isomers from 2′FL and lacto‐N‐tetraose (LNT). Alkaline pH also affected the transglycosylation product regioselectivity of SeGH29, which was also affected by a Leu/Phe exchange in the acceptor binding site. New crystal structures of TfFuc1 and BF3242 showed congruence in active site topology between these two enzymes and contributed to understanding the function of GH29A α‐l‐fucosidases. Notably, the structural data provide new insight into the role of an Asn residue located between the two catalytic residues in the active site. [ABSTRACT FROM AUTHOR]

Inefficient conversion of small molecule maltooligosaccharides into trehalose greatly affects the cost of the production of trehalose by double enzyme method [maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohyrolase (MTHase)]. This study used directed evolution to increase oligosaccharide utilization by the cyclomaltodextrin glucanotransferase (CGTase) from Bacillus species G1. This enzyme was chosen for its adaptability and stability in trehalose production. Model analysis revealed that the hydrogen bond distance between the N33K mutant and maltose reduced from 2.6 Å to 2.3 Å, increasing maltose affinity and boosting transglycosylation activity by 2.1-fold compared to the wild type. Further mutations improved thermal stability and optimum temperature, resulting in the N33K/S211G mutant. Consistent results from repeated experiments showed that the N33K/S211G mutant increased trehalose yield by 32.6% using maltodextrin. The results enhanced the double-enzyme method formed by MTSase and MTHase for trehalose production. Overall, we have identified optimal catalytic conditions, demonstrating significant potential for industrial-scale trehalose production with enhanced efficiency and cost-effectiveness. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. The transgalactosylase activity of β-galactosidases offers a convenient and promising strategy for conversion of lactose into high-value oligosaccharides, such as galactooligosaccharides (GOS) and human milk oligosaccharides. In this study, we cloned and biochemically characterized a novel C-terminally truncated β-galactosidase (PaBgal2A-D) from Paenibacillus antarcticus with high transglycosylation activity. PaBgal2A-D is a member of glycoside hydrolase family 2. The optimal pH and temperature of PaBgal2A-D were determined to be pH 6.5 and 50°C, respectively. It was relatively stable within pH 5.0–8.0 and up to 50°C. PaBgal2A-D showed high transglycosylation activity for GOS synthesis, and the maximum yield of 50.8% (wt/wt) was obtained in 2 h. Moreover, PaBgal2A-D could synthesize lacto- N -neotetraose (LNnT) using lactose and lacto- N -triose II, with a conversion rate of 16.4%. This study demonstrated that PaBgal2A-D could be a promising tool to prepare GOS and lacto- N -neotetraose. [ABSTRACT FROM AUTHOR]

In order to further investigate the structural properties and catalytic function of acarviosyltransferase (ATase), a key enzyme in the biosynthesis of acarbose, its gene (acbD) was cloned from Actinoplanes sp. SE50 genome and heterologously expressed in Escherichia coli. Bioinformatics analysis showed that the conserved domains of ATase, the expression product of acbD, were highly similar to those of cyclodextrin glycosyltransferase, which belongs to the glycoside hydrolase 13 (GH13) family, and ATase possessed a signal peptide and a transmembrane domain. After removal of the coding sequences in the signal peptide, the soluble expression level of acbD increased by 23.4 times as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal catalytic temperature and pH for the recombinant ATase were 30 ℃ and 7.0, respectively. The substrate spectrum showed that the recombinant ATase had the highest catalytic activity toward D-salicin (82.85 U/mL), followed by that (63.75 U/mL) toward L-sorbose. This is the first finding that L-sorbose can serve as an excellent glycosyl donor for ATase. The above results lay the foundation for further clarifying the catalytic mechanism of ATase.

A systematic study on the enzymatic transglycosylation of 3′‐amino nucleosides has been reported. Using the commercially available 3′‐amino‐3′‐deoxythymidine as the sugar donor, 13 pyrimidine and purine nucleobases were shown as the viable sugar acceptors in the nucleoside phosphorylase EcPPNP‐ and/or nucleoside 2′‐deoxyribosyltransferase LlNDT‐catalyzed reactions, furnishing the corresponding 3′‐amino containing nucleosides in 23%–73% NMR yields at an analytical scale. The synthetic utility of this diversification approach was further showcased by the successful synthesis of 3′‐amino‐2′,3′‐dideoxy‐5‐bromouridine in 29% isolated yield at a preparative scale. The current work demonstrates the transglycosylation activity of EcPPNP for the first time, expands the substrate scope of LlNDT, and provides an efficient, direct access to various 3′‐amino nucleosides of high value. [ABSTRACT FROM AUTHOR]

Cello-oligosaccharides (COS) become a new type of functional oligosaccharides. COS transglycosylation reactions were studied to enhance COS yield production. Seeking the ability of the free form of Fusarium solani β-glucosidase (FBgl1) to synthesize COS under low substrate concentrations, we found out that this biocatalyst initiates this reaction with only 1 g/L of cellobiose, giving rise to the formation of cellotriose. Cellotriose and cellopentaose were detected in biphasic conditions with an immobilized FBgl1 and when increased to 50 g/L of cellobiose as a starter concentration. After the biocatalyst recycling process, the trans-glycosylation yield of COS was maintained after 5 cycles, and the COS concentration was 6.70 ± 0.35 g/L. The crude COS contained 20.15 ± 0.25 g/L glucose, 23.15 ± 0.22 g/L non-reacting substrate cellobiose, 5.25 ± 0.53 g/L, cellotriose and 1.49 ± 0.32 g/L cellopentaose. A bioprocess was developed for cellotriose enrichment, using whole Bacillus velezensis cells as a microbial purification tool. This bacteria consumed glucose, unreacted cellobiose, and cellopentaose while preserving cellotriose in the fermented medium. This study provides an excellent enzyme candidate for industrial COS production and is also the first study on the single-step COS enrichment process. [ABSTRACT FROM AUTHOR]

The biochemical and structural characteristics of PtLam, a laminarinase from deep-sea Planctomycetota, have been extensively elucidated, unveiling the fundamental molecular mechanisms governing substrate recognition and enzymatic catalysis. PtLam functions as an exo-laminarinase with the ability to sequentially hydrolyze laminarin, cleaving glucose units individually. Notably, PtLam exhibits proficient transglycosylation capabilities, utilizing various sugar alcohols as acceptors, with lyxose, in particular, yielding exclusively transglycosylated products. Structural analysis of both apo-PtLam and its laminarin oligosaccharide-bound complex revealed significant conformational alterations in active residues upon substrate binding. Moreover, pivotal residues involved in substrate recognition were identified, with subsequent mutation assays indicating the contribution of positive subsites in modulating exo-hydrolysis and transglycosidic activities. These results enhance our comprehension of laminarin cycling mechanisms by marine Planctomycetota, while also providing essential enzyme components for laminarin hetero-oligosaccharide synthesis. IMPORTANCE The ubiquitous Planctomycetota, with distinctive physiological traits, exert a significant influence on global carbon and nitrogen fluxes. Their intimate association with algae suggests a propensity for efficient polysaccharide degradation; however, research on glycoside hydrolases derived from Planctomycetota remains scarce. Herein, we unveil the GH16 family laminarinase PtLam from deep-sea Planctomycetota, shedding light on its catalytic mechanisms underlying hydrolysis and transglycosylation. Our findings elucidate the enzymatic pathways governing the marine laminarin cycle orchestrated by Planctomycetota, thereby fostering the exploration of novel polysaccharide hydrolases with promising practical implications. [ABSTRACT FROM AUTHOR]

Endo-β-N-acetylglucosaminidases (ENGases) are pivotal enzymes in the degradation and remodeling of glycoproteins, which catalyze the cleavage or formation of β-1,4-glycosidic bond between two N-acetylglucosamine (GlcNAc) residues in N-linked glycan chains. It was investigated that targeted mutations of amino acids in ENGases active site may modulate their hydrolytic and transglycosylation activities. Endo-Tb, the ENGase derived from Trypanosoma brucei, belongs to the glycoside hydrolase family 85 (GH85). Our group previously demonstrated that Endo-Tb exhibits hydrolytic activity toward high-mannose and complex type N-glycans and preliminarily confirmed its transglycosylation potential. In this study, we further optimized the transglycosylation activity of recombinant Endo-Tb by focusing on the N536A, E538A and Y576F mutants. A comparative analysis of their transglycosylation activity with that of the wild-type enzyme revealed that all mutants exhibited enhanced transglycosylation capacity. The N536A mutant exhibited the most pronounced improvement in transglycosylation activity with a significant reduction in hydrolytic activity. It is suggested that Endo-Tb N536A possesses the potential as a tool for synthesizing a wide array of glycoconjugates bearing high-mannose and complex type N-glycans. [ABSTRACT FROM AUTHOR]

AbstractAlpha glucosidases are multifunctional glycoside hydrolases with hydrolysis and transglycosylation ability. It can be utilized for the glycosidic bond synthesis or glycosylation of Ascorbic acid/Vitamin C to its stable analogue, Ascorbic acid 2 glucoside (AA2G), a compound with wide applications in cosmetics and pharma. The application of α-glucosidases for industrial scale transglycosylation is limited due to the low transglycosylation yield of free enzymes. Enzyme immobilization techniques could enable the development of efficient, reusable catalysts. Only a few glycoside hydrolases have been studied in immobilized form for transglycosylation reactions, and α-glucosidases are probably the least explored in this form. Transglycosylation activity of immobilized α-glucosidase from Aspergillus carbonarius BTCF 5 was studied for AA2G synthesis, where different immobilization techniques like calcium alginate encapsulation, adsorption on chitosan beads, covalent cross-linking on magnetic nanoparticles, and cross-linked enzyme aggregates (CLEA) were employed for the immobilization. The immobilization yield of calcium alginate encapsulated enzyme, enzyme immobilized on Fe-MNP support, enzyme immobilized on chitosan beads and as CLEA were 107%, 99%, 46% and 486%, respectively. CLEA was identified as the best immobilization technique for this bi-substrate reaction due to the high immobilization yield and activity retention (30% activity retained after 5 consecutive cycles). Enzyme immobilization increased the transglycosylation activity by 38%, yielding 118 mM AA2G against 72 mM by the free enzyme. This indicates the potential of immobilized α-glucosidase as a catalyst for synthesizing AA2G at an industrial scale. [ABSTRACT FROM AUTHOR]

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application. [ABSTRACT FROM AUTHOR]

The α-glucosidase from Schwanniomyces occidentalis (GAM1p) was expressed in Komagataella phaffii to about 70 mg/L, and its transferase activity studied in detail. Several isomaltooligosaccharides (IMOS) were formed using 200 g/L maltose. The major production of IMOS (81.3 g/L) was obtained when 98% maltose was hydrolysed, of which 34.8 g/L corresponded to isomaltose, 26.9 g/L to isomaltotriose, and 19.6 g/L to panose. The addition of glucose shifted the IMOS synthesis towards products containing exclusively α(1 → 6)-linkages, increasing the production of isomaltose and isomaltotriose about 2–4 fold, enabling the formation of isomaltotetraose, and inhibiting that of panose to about 12 times. In addition, the potential of this enzyme to glycosylate 12 possible hydroxylated acceptors, including eight sugars and four phenolic compounds, was evaluated. Among them, only sucrose, xylose, and piceid (a monoglucosylated derivative of resveratrol) were glucosylated, and the main synthesised products were purified and characterised by MS and NMR. Theanderose, α(1 → 4)-D-glucosyl-xylose, and a mixture of piceid mono- and diglucoside were obtained with sucrose, xylose, and piceid as acceptors, respectively. Maximum production of theanderose reached 81.7 g/L and that of the glucosyl-xylose 26.5 g/L, whereas 3.4 g/L and only 1 g/L were produced of the piceid mono- and diglucoside respectively. Key points: • Overexpression of a yeast α-glucosidase producing novel molecules. • Yeast enzyme producing the heterooligosaccharides theanderose and glucosyl-xylose. • Glycosylation of the polyphenol piceid by a yeast α-glucosidase. [ABSTRACT FROM AUTHOR]

Rutinosidase is a diglycosidase that catalyzes the cleavage of rutinose (α‐l‐Rhap‐(1→6)‐β‐d‐Glcp) from rutin or other rutinosides. It is also able to cleave β‐glucopyranosides, e. g., isoquercitrin. This enzyme has a strong transglycosylation activity and a remarkable substrate specificity. We have shown that rutinosidase from Aspergillus niger (AnRut) is able to cleave β‐glucopyranosides acylated at C‐6 of glucose (6′‐O‐acylisoquercitrin) with acetyl, benzoyl, phenylacetyl, phenylpropanoyl, cinnamoyl, vanillyl, galloyl, 4‐hydroxybenzoyl and 3‐(4‐hydroxy‐3‐methoxyphenyl)propanoyl. The release of the respective 6‐acylglucopyranoses was confirmed by HPLC/MS and NMR methods. Selected compounds, i. e., 6′‐O‐acetyl, 6′‐O‐benzoyl, and 6′‐O‐cinnamyl derivatives of isoquercitrin, were also tested as transglycosylation substrates. Only 6′‐acetylisoquercitrin and 6′‐O‐benzoylisoquercitrin underwent transglycosylations by AnRut to produce n‐butyl 6‐acetyl‐β‐d‐glucopyranoside and n‐butyl 6‐benzoyl‐β‐d‐glucopyranoside. Isoquercitrin 6′‐O‐cinnamate yielded on hydrolytic product. Molecular modeling based on the crystal structure of AnRut showed that large aromatic moieties at C‐6′ of isoquercitrin block the side tunnel of AnRut leading into its active site and thus hinder the entry of the acceptor substrate for transglycosylation. This study demonstrates the great substrate flexibility of rutinosidase at the glycone site. [ABSTRACT FROM AUTHOR]

Inefficient conversion of small molecule maltooligosaccharides into trehalose greatly affects the cost of the production of trehalose by double enzyme method [maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohyrolase (MTHase)]. This study used directed evolution to increase oligosaccharide utilization by the cyclomaltodextrin glucanotransferase (CGTase) from Bacillus species G1. This enzyme was chosen for its adaptability and stability in trehalose production. Model analysis revealed that the hydrogen bond distance between the N33K mutant and maltose reduced from 2.6 Å to 2.3 Å, increasing maltose affinity and boosting transglycosylation activity by 2.1-fold compared to the wild type. Further mutations improved thermal stability and optimum temperature, resulting in the N33K/S211G mutant. Consistent results from repeated experiments showed that the N33K/S211G mutant increased trehalose yield by 32.6% using maltodextrin. The results enhanced the double-enzyme method formed by MTSase and MTHase for trehalose production. Overall, we have identified optimal catalytic conditions, demonstrating significant potential for industrial-scale trehalose production with enhanced efficiency and cost-effectiveness.

Abstract Background Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. Results Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-β-glucosidase (αRβG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRβG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. Conclusions αRβG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies.

Background: Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. Results: Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-β-glucosidase (αRβG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRβG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. Conclusions: αRβG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies. [ABSTRACT FROM AUTHOR]

The present study reports a highly thermostable β-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular β-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified β-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50–70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of β-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of β-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal β-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant β-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted β-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme. [ABSTRACT FROM AUTHOR]

ABSTRACT: Lacto-N-tetraose (LNT) is one of the most important components of human milk oligosaccharides, which has various beneficial health effects. β-Galactosidase is an important enzyme used in dairy processing. The transglycosylation activity of β-galactosidases offers an attractive approach for LNT synthesis. In this study, we reported for the first time the biochemical characterization of a novel β-galactosidase (LzBgal35A) from Lacticaseibacillus zeae. LzBgal35A belongs to glycoside hydrolases (GH) family 35 and shared the highest identity of 59.9% with other reported GH 35 members. The enzyme was expressed as soluble protein in Escherichia coli. The purified LzBgal35A displayed optimal activity at pH 4.5 and 55°C. It was stable within the pH range of 3.5 to 7.0 and up to 60°C. Moreover, LzBgal35A could catalyze the synthesis of LNT via transferring the galactose residue from o-nitrophenyl-β-galactopyranoside to lacto-N-triose II. Under optimal conditions, the conversion rate of LNT reached 45.4% (6.4 g/L) within 2 h, which was by far the highest yield of LNT synthesized through a β-galactosidase-mediated transglycosylation reaction. This study demonstrated that LzBgal35A has great potential application in LNT synthesis.

Transglycosylation has been used to modify the physicochemical properties of original compounds. As a result, transglycosylated compounds can form molecular aggregates in size ranges of a few nanometers in an aqueous medium when their concentrations exceed a specific level. Incorporating these hydrophobic compounds has been observed to enhance the solubility of hydrophobic compounds into aggregate structures. Thus, this review introduces four transglycosylated compounds as food additives that can enhance the solubility and oral absorption of hydrophobic compounds. Here, transglycosylated hesperidin, transglycosylated rutin, transglycosylated naringin, and transglycosylated stevia are the focus as representative substances. Significantly, we observed that amorphous formations containing hydrophobic compounds with transglycosylated compounds improved solubility and oral absorption compared to untreated hydrophobic compounds. Moreover, combining transglycosylated compounds with hydrophilic polymers or surfactants enhanced the solubilizing effects on hydrophobic compounds. Furthermore, the enhanced solubility of hydrophobic compounds improved their oral absorption. Transglycosylated compounds also influenced nanoparticle preparation of hydrophobic compounds as a dispersant. This study demonstrated the benefits of transglycosylated compounds in developing supplements and nutraceuticals of hydrophobic compounds with poor aqueous solubility. [ABSTRACT FROM AUTHOR]

Abstractβ-Galactosidases are crucial carbohydrate-active enzymes that naturally catalyze the hydrolysis of galactoside bonds in oligo- and disaccharides. These enzymes are commonly used to degrade lactose and produce low-lactose and lactose-free dairy products that are beneficial for lactose-intolerant people. β-galactosidases exhibit transgalactosylation activity, and they have been employed in the synthesis of galactose-containing compounds such as galactooligosaccharides. However, most β-galactosidases have intrinsic limitations, such as low transglycosylation efficiency, significant product inhibition effects, weak thermal stability, and a narrow substrate spectrum, which greatly hinder their applications. Enzyme engineering offers a solution for optimizing their catalytic performance. The study of the enzyme’s structure paves the way toward explaining catalytic mechanisms and increasing the efficiency of enzyme engineering. In this review, the structure features of β-galactosidases from different glycosyl hydrolase families and the catalytic mechanisms are summarized in detail to offer guidance for protein engineering. The properties and applications of β-galactosidases are discussed. Additionally, the latest progress in β-galactosidase engineering and the strategies employed are highlighted. Based on the combined analysis of structure information and catalytic mechanisms, the ultimate goal of this review is to furnish a thorough direction for β-galactosidases engineering and promote their application in the food and dairy industries. [ABSTRACT FROM AUTHOR]

Tyrosol robinobioside was prepared under catalysis of robinobiosidase-containing seed meal from common buckthorn Rhamnus cathartica. Robinin, a flavonoid isolated from the flowers of black locust (Robinia pseudoacacia) served as a robinobiosyl donor. The glycosylation proceeded predominantly on the primary hydroxyl of tyrosol, typically yielding mixtures of isomeric glycosides in ratios of 5:1 to 8:1 with overall yields of robinobiosides higher than 20%. This is the first robinobiosylation promoted under enzymatic catalysis. [ABSTRACT FROM AUTHOR]

Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1–110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region’s impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%–97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT.

Prebiotics play an important role in biological processes, which has stimulated great interest in their synthesis. This paper reports a levansucrase from Bacillus subtilis , demonstrating high catalytic selectivity for the synthesis of prebiotic lactosucrose with sucrose as a donor and lactose as an acceptor. The product was purified and the structure was characterized. The effects of lactosucrose on the proliferation and acid production of bifidobacterium and lactobacillus were better than that of lactulose and galactooligosaccharides(GOS). The short chain fatty acid (SCFA) content in the fermentation broth was also measured. It contains large amounts of SCFAs that act roles in intestinal homeostasis. The lactosucrose affected inflammatory responses in lipopolysaccharide-induced RAW 264.7 macrophages since they suppressed inflammatory cytokines TNF-α, IL-1β, and IL-6 well as compared to the control. Also, lactosucrose had no cytotoxicity in the concentration range of 10 mg/mL. These results indicate that lactosucrose was effective in promoting the proliferation of intestinal probiotics and have anti-inflammatory activity. Therefore, lactosucrose has positive prebiotic activity. [Display omitted] • Enzymatic production is a promising strategy for preparing functional lactosucrose. • Lactosucrose exhibits high prebiotic activity. • Lactosucrose has an obvious anti-inflammatory effect and low cytotoxicity. [ABSTRACT FROM AUTHOR]

Naringenin, a flavanone (flavonoid family) has bioactive effects, such as antioxidant, free radical scavenging, anti-inflammatory, carbohydrate metabolism, and immunoregulatory effects on the human body. However, its low water solubility and bioavailability limit its use in food, pharmaceutical, cosmetic, and drug industries. Amylosucrase (ASase, E. C. 2.4.1.4), uses sucrose as a substrate and is a glycosyltransferase that hydrolyzes glucose and fructose generating a glycosylated product. In this study, the putative ASase gene (accession number: WP_102128133.1) from Deinococcus planocerae KCTC 33809 (DplAS) was expressed in Escherichia coli. The optimum reaction temperature and pH for sucrose hydrolytic capacity of DplAS were determined to be 30 ℃ and 7.0, respectively. DplAS was determined to have a half-life of 256.7 and 42.8 min at 40 ℃ and 45 ℃, respectively. Although it exhibited high similarity with DgAS (80%), DplAS displayed different acceptor specificities to that of DgAS. Uniquely, DplAS generated one major product (naringenin-maltoside) via transglycosylation using sucrose and naringenin as donor and acceptor molecules, respectively. Based on NMR, naringenin-maltoside was identified as naringenin-4'-O-a-D-maltopyranoside, and its conversion rate was optimized using the response surface method (RSM) with central composite design (CCD). This resulted in a donor to acceptor ratio of 25.9:1 at 27.64 ℃, and a 55.69% conversion of sucrose. [ABSTRACT FROM AUTHOR]

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas. [ABSTRACT FROM AUTHOR]

Chitooligosaccharides (COSs) are b-1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as antimicrobial, anti-inflammatory, antioxidant and antitumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COSs used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COSs have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure–bioactivity relationship of COSs has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COSs with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COSs, leading to the enhanced utilization of chitin biomass. [ABSTRACT FROM AUTHOR]

β-Mannanases hydrolyze β-mannans, important components of plant and microalgae cell walls. Retaining β-mannanases can also catalyze transglycosylation, forming new β-mannosidic bonds that are applicable for synthesis. This study focused on the blue mussel (Mytilus edulis) GH5_10 β-mannanase MeMan5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of MeMan5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme's catalytic efficiency (kcat/Km using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by 18O labeling of hydrolysis products and mass spectrometry. Results show that the substitution of both tryptophans was required to shift away from the dominant binding mode of mannopentaose (spanning subsites −3 to +2), suggesting that both tryptophans contribute to glycan binding. NMR spectroscopy and molecular dynamics simulations were conducted to analyze protein flexibility and glycan binding. We suggest that W240 is rigid and contributes to +2 subsite mannosyl specificity, while W281 is flexible, which enables stacking interactions in the +2 subsite by loop movement to facilitate binding. The substitutions significantly reduced or eliminated transglycosylation with saccharides as glycosyl acceptors but had no significant effect on reactions with alcohols. [ABSTRACT FROM AUTHOR]

Alternative sweeteners, such as steviol glucosides from the plant Stevia rebaudiana Bertoni, are becoming increasingly popular for the design of next-generation foodstuffs. However, the bitter aftertaste of native steviol glucosides is one of the main reasons behind consumer reluctance towards stevia-containing products. Biocatalysis could be a sustainable solution to this problem, through addition of glucosyl moieties to the molecule. Glycoside hydrolases are enzymes performing transglycosylation reactions, and they can be exploited for such modifications. In the present work, the commercial β-glucanase Finizym 250L® was employed for the transglycosylation of stevioside. After optimization of several reaction parameters, the maximal reaction yield obtained was 19%, with barley β-glucan as the glycosyl donor. With the aim to develop a sustainable process, β-glucan extracts from different fungal sources were prepared. Pulsed Electric Field pretreatment of mycelial biomass resulted in extracts with higher β-glucan content. The extracts were tested as alternative glucosyl donors, reaching up to 15.5% conversion yield, from Pleurotus-extracted β-glucan. Overall, in the present work a novel enzymatic process for the modification of stevioside is proposed, with concomitant valorization of β-glucans extracted from fungal biomass, potentially generated as a byproduct from other applications, in concert with the principles of circular economy. [ABSTRACT FROM AUTHOR]

Objectives: To improve the biochemical characteristics of the GH2 family β-galactosidases using a family shuffling method based on degenerate oligonucleotide gene shuffling. Results: Four β-galactosidase genes from the genus Alteromonas were divided into 14 gene segments, and each included the homologous sequence in the adjacent segments. The gene segments were regenerated into complete β-galactosidase genes and amplified by PCR. The obtained chimeric genes were cloned into a plasmid and screened for β-galactosidase activity. Approximately 320 positive clones were observed on the screening plate, of which nine sequenced genes were chimera. Additionally, the M22 and M250 mutants were expressed, purified, and characterized. The optimal temperature and substrate specificity of the recombinant M22 and M250 were consistent with those of the wild-type enzymes. The catalytic efficiency of recombinant M22 enzyme was higher than that of the wild-type enzymes, and the recombinant M250 displayed weak transglycosylation activity. Conclusions: The chimeric genes of GH2 β-galactosidase were obtained using a controlled family shuffling that will provide an enzyme evolutionary method to obtain the β-galactosidases with excellent characteristics for laboratory and industrial purposes. [ABSTRACT FROM AUTHOR]

Mannan, a highly abundant and cost-effective natural resource, holds great potential for the generation of high-value compounds such as bioactive polysaccharides and biofuels. In this study, we successfully enhanced the expression of constructed GH5 β-mannanase (TaMan5) from Trichoderma asperellum ND-1 by employing propeptide in Pichia pastoris. By replacing the α-factor with propeptide (MGNRALNSMKFFKSQALALLAATSAVA), TaMan5 activity was significantly increased from 67.5 to 91.7 U/mL. It retained higher activity in the presence of 20% ethanol and 15% NaCl. When incubated with a high concentration of mannotriose or mannotetraose, the transglycosylation action of TaMan5 can be detected, yielding the corresponding production of mannotetraose or mannooligosaccharides. Moreover, the unique mechanism whereby TaMan5 catalyzes the degradation of mannan into mannobiose involves the transglycosylation of mannose to mannotriose or mannotetraose as a substrate to produce a mannotetraose or mannopentose intermediate, respectively. Additionally, the production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaMan5 could synergistically yield fermentable sugars from corn stover and bagasse. These findings offered valuable insights and strategies for enhancing β-mannanase expression and efficient conversion of lignocellulosic biomass, providing cost-effective and sustainable approaches for high-value biomolecule and biofuel production.

Consumption of oligosaccharides which can evade host digestive enzymes but can be selectively taken-up by populations of bacteria native to the host colon in situ, improves the gut health of host through various mechanisms. However, their consumption from natural sources is constrained by adequacy, bioavailability and geography. Here in the present review, we detail the enzymatic production and applications of promising multifunctional dietary molecule “isomaltooligosaccharides (IMO)”. Although presently IMO are not considered as dietary fibers (vide FDA-2019-P-2239), these are proven as slowly digestible carbohydrates with multiple food applications and health benefits. Commercially IMO are synthesized using transglycosylating α-glucosidase (tAG) with pretreated starch as the substrate. Usually, the tAG obtained from Aspergillus spp. are widely used to produce IMO. The present review provides comprehensive information on tAG from different sources and their applications. Strategies to increase the transglycosylation to hydrolysis ratio by tAG is of both scientific and commercial interest. Some of the existing strategies to improve this ratio are detailed in this review. This review describes the use and application of IMO in food industry and its potential as functional oligosaccharides. Apart from production strategies, both food applications of IMO and their health benefits are discussed.

Galectins are carbohydrate-binding lectins that modulate the proliferation, apoptosis, adhesion, or migration of cells by cross-linking glycans on cell membranes or extracellular matrix components. Galectin-4 (Gal-4) is a tandem-repeat-type galectin expressed mainly in the epithelial cells of the gastrointestinal tract. It consists of an N- and a C-terminal carbohydrate-binding domain (CRD), each with distinct binding affinities, interconnected with a peptide linker. Compared to other more abundant galectins, the knowledge of the pathophysiology of Gal-4 is sparse. Its altered expression in tumor tissue is associated with, for example, colon, colorectal, and liver cancers, and it increases in tumor progression, and metastasis. There is also very limited information on the preferences of Gal-4 for its carbohydrate ligands, particularly with respect to Gal-4 subunits. Similarly, there is virtually no information on the interaction of Gal-4 with multivalent ligands. This work shows the expression and purification of Gal-4 and its subunits and presents a structure–affinity relationship study with a library of oligosaccharide ligands. Furthermore, the influence of multivalency is demonstrated in the interaction with a model lactosyl-decorated synthetic glycoconjugate. The present data may be used in biomedical research for the design of efficient ligands of Gal-4 with diagnostic or therapeutic potential. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 29 (GH29) encompasses α-L-fucosidases, i.e. enzymes that catalyze the hydrolytic release of fucose from fucosylated glycans, including N - and O -linked glycans on proteins, and these α-L-fucosidases clearly play important roles in biology. GH29 enzymes work via a retaining exo-action mechanism, and some can catalyze transfucosylation. There is no formal subfamily division of GH29 α-L-fucosidases, but they are nonetheless divided into two subfamilies: GH29A having a range of substrate specificities and GH29B having narrower substrate specificity. However, the sequence traits that determine the substrate specificity and transglycosylation ability of GH29 enzymes are not well characterized. Here, we present a new functional map of family GH29 members based on peptide-motif clustering via CUPP (conserved unique peptide patterns) and compare the substrate specificity and transglycosylation activity of 21 representative α-L-fucosidases across the 53 CUPP groups identified. The 21 enzymes exhibited different enzymatic rates on 8 test substrates, CNP-Fuc, 2'FL, 3FL, Lewisa, Lewisx, Fuc-α1,6-GlcNAc, Fuc-α1,3-GlcNAc, and Fuc-α1,4-GlcNAc. Certain CUPP groups clearly harbored a particular type of enzymes, e.g. the majority of the enzymes having activity on Lewisa or Lewisx categorized in the same CUPP clusters. In general, CUPP was useful for resolving GH29 into functional diversity subgroups when considering hydrolytic activity. In contrast, the transglycosylation capacity of GH29 α-L-fucosidases was distributed across a range of CUPP groups. Transglycosylation thus appears to be a common trait among these enzymes and not readily predicted from sequence comparison. [ABSTRACT FROM AUTHOR]

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on β→α loop 5 of the catalytic (β/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation. [ABSTRACT FROM AUTHOR]

β- d -Glucan glucohydrolases from glycoside hydrolase family 3 (GH3) act to break-down β-glucans, which is critical to growth and cell wall remodeling in plants. To investigate the function and application of the rice GH3 β- d -glucan glucohydrolases OsExoI, we produced the protein from a codon-optimized cDNA in the heterogenous expression host Pichia pastoris. After purification from the medium, OsExoI was found to be heterogeneously glycosylated and could be separated into pools with different glycosylation levels and specific activities by size exclusion chromatography (SEC). The total glycosylated protein pool had specific activity similar to that of protein from which N-linked carbohydrate had been removed with endoglycosidase H. Purified enzyme hydrolyzed β-1,3- and 1,4- linked oligosaccharides, polysaccharides, and synthetic p -nitrophenyl-β- d -glucopyranoside. The deglycosylated OsExoI had catalytic efficiency (k cat / K m) slightly higher than pooled glycosylated OsExoI, although their overall biochemical properties were similar. OsExoI also exhibits transglycosylation activity using p NP-β- d -glucopyranoside, oligosaccharides, or polysaccharides as glucosyl donor to glycosylate a variety of alcohols. These results suggest that the OsExoI can be applied to alkyl glycoside production using natural polysaccharides or oligosaccharides released during biomass degradation as a source of glucosyl moieties. [Display omitted] • Rice OsExoI is successfully produced via expression in Pichia and purification. • OsExoI heterogeneous glycosylation gives heterogenous activity levels. • OsExoI can transglycosylate primary alcohols of varying length. • OsExoI can transfer glucose from oligo- and polysaccharides to produce glycosides. [ABSTRACT FROM AUTHOR]

Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods, the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continuous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation reactions. Over a period of 55 days, 2′-deoxyadenosine was produced continuously, with a product yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2′-deoxyribonucleoside and 6-chloro-2-fluoro-2′-deoxyribonucleoside were also produced, with high conversion, but for shorter operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic unit was produced, for 2′-deoxyadenosine, 2,6-dichloropurine-2′-deoxyribonucleoside, and 6-chloro-2-fluoro-2′-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the overall cost of the biocatalyst and enhances the downstream processing of nucleoside production. [ABSTRACT FROM AUTHOR]

Bacillus cereus is a rod-shaped, gram-positive, motile, and β-hemolytic soil bacterium. B. cereus is an opportunistic pathogen, often responsible for human foodborne illness that is caused by ingestion of starchy foods with symptoms of diarrhea and vomiting. Among the numerous amylolytic enzymes in the genome of the pathogen, the one annotated as a putative neopullulanase (NPase) was cloned and its biochemical properties were characterized in this study. The corresponding gene encoded an enzyme of 586 amino acids with a predicted molecular mass of 68.25 kDa. The putative NPase shared 43.7–59.2% of identity with NPases, cyclomaltodextrinases (CDases), and maltogenic amylases from various bacteria, but shared very low similarity with other amylolytic enzymes of B. cereus. The optimal pH and temperature of the enzyme was 6.5 and 37 ℃, respectively. The enzyme activity was decreased by the cations tested in this study and completely inhibited by Co2+ and Cu2+. The purified enzyme showed substrate preference in the order of α-CD > β-CD > starch > maltodextrin > γ-CD and hydrolyzed them mainly to maltose. However, it did not hydrolyze maltose, pullulan, and glycogen. The enzyme was designated herein as a CDase of B. cereus (BcCDase). Furthermore, the enzyme could transfer the sugars released from CDs and maltotriose to acceptor molecules. BcCDase was likely to be involved in the maltodextrin metabolism in B. cereus. [ABSTRACT FROM AUTHOR]

This study presents the enzymatic synthesis of resveratrol-3,4′-O-α-diglucoside (RDG) using a hyperactive O-α-glycoligase (MalA-D416R/Q450S) and α-glucopyranosyl fluoride as the donor substrate. The transglycosylation rate for resveratrol by MalA-D416R/Q450S was maximized in 100 mM Tris-HCl (pH 9.5) containing 20 % DMSO at 45°C. Because the p K a of the 4′-OH group of resveratrol is lower than that of the 3-OH group, the 4′-OH group is more nucleophilic at the alkaline pH, leading to a preference for glycosylation at the 4′-OH site rather than the 3-OH site. This preference makes resveratrol 3-O-α-glucoside (R3G) as the more efficient acceptor than resveratrol 4′-O-α-glucoside (R4′G), resulting in negligible production of resveratrol 3-O-α-glucoside (R3G) due to its complete consumption in the second transglycosylation reaction when using a 2:1 ratio of donor to acceptor substrates. From a preparative scale reaction, R4′G and RDG were isolated with yields of 41.2 % and 43.3 %, respectively. The water solubility of RDG exceeded 1.67 M, which represents more than a 9,800-fold improvement compared to resveratrol. In a hydrolysis experiment using intestinal α-glycosidase from rat, the α-glucosides of resveratrol (R4′G and RDG) were completely deglycosylated to the aglycone. [Display omitted] • The MalA-derived engineered O-α-glycoligase produced resveratrol 3,4-O-α-diglucoside (RDG). • The 4'-OH group, with a lower p K a , is preferable to the 3-OH as a glucosylation site. • The conversion yield of RDG was the highest compared to reported enzymatic syntheses. • The water solubility of RDG was improved by 9,800-fold compared to resveratrol. • The facile hydrolysis of RDG by α-glucosidase would lead to efficient absorption in the intestine. [ABSTRACT FROM AUTHOR]

The enzymatic transglycosylation of steviol glycosides can improve the edulcorant quality of steviol glycosides. Cyclodextrin glucanotransferase (CGTase) is one of the most popular glucanotransferases applied in this reaction. Herein, the CGTase-producing strain Alkalihalobacillus oshimensis CGMCC 23164 was isolated from Stevia planting soil. Using mass spectrometry-based secretome profiling, a high-efficiency CGTase that converted steviol glycosides to glucosylated steviol glycosides was identified and termed CGTase-13. CGTase-13 demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at <40 °C. Under the above conditions, the conversion rate of stevioside and rebaudioside A, two main components of steviol glycosides, reached 86.1% and 90.8%, respectively. To the best of our knowledge, this is the highest conversion rate reported to date. Compared with Toruzyme® 3.0 L, the commonly used commercial enzyme blends, glucosylated steviol glycosides produced using CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, and stronger sweetness intensity. Thus, CGTase provides a novel option for producing high-quality glucosylated steviol glycoside products and has great potential for industrial applications. [ABSTRACT FROM AUTHOR]

β-galactosidase is the principal enzyme for dairy and pharmaceutical industries owing to its multiple applications. Different sources of β-galactosidase such as plants, microbes, mammals have been explored by far. Due to its high industrial demands, recombinant strains have also been developed and well-characterized for industrial applications apart from the already existing natural sources. Immobilization strategies such as entrapment, cross-linking, covalent binding and physical absorption are also performed to increase the stability and durability, therefore reducing the process cost. This review provides systematic information about the recent advancements in the production of β-galactosidase and its industrial applications. Recombinant strains are observed to be dominant in the industry along with immobilization strategies and used predominantly for prebiotic galactooligosaccharides production. These developments on novel β-galactosidases derived from microbial sources or by engineering means together have converted the enzyme into a relevant synthetic tool with the use of efficient recombinant systems. [ABSTRACT FROM AUTHOR]

Glycogen is important for transmission of V. vulnificus undergoing disparate environments of nutrient-rich host and nutrient-limited marine environment. The malZ gene of V. vulnificus encoding a maltodextrin glucosidase was cloned and over-expressed in E. coli to investigate its roles in glycogen/maltodextrin metabolism in the pathogen. The malZ gene encoded a protein with a predicted molecular mass of 70 kDa. The optimal pH and temperature of MalZ was 7.0 and 37 °C, respectively. MalZ hydrolyzed maltodextrin to glucose and maltose most efficiently, while hydrolyzed other substrates such as starch, maltose, β-cyclomaltodextrin, and glycogen less efficiently. The activity was enhanced greatly by Mn2+. It also exhibited transglycosylation activity toward excessive maltotriose. The malZ knock-out mutant accumulated 2.3–5.6-fold less glycogen than the wild type when excessive maltodextrin or glucose was added to LB medium, while it accumulated more glycogen than the wild type (3.5-fold) in the presence of excessive maltose. Growth and glycogen accumulation of the mutant were retarded most significantly in the M63 minimal medium supplemented with 0.5% maltodextrin. Side chain length distributions of glycogen molecules were varied by the malZ mutation and types of the excessive carbon source. Based on the results, MalZ of V. vulnificus was likely to be involved in maltose/maltodextrin metabolism, thereby balancing synthesis of glycogen and energy generation in the cell. The bacterium seemed to have multiple and unique pathways for glycogen metabolism according to carbon sources. [ABSTRACT FROM AUTHOR]

Oligosaccharides are low molecular weight carbohydrates with a wide range of health benefits due to their excellent bio-preservative and prebiotic properties. The popularity of functional oligosaccharides among modern consumers has resulted in impressive market demand. Organoleptic and prebiotic properties of starch-derived oligosaccharides are advantageous to food quality and health. The extensive health benefits of oligosaccharides offered their applications in the food, pharmaceuticals, and cosmetic industry. Maltooligosaccharides and isomaltooligosaccharides comprise 2–10 glucose units linked by α-1-4 and α-1-6 glycoside bonds, respectively. Conventional biocatalyst-based oligosaccharides processes are often multi-steps, consisting of starch gelatinization, hydrolysis and transglycosylation. With higher production costs and processing times, the current demand cannot meet on a large-scale production. As a result, innovative and efficient production technology for oligosaccharides synthesis holds paramount importance. Malto-oligosaccharide forming amylase (EC 3.2.1.133) is one of the key enzymes with a dual catalytic function used to produce oligosaccharides. Interestingly, Malto-oligosaccharide forming amylase catalyzes glycosidic bond for its transglycosylation to its inheritance hydrolysis and alternative biocatalyst to the multistep technology. Genetic engineering and reaction optimization enhances the production of oligosaccharides. The development of innovative and cost-effective technologies at competitive prices becomes a national priority. [ABSTRACT FROM AUTHOR]

β-Galactosidase is a widely adopted enzyme in the food and pharmaceutical industries. Metagenome techniques have the advantage of discovering novel functional genes, particularly potential genes from uncultivated microbes. In this study, a novel GH42 β-galactosidase isolated from a deep-sea metagenome was overexpressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. The optimal temperatures and pH of the enzyme for o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose were 40 ℃, 6.5 and 50 ℃, 7, respectively. The enzyme was stable at temperatures between 4 and 30 ℃ and within the pH range of 6–9. Moreover, it was highly tolerant to salt and inhibited by Zn2+ and Cu2+. The kinetic values of Km and kcat of the enzyme against oNPG were 1.1 mM and 57.8 s−1, respectively. Furthermore, it showed hydrolysis and transglycosylation activity to lactose and the extra monosaccharides could improve the productivity of oligosaccharides. Overall, this recombinant β-galactosidase is a potential biocatalyst for the hydrolysis of milk lactose and synthesis of functional oligosaccharides. [ABSTRACT FROM AUTHOR]

Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its kcat/KmA is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. [ABSTRACT FROM AUTHOR]