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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.

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.

The total phenolic and flavonoid content and antioxidative activities during production of pear juice concentrate (PJC) from two cultivars, Hwasan and Niitaka, were investigated. The main processing steps in PJC production are washing, pressing, pasteurization, clarification, filtration, evaporation, and packaging. Total phenolic and flavonoid content of end-product PJC from Niitaka decreased by 53.11 and 46.47%, respectively, while those from Hwasan decreased by 55.46 and 36.09%, respectively, compared to the phenolic and flavonoid content of original fresh fruit. DPPH radical-scavenging activities, reducing power and nitrate radical-scavenging activities showed a similar tendency as total phenolic and flavonoid content; that is, they decreased in juice concentrate made from both cultivars. Also, antioxidant activities of press cake waste (skin and seeds) from Niitaka and Hwasan pears were higher than fresh pears. In conclusion, antioxidant levels were significantly affected during processing of PJC, especially during the pressing step in which press cake waste retains the seeds and skin.

This study was performed to characterize pear protease proteolytic activity and investigate the use of pear protease as a meat tenderizer. Pear protease was purified and stabilized by 5% dextrin during lyophilization (dry) or concentration (liquid). Pear protease was further characterized with respect to pH, thermodynamics, and enzyme kinetics. Pear protease was stable at a pH range of 5-8 with an optimum pH of 6.5. From Arrhenius plots, liquid protease showed higher temperature dependency (23.49 kJ/mol) than dry protease (18.62 kJ/mol) due to its higher activation energy. The kcat/Km, catalytic efficiency of enzyme, was similar with 2.9 and 2.7 µM/min with dry and liquid proteases. Pear protease was evaluated for its proteolytic activities with casein and beef myofibrillar proteins by individually and combination with fig and kiwifruit proteases. These result indicated that pear and kiwifruit proteases could be complementary to be a desirable product for meat tenderization.

Different ascorbic acid (AA) concentrations of 0.16, 0.20, and 0.24% (w/v) were added to pear juice from the new cultivar Pyrus pyrifolia Nakai cv. "Sinhwa". Enzymatic browning reduction and antioxidant activity were analyzed after 24 h at 37°C. Juices with 0.20% added AA showed the highest inhibition of 78.8% of polyphenol oxidase (PPO) activity. L* values of juices a with 0.20 and 0.24% added AA decreased more slowly than controls lacking AA addition and juice with 0.16% added AA after storage for 24 h. Browning indices of juices with added AA were lower than for controls. However, indices increased after storage for 24 h. The DPPH radical-scavenging activity, reducing power, and nitrite scavenging activity of all juices with added AA were higher than for controls and decreased after storage for 24 h. Addition of 0.20% AA to pear juice from the new "Sinhwa" cultivar showed the highest browning activity reduction.

Pear fruit is one of the most popular fruits, extensively exploited for its protease activity commercially in the food, cosmetic, and pharmacological industries. The aim of this study was to compare four different purification methods for protease purification from an Asian pear cultivar. Pear proteases were purified by ethanol precipitation, two phase partitioning, or ion exchange chromatography (DEAE Sepharose alone or followed by Mono Q). Purified proteases from each method were further evaluated for purification efficiency by SDS-PAGE analysis and enzyme activity with succinlyated casein substrate. All methods produced two protease bands with molecular weights of 36 kDa and 38 kDa. Among the methods, 15% PEG 1000–20% MgSO4 system and DEAE followed by Mono Q chromatography showed efficient separation of protease with high recovery of enzymatic activity (91–98%). DEAE followed by Mono Q chromatography achieved the highest purification fold (15). These result suggested that a two phase partitioning technique is effective as a single purification step for pear protease.

This study was performed to compare the total protein and protease content among the whole fruit, flesh, and peel of nine different pear cultivars. Pear proteases were functionally characterized with respect to three enzyme assays. Proteases from pears were further identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in gel activity staining, and matrix assisted laser desorption/ionization-time of flight mass spectrometry analysis. Flesh from Whasan, Nikita, and Hanareum cultivars contained relatively more total protein and protease and showed high enzyme activities, while Chuwhang contained the lowest amount of protein and protease activity. Protease content and enzyme activities found in the pear flesh or whole fruits were two to six times higher than those in the pear peel. Pear cultivars contained one or two protease bands with molecular weights of 36 kDa and/or 38k Da. The larger band was further identified as a cysteine proteinase with 70% homology to the pear cysteine protease f...

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 141 mM, as determined by response surface methodology. CHG possessed a 65% increase in water solubility and a 2-fold browning resistance and 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.

The effects of treatment with various complex carbohydrases such as Pectinex, Celluclast, Viscozyme, and Ultraflo on the physiochemical properties, polyphenol extraction yields and antioxidant activities of pear peel and pear core were investigated. When pear peel and pear core were treated with complex carbohydrases, the soluble solid content of peel increased, whereas it did not change significantly in the case of pear core. When pear peel and pear core were treated with Pectinex, significant improvement of soluble solid content was observed along with the highest extraction yield of reducing sugar content. Total sugar content increased in most of the enzyme treatment groups. In the case of pear peel, the Viscozyme treatment group showed the highest total polyphenol contents, total flavonoid contents, DPPH radical scavenging activity, ABTS radical scavenging activity, and SOD-like activity. When the flesh and core of pear were treated with Celluclast, total polyphenol contents increased. All enzyme treatment groups except for the Ultraflo treatment group showed increases in total flavonoid contents. With regard to pear flesh, the Celluclast group showed the highest DPPH radical scavenging activity. When pear core was treated with the four complex carbohydrases, DPPH radical scavenging activity and ABTS radical scavenging activity did not increase significantly. However, the SOD-like activity of all enzyme treatment groups significantly increased. Consequently, dry matter and soluble solid contents, polyphenol content, and antioxidant activity of pear peel and core could be improved by complex carbohydrase treatment.

This study was performed to confirm physiological activities according to parts of new pear cultivars (Gamcheonbae, Manpungbae, Chuwhangbae, Hanareum) and Niitaka pear. The total polyphenol compound contents of pear peel, flesh and core were 178~235, 95~113, 177~229 mg/100 g as tannic acid equivalent, respectively. There were differences in the contents by cultivars, Chuwhangbae and Hanareum cultivars showed high contents. The total flavonoid contents of the pear peel, flesh and core were 29.2~40.2, 24.3~34.3, 26.9~38.8 mg/100 g, respectively and those of Chuwhangbae and Gamcheonbae cultivars showed comparatively high values. The electron-donating ability was high in Chuwhangbae, Gamcheonbae and in the pear peel (29.7~57.7%), core (29.1~38.2%), flesh (7.6~17.7%), in that order. The nitrate scavenging activity was highest in that pear peel (21.0~49.8%), followed by the core (11.8~16.2%) and flesh (7.8~9.7%), but there was little difference by cultivar.

The human fibroblast collagenase catalytic domain (MMP1ca) that is considered a prototype for all interstitial collagenase and plays an important role in the turnover of collagen fibrils in the matrix was expressed as an inclusion body in the Escherichia coli. The purified enzyme displayed activity with substrate Dnp-Pro-Leu-Ala-Leu-Trp-Ala-Arg-OH with a K(m) value of 26.61±1.42 μM. The inhibition activity of the nine flavonoid compounds and gallic acid against MMP1ca was examined. Among the compounds tested, the IC(50) of seven flavonoid compounds were determined and ranged from 14.13 to 339.21 μM. Epigallocatechin gallate (EGCG) showed the highest inhibition toward MMP1ca with IC(50) values of 14.13±0.49 μM. EGCG showed a competitive inhibition pattern with a K(i) value of 10.47±0.51 μM. The free binding energy of EGCG against MMP1ca was -13.07 kcal mol(-1), which was calculated by using Autodock 3.0.5 software and showed numerous hydrophobic and hydrogen bond interactions. The galloyl group of EGCG, gallocatechin gallate and epicatechin gallate was determined to be important for inhibitory activity against MMP1ca.

Acarbose-fructoside (acarbose-Fru) was newly synthesized via the acceptor reaction of a levansucrase from Leuconostoc mesenteroides B-512 FMC with acarbose and sucrose. The resultant product was separated with 10.5% purification yield via Bio-gel P-2 column chromatography and HPLC. Its structure was determined to be 1 I -β- d -fructofuranosyl α-acarbose, according to the results of 1 H, 13 C, HSQC, and HMBC analyses. Acarbose-Fru was inhibited competitively on α-glucosidase ( A. niger and baker's yeast) but mixed noncompetitively on α-amylases ( A. oryzae and porcine pancreatic). Compared to acarbose, acarbose-Fru exhibited inhibition potency of 1.12 or 1.52 on A. niger α-glucosidase or A. oryzae α-amylase, respectively. Additionally, acarbose-Fru was identified as a novel substrate for dextransucrase with K m and V max values of 189.0 mM and 8.51 μmol/(mg min), respectively. Therefore, acarbose-Fru as a substrate might be synthesized novel acarbose derivatives by using dextransucrase.

Leuconostoc mesenteroides NRRL B-1149 sucrose phosphorylase (SPase) gene, 1149sp, was isolated and characterized. It is composed of 1479 bp nucleotides and encodes a 1149SPase of 492 amino acid residues with a calculated molecular mass of 56.1 kDa. It has unique C-terminal amino acid sequence (439DVETPSDTTIKITRKDKSGENVAVLVANAADKTFTITANGEEILANTEADKQQL492). 1149sp was expressed in Escherichia coli and the purified 1149SPase specific activity was 1.49 U/mg for sucrose. The optimum temperature and pH for SPase activities were ranged broad between 20 and 50 °C, between pH 6.0 and 7.5, respectively. The optimum temperature and pH were 37 °C at pH 6.7 and it showed Km of 6.3 mM and kcat of 1.59 s−1 for sucrose. It had a broad range of acceptor specificity and transferred the glucosyl moiety of sucrose or glucose-1-phosphate to various acceptors.

In this study, three epigallocatechin gallate glycosides were synthesized by the acceptor reaction of a glucansucrase produced by Leuconostoc mesenteroides B-1299CB with epigallocatechin gallate (EGCG) and sucrose. Each of these glycosides was then purified, and the structures were assigned as follows: epigallocatechin gallate 7-O-α-d-glucopyranoside (EGCG-G1); epigallocatechin gallate 4‘-O-α-d-glucopyranoside (EGCG-G1‘); and epigallocatechin gallate 7,4‘-O-α-d-glucopyranoside (EGCG-G2). One of these compounds (EGCG-G1) was a novel compound. The EGCG glycosides exhibited similar or slower antioxidant effects, depending on their structures (EGCG ≥ EGCG-G1 > EGCG-G1‘ > EGCG-G2), and also manifested a higher degree of browning resistance than was previously noted in EGCG. Also, EGCG-G1, EGCG-G1‘, and EGCG-G2 were 49, 55, and 114 times as water soluble, respectively, as EGCG. Keywords: Leuconostoc mesenteroides glucansucrase; EGCG; glycoside; acceptor reaction

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB). Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized beta-casein (30 mg/g beads) column. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP. The cold water and Mg-ATP eluates were analyzed by SDS-PAGE. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB. The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented.

A non-positional (or suspension) cell microarray was developed using shape-coded SU-8 photoresist microboards for potential application in multiplex and high-throughput cell-based assays. A conventional photolithography process on glass slides produced various shapes of SU-8 micropatterns that had a lateral dimension of 200 μm and a thickness of 40 μm. The resultant micropatterns were detached from the slides by sonication and named “microboards” due to the fact that had a much larger lateral dimension than thickness. The surfaces of the SU-8 microboards were modified with collagen to promote cell adhesion, and it was confirmed that collagen-coated SU-8 microboards supported cell adhesion and proliferation. Seeding of cells into poly(ethylene glycol)(PEG) hydrogel-coated well plates containing collagen-modified microboards resulted in selective cell adhesion onto the microboards due to the non-adhesiveness of PEG hydrogel toward cells, thereby creating non-positional arrays of microboards carrying cells. Finally, two different cell types (fibroblasts and HeLa cells) were separately cultured on different shapes of microboards and subsequently mixed together to create a non-positional cell microarray consisting of multiple cell types where each cell could be easily identified by the shape of the microboard to which they had adhered. Because numerous unique shapes of microboards can be fabricated using this method by simply changing the photomask designs, high throughput and multiplex cell-based assays would be easily achieved with this system in the future.