Monitoring and evaluation of provincial classical swine fever immunization implementation with an E2 subunit vaccine in Jeju Island, South Korea.

Purpose: Accidental vaccination with a live attenuated low-virulence strain of Miyagi (LOM) vaccine led to the reemergence of classical swine fever virus (CSFV) in Jeju province, South Korea in 2014. To control the continual outbreaks of LOM-derived CSFV, the provincial government launched a provincial mass vaccination project using a CSF-E2 subunit vaccine. We conducted this study to assess the herd immunity level and outcomes of E2 vaccine-based immunization in breeding and growing herds on Jeju Island during 2020-2021. Materials and Methods: A large-scale vaccination trial using the Bayovac CSF-E2 vaccine investigated its efficacy in breeding and growing herds under farm application conditions (10 CSFV-affected and three CSFV-naïve swine farms). Results: The level of herd immunity in each farm was classified into three (S1-S3) and six (G1-G6) profiles in breeding and growing herds, respectively. Immunity monitoring revealed a remarkable improvement in the herd immunity status in all farms. The majority (10/13) of farms, including CSFV-free farms, showed the S1G1 immunity profile in 2021, indicating the appropriate implementation of the advised vaccination regime. Moreover, there were significant decreases in Erns seropositivity from 100% to 50% and 25.9% to 4.3% at farm and pig levels, respectively. In particular, all farms were confirmed as CSFV free in the growing-finishing herds. Conclusion: Our large-scale trial demonstrated the effectiveness of the E2 subunit vaccine in establishing herd immunity stabilization and eliminating CSFV circulation in the affected farms and highlighted the need for a provincial vaccination policy to regain the CSF-free status on Jeju Island.

Characterization of a lactic acid bacterium-derived ß-glucosidase for the production of rubusoside from stevioside.

Rubusoside, which is used as a natural sweetener or a solubilizing agent for water-insoluble functional materials, is currently expensive to produce owing to the high cost of the membrane-based technologies needed for its extraction and purification from the sweet tea plant (Rubus suavissimus S. Lee). Therefore, this study was carried out to screen for lactic acid bacteria that possess enzymes capable of bio-transforming stevioside into rubusoside. Subsequently, one such rubusoside-producing enzyme was isolated from Lactobacillus plantarum GS100. Located on the bacterial cell surface, this enzyme was stable at pH 4.5-6.5 and 30-40 °C, and it produced rubusoside as a major product through its stevioside-hydrolyzing activity. Importantly, the enzyme showed higher ß-glucosidase activity toward the ß-linked glucosidic bond of stevioside than toward other ß-linked glucobioses. Under optimal conditions, 70 U/L of the rubusoside-producing enzyme could produce 69.03 mM rubusoside from 190 mM stevioside. The ß-glucosidase activity on the cell surface was high at 35 h of culture. This is the first report detailing the production of rubusoside from stevioside by an enzyme derived from a food-grade lactic acid bacterium. The application of this ß-glucosidase could greatly reduce the cost of rubusoside production, hence benefiting all industries that use this natural product.

Optimization of Microwave-Assisted Green Method for Enhanced Solubilization of Water-Soluble Curcuminoids Prepared Using Steviol Glycosides.

In this study, the optimization and modeling of microwave-assisted extraction (MAE) of water-soluble curcuminoids prepared using novel steviol glycosides (SGs) was carried out using four independent process variables at varying levels-X1: microwave power (50-200 W), X2: stevioside concentration (50-200 mg/mL), X3: curcumin concentration (20-200 mg/mL), and X4: time (1-10 min)-in response surface methodology configuration. Moreover, the effects of stevioside, as the most cost-effective natural solubilizer, were also evaluated. The water solubility of curcuminoids increased from 11 to 1320 mg/L with the addition of stevioside as a natural solubilizer. Moreover, microwave heating synergistically with stevioside addition significantly (p < 0.05) increased the solubility up to 5400 mg/L. Based on the results, the optimum conditions providing the maximum solubilization of 16,700 mg/L were 189 W microwave power, 195 g/L stevioside concentration, 183 g/L curcuminoid concentration, and 9 min of incubation time. Moreover, MAE of curcuminoids using SGs might render a significant advantage for its wide-scale application to solubilizing the multitude of insoluble functional flavonoids in fruits, plants, and food materials.

Enzymatic Synthesis of Glucosyl Rebaudioside A and its Characterization as a Sweetener.

Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001 in Escherichia coli BL21 (DE3), forming single O-α-D-glucosyl-(1″→6') rebaudioside A with yield of 86%. O-α-D-glucosyl-(1″→6') rebaudioside A was purified using HPLC and Diaion HP-20 and its properties were characterized for possible use as a food ingredient. Almost 98% of O-α-D-glucosyl-(1″→6') rebaudioside A was dissolved after 15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O-α-D-glucosyl-(1″→6') rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus, O-α-D-glucosyl-(1″→6') rebaudioside A could be used as a highly pure and improved sweetener with high stability in commercial drinks. PRACTICAL APPLICATION: The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation. Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks. This method can be applied to obtain high value-added bioactive compounds by enzymatic modification.

Biochemical Characterization of Alkaliphilic Cyclodextran Glucanotransferase from an Alkaliphilic Bacterium, Paenibacillus daejeonensis.

Cycloisomaltooligosaccharide glucanotransferase (CITase) was isolated from alkaliphilic Paenibacillus daejeonensis via an amino acid homology search for the reported CITase. The recombinant alkaliphilic CITase (PDCITase) from P. daejeonensis was expressed in an Escherichia coli expression system and purified as a single protein band of 111 kDa. PDCITase showed optimum activity at pH 8.0 and retained 100% of activity within a broad pH range (7.0-11.5) after 18 h, indicating alkaliphilic or alkalistable CITase properties. In addition, PDCITase produced CI-7 to CI-17, CI-18, and CI-19, which are relatively large cycloisomaltooligosaccharides yet to be reported. Therefore, these large cycloisomaltooligosaccharides can be applied to the improvement of water solubility of pharmaceutical biomaterials.

Transglycosylation of gallic acid by using Leuconostoc glucansucrase and its characterization as a functional cosmetic agent.

Gallic acid glycoside was enzymatically synthesized by using dextransucrase and sucrose from gallic acid. After purification by butanol partitioning and preparative HPLC, gallic acid glucoside was detected at m/z 355 (C13, H16, O10, Na)+ by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The yield of gallic acid glucoside was found to be 35.7% (114 mM) by response surface methodology using a reaction mixture of 319 mM gallic acid, 355 mM sucrose, and 930 mU/mL dextransucrase. The gallic acid glucoside obtained showed 31% higher anti-lipid peroxidation and stronger inhibition (Ki = 1.23 mM) against tyrosinase than that shown by gallic acid (Ki = 1.98 mM). In UVB-irradiated human fibroblast cells, gallic acid glucoside lowered matrix metalloproteinase-1 levels and increased the collagen content, which was indicative of a stronger anti-aging effect than that of gallic acid or arbutin. These results indicated that gallic acid glucoside is likely a superior cosmetic ingredient with skin-whitening and anti-aging functions.

Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties.

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization-time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141mM, as determined by response surface methodology. CHG possessed a 65% increased water solubility and 2-fold browning resistance while it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation.

Synthesis and Functional Characterization of Caffeic Acid Glucoside Using Leuconostoc mesenteroides Dextransucrase.

Caffeic acid was modified via transglucosylation using sucrose and dextransucrase from Leuconostoc mesenteroides B-512FMCM. Following enzymatic modification, a caffeic acid glucoside was isolated by butanol separation, silica gel chromatography, and preparative HPLC. The synthesized caffeic acid glucoside had a molecular mass-to-charge ratio of 365 m/z, and its structure was identified as caffeic acid-3-O-α-d-glucopyranoside. The production of this caffeic acid-3-O-α-d-glucopyranoside at a concentration of 153 mM was optimized using 325 mM caffeic acid, 355 mM sucrose, and 650 mU mL-1 dextransucrase in the synthesis reaction. In comparison with the caffeic acid, the caffeic acid-3-O-α-d-glucopyranoside displayed 3-fold higher water solubility, 1.66-fold higher antilipid peroxidation effect, 15% stronger inhibition of colon cancer cell growth, and 11.5-fold higher browning resistance. These results indicate that this caffeic acid-3-O-α-d-glucopyranoside may be a suitable functional component of food and pharmaceutical products.

Synthesis and characterization of glucosyl stevioside using Leuconostoc dextransucrase.

Glucosyl stevioside was synthesized via transglucosylation by dextransucrase from Leuconostoc citreum KM20 (LcDexT), forming α-d-glucosyl stevioside. A production yield of 94% was reached after 5days of LcDexT reaction at 30°C. Glucosyl stevioside induced a 2-fold improved quality of taste and sweetness, compared to stevioside. After 15days of storage at 25°C, 98% of glucosyl stevioside in an aqueous solution was present in a soluble form, compared to only 11% for stevioside or rebaudioside A. Furthermore, glucosyl stevioside exhibited a similar or improved stability in commercially available soft drinks, when compared to stevioside and rebaudioside A. These results suggest that glucosyl stevioside could serve as a highly pure and stable sweetener in soft drinks.

Identification of Catalytic Amino Acid Residues by Chemical Modification in Dextranase.

A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3- (dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.

Glucosyl Rubusosides by Dextransucrases Improve the Quality of Taste and Sweetness.

Glucosyl rubusosides were synthesized by two dextransucrases. LcDexT was obtained from Leuconosotoc citreum, that LlDexT was obtained from Leuconostoc lactis. LcDexT and LlDexT regioselectively transferred a glucosyl residue to the 13-O-glucosyl moiety of rubusoside with high yield of 59-66% as analyzed by TLC and HPLC. Evaluation of the sweetness of these glucosyl rubusosides showed that their quality of taste, in particular, was superior to that of rubusoside. These results indicate that transglucosylation at the 13-O-glucosyl moiety of rubusoside by different regioselective dextransucrases can be applicable for increasing its sweetness and quality of taste.

Crystal structure of thermophilic dextranase from Thermoanaerobacter pseudethanolicus.

The crystal structures of the wild type and catalytic mutant Asp-312→Gly in complex with isomaltohexaose of endo-1,6-dextranase from the thermophilic bacterium Thermoanaerobacter pseudethanolicus (TpDex), belonging to the glycoside hydrolase family 66, were determined. TpDex consists of three structural domains, a catalytic domain comprising an (ß/α)8-barrel and two ß-domains located at both N- and C-terminal ends. The isomaltohexaose-complex structure demonstrated that the isomaltohexaose molecule was bound across the catalytic site, showing that TpDex had six subsites (-4 to +2) in the catalytic cleft. Marked movement of the Trp-376 side-chain along with loop 6, which was the side wall component of the cleft at subsite +1, was observed to occupy subsite +1, indicating that it might expel the cleaved aglycone subsite after the hydrolysis reaction. Structural comparison with other mesophilic enzymes indicated that several structural features of TpDex, loop deletion, salt bridge and surface-exposed charged residue, may contribute to thermostability.

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV.

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL(pro)) and a papain-like protease (PL(pro)) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1-9) and four coumarins (10-13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL(pro) and PL(pro)) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL(pro) and PL(pro) inhibitory activity with IC50 values of 11.4 and 1.2 µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL(pro), whereas noncompetitive inhibition was observed with the SARS-CoV PL(pro).

Purification and functional characterization of the first stilbene glucoside-specific ß-glucosidase isolated from Lactobacillus kimchi.

This study aimed to develop viable enzymes for bioconversion of resveratrol-glucoside into resveratrol. Out of 13 bacterial strains tested, Lactobacillus kimchi JB301 could completely convert polydatin into resveratrol. The purified enzyme had an optimum temperature of 30-40°C and optimum pH of pH 5.0 against polydatin. This enzyme showed high substrate specificities towards different substrates in the following order: isorhaponticin>>polydatin>>mulberroside A>oxyresveratrol-3-O-glucoside. Additionally, it rarely hydrolyzed astringin and desoxyrhaponticin. Based on these catalytic specificities, we suggest this enzyme be named stilbene glucoside-specific ß-glucosidase. Furthermore, polydatin extracts from Polygonum cuspidatum were successfully converted to resveratrol with a high yield (of over 99%). Stilbene glucoside-specific ß-glucosidase is the first enzyme isolated from lactic acid bacteria capable of bio-converting various stilbene glucosides into stilbene.

Characterization of a novel steviol-producing ß-glucosidase from Penicillium decumbens and optimal production of the steviol.

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing ß-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3-6.0 and at 40-60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and (1)H- and (13)C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other ß-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 µl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel ß-glucosidase, which may be useful for the pharmaceutical and agricultural areas.

Enzymatic synthesis of puerarin glucosides using Leuconostoc dextransucrase.

Puerarin (P), an isoflavone derived from kudzu roots, has strong biological activities, but its bioavailability is often limited by its low water solubility. To increase its solubility, P was glucosylated by three dextransucrases from Leuconostoc or Streptococcus species. Leuconostoc lactis EG001 dextransucrase exhibited the highest productivity of puerarin glucosides (P-Gs) among the three tested enzymes, and it primarily produced two P-Gs with a 53% yield. Their structures were identified as alpha-D-glucosyl-(1-->6)-P (P-G) by using LC-MS or (1)H- or (13)C-NMR spectroscopies and alpha-D-isomaltosyl-(1-->6)-P (P-IG2) by using specific enzymatic hydrolysis, and their solubilities were 15- and 202-fold higher than that of P, respectively. P-G and P-IG2 are easily applicable in the food and pharmaceutical industries as alternative functional materials.

Biochemical characterization of thermophilic dextranase from a thermophilic bacterium, Thermoanaerobacter pseudethanolicus.

TPDex, a putative dextranase from Thermoanaerobacter pseudethanolicus, was purified as a single 70 kDa band of 7.37 U/mg. Its optimum pH was 5.2 and the enzyme was stable between pH 3.1 and 8.5 at 70 degrees C. A half-life comparison showed that TPDex was stable for 7.4 h at 70 degrees C, whereas Chaetominum dextranase (CEDex), currently used as a dextranase for sugar milling, was stable at 55 degrees C. TPDex showed broad dextranase activity regardless of dextran types, including dextran T2000, 742CB dextran, and alternan. TPDex showed the highest thermostability among the characterized dextranases, and may be a suitable enzyme for use in sugar manufacture without decreased temperature.

Large increase in Leuconostoc citreum KM20 dextransucrase activity achieved by changing the strain/inducer combination in an E. coli expression system.

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction (OD600) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of 17 degrees C. These results may effectively apply to the heterologous expression of other large DexT genes.

Mass production of rubusoside using a novel stevioside-specific ß-glucosidase from Aspergillus aculeatus.

Rubusoside (R) is a natural sweetener and a solubilizing agent with antiangiogenic and antiallergic properties. However, currently, its production is quite expensive, and therefore, we have investigated nine commercially available glycosidases to optimize an economically viable R-production method. A stevioside (ST)-specific ß-glucosidase (SSGase) was selected and purified 7-fold from Aspergillus aculeatus Viscozyme L by a two-step column chromatography procedure. The 79 kDa protein was stable from pH 3.0 to pH 7.0 at 50-60 °C. Hydrolysis of ST by SSGase produced R and steviol monoglucosyl ester as determined by (1)H and (13)C nuclear magnetic resonance (NMR). Importantly, SSGase showed higher activity toward ST than other ß-linked glucobioses. The optimal conditions for R production were 280 mM ST and 16.6 µL of SSGase at pH 5.1 and 63 °C. This is the first discussion detailing the production of R by enzymatic hydrolysis of ST and is useful for the food additive and pharmaceutical industries.