• Journal of Life Science
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• v.26 no.12
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• pp.1477-1486
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• 2016

Lactulose (4-O-${\beta}$-D-galactopyranosyl-D-fructose) is a non-digestible synthetic ketose disaccharide which can used in food and pharmaceutical fields due to its useful functions for encephalopathy, chronic constipation, hyperammonemia, etc. Therefore, the lactulose is regarded as one of the most important disaccharides and have been concentrated much interesting as an attractive functional material in the current industry. From this reason, the research related on the production of lactulose has been carried out various academic and industrial research groups. To produce lactulose, two main methods, chemical production and enzymatic production have been used. Commercially lactulose produced by alkaline isomerization of lactose as chemical production method but it has many disadvantages such as rapid lactulose degradation, purification, and waste management. From these reasons, lactulose produced by enzymatic method which solves these problems has been suggested as a proper method for lactulose production. Two different enzymatic methods have been reported as methods for lactulose production. Lactulose can be obtained through hydrolysis and transfer reaction catalyzed by a ${\beta}$-galactosidase which requires fructose as co-substrate and exhibits a low conversion. Alternatively, lactulose can be produced by direct isomerization of lactose to lactulose catalyzed by cellobiose 2-epimerase which requires lactose as a single substrate and achieves a high lactulose yield. This review summarizes the current state of lactulose production by chemical and biological methods.

• Applied Biological Chemistry
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• v.13 no.1
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• pp.65-72
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• 1970

Two peptides (Im pr-M, Im ch-M) derived from Im ${\lambda}-type$ of Bence Jones Protein and one peptide (Ikch-M) from Ik were separated and purified using the Dowex $50{\times}2$ column $(1{\times}20\;cm)$ and Dowex $1{\times}2(0.9{\times}50\;cm)$. The buffer solution was composed of 1% pyridine and IM formic acid in Dowex $1{\times}2$ column. The blocked N-terminal was examined with ninhydrin reaction before and after alkaline hydrolysis, which was fractionated by Dowex $1{\times}2$ column. Pyrro-glutamic acid in N-terminal residue was identified by comparing with the authentic pyrro-glutamic acid through a high voltage electrophoresis (pH 3.5, 3000 V.) after the peptide Im pr-M (PCA. Ser) was cleavaged at the position of serine with cone. (12 N) HCl and the pyrro-glutamic acid was converted to glutamic acid by treating it with N-NaOH for 116 hours at $27^{\circ}C$. The substractive method was applied to find out the sequence of peptides and carboxypeptidase A was employed to release C-terminal residue from the peptide. In present study PCA. Ser in Im Pr-M was isolated from the pronase digested ${\lambda}$-type Bence Jones protein. The yield of the Im Pr-M was 79.6 percent of its theoretical value, based on the molecular weight of Bence Jones Protein. Im ch-M (PCA. Ser Val. Leu) was isolated from the chymotrypsin digested ${\lambda}$-type Bence Jones Protein. The yield of the Im ch-M was 72.2 percent. based on the molecular weight of Bence Jones Protein. Ik ch-M (PCA. Ser. Ala. Leu) was isolated from the chymotrypsin digested ${\lambda}$-type Bence Jones Protein and its yield was 42% based on the molecular weight of Bence Jones Protein.

• Korean Journal of Food Science and Technology
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• v.42 no.5
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• pp.593-597
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• 2010

This study investigated the properties and antioxidative activities of phenolic acid concentrates of rice bran. Rice bran contains bioactive substances such as phenolic compounds, which can provide health benefits as natural antioxidants. This study examined how levels of phenolic acids can be obtained efficiently through various extraction methods. The extractions of defatted rice bran were followed by using ethylacetate (RBE-I), ethylacetate after alkaline hydrolysis (RBE-II), and 80% methanol (RBE-III). For all extracts, yields (%), total polyphenol contents (TPC), various phenolic acids and antioxidative activities were estimated. RBE-II had the highest total polyphenol contents (526.72 mg/100 g rice bran) and showed high antioxidative activity (74.7%). To concentrate the phenolic acids, RBE-II was passed through Sep-pak $C_{18}$ Vac cartridge and F1-RBE-II was collected by the elution of 50% methanol. The total phenolic content of F1-RBE-II (736.8 mg/100 g rice bran) was higher than that of RBE-II (367.1 mg/100 g rice bran), and the ratios of ferulic acid (73%) and sinapic acid (14%) increased. As RBE-II was analysed by HPLC, 6 different phenolic acids were found via chromatography, whereas F1-RBE-II showed 5 different peaks and the major phenolic acid was identified as ferulic acid. The ABTS radical scavenging activity of F1-RBE-II was the highest among the rice bran extracts. In a ${\beta}$-carotene-linoleic acid model system, linoleic acid oxidation was reduced by F1-RBE-II (73%) and RBE-II (35%).

• Food Science and Biotechnology
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• v.18 no.1
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• pp.95-102
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• 2009

The aerial parts of garland (Chrysanthemum coronarium L.) were extracted in 80% aqueous methanol (MeOH) and the concentrated extract was then partitioned using ethyl acetate (EtOAc), n-butanol (n-BuOH), and $H_2O$, successively. EtOAc and n-BuOH fractions resulted in 4 glycerides with the application of octadecyl silica gel and silica gel column chromatography. The chemical structures of the glycerides were determined using several spectroscopic methods, including nuclear magnetic resonance (NMR) and mass spectrometry (MS) as (2S)-1-O-palmitoyl-sn-glycerol (1), (2S)-1-O-oleoyl-2-O-oleoyl- 3-O-$\beta$-D-galactopyranosyl-sn-glycerol (2), (2S)-1-O-palmitoyl-2-O-linoleoyl-3-O-phosphorouscholine-sn-glycerol (3), and (2S)-1-O-linolenoyl-2-O-palmitoyl-3-O-[$\alpha$-D-galactopyrasyl-($1{\rightarrow}6$)-$\beta$-D-galactopyranosyl]-sn-glycerol (4). The free fatty acids of these glycerides were determined with gas chromatography (GC)-MS analysis following alkaline hydrolysis and methylation. These glycerides demonstrated an inhibitory effect on acyl-CoA: cholesterol acyltransferase (ACAT, compound 1: $45.6{\pm}0.2%$ at $100{\mu}g/mL$), diacylglycerol acyltransferase (DGAT, compound 1: $59.1{\pm}0.1%$ at $25{\mu}g/mL$), farnesyl protein transferase (FPTase, compound 2: $98.0{\pm}0.1%$; compound 3: $55.2{\pm}0.1%$ at $100{\mu}g/mL$), and $\beta$-secretase ($IC_{50}$, compound 4: $2.6{\mu}g/mL$) activity. This paper is the first report on the isolation of these glycerides from garland and their inhibitory activity on ACAT, DGAT, FPTase, and $\beta$-secretase.

• Journal of the Korean Wood Science and Technology
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• v.37 no.3
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• pp.245-254
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• 2009

As a substitute for CCA, which is inhibited due to its environmental pollution and human harmfulness, and CuAz and ACQ with a high cost, okara-based wood preservatives were formulated with okara hydrolyzates using copper sulfate and/or borax as a metal salt. The efficacy of the preservatives and X-ray microanalysis of wood specimens treated with the preservatives were examined to confirm the potential of the okara-based wood preservatives. Most of the preservatives showed excellent decay resistance against brown-rot fungi, Postia placenta and Gloeophyllum trabeum. The efficacy was improved when the acid concentration and temperature used for the hydrolysis of okara increased. In addition, when borax was added into copper sulfate/okara hydrolyzates preservative formulations, any decay was not found in the specimens. From the microscopic observation of the specimens treated with okara-based wood preservatives, it seems that okara is contributed to the fixing of metal salts in wood blocks. Therefore, it is speculated that okara-based wood preservatives can effectively protect wood against fungal attack as CuAz, and that the preservatives are sufficient to use as an alternative wood preservative of CCA, ACQ and CuAz.

• Applied Biological Chemistry
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• v.41 no.5
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• pp.399-404
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• 1998

Genipin was obtained from hydrolysis of geniposide isolated from gardenia fruits with ${\beta}-glucosidase$. Reaction of genipin with glycine, alanine, histidine, lysine, phenylalanine and glutamate in aqueous buffer solution converted colorless starting materials to blue pigments. Effect of pH for the formation of blue pigments was tested using UV/Vis spectrophotometer. The optimum pH for the formation of blue pigments was 7.0. No pigment and trace amounts were formed at acidic (pH 3.0) and alkaline (pH 12.0) conditions, respectively. The amount and tincture of blue color were distinct with different amino acids. In contrast with lysine $({\lambda}_{max}=573\;nm)$, glycine $({\lambda}_{max}=595\;nm)$, phenylalanine $({\lambda}_{max}=602\;nm)$ and alanine $({\lambda}_{max}=595\;nm)$, the reaction of genipin with histidine $({\lambda}_{max}=601\;nm)$ and glutamate $({\lambda}_{max}=601\;nm)$ produced relatively small amounts of blue pigments. Rate constants for the formation of blue pigments from genipin with amino acids at various temperatures $(60,\;70,\;80,\;90^{\circ}C,\;pH\;7.0\;phosphate\;buffer)$ were obtained. Rate constants of genipin with basic amino acids were larger than neutral or acidic amino acids. Arrhenius activation energies of the formation of blue pigments indicated that activation energy of glycine $(E_A=9.8\;kcal/mol)$ was especially lower than those of other amino acids $(E_A=13.3{\sim}15.4\;kcal/mol)$.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.87-94
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• 2007

A bacterium which has high enzymatic activities such as amylase, cellulase and protease was isolated from Korean traditional soybean food, doenjang. The isolated bacterium was identified to Bacillus subtilis HS25 by the test of morphological and biochemical properties according to Bergey's Manual of Systematic Bacteriology and API 50 CHL kit, and by the 16S rDNA sequence. The isolated B. subtilis HS25 had a potent antibacterial activity against food born causative or pathogenic bacteria. B. subtilis HS25 is endospore forming cell and contained flagella and abundant viscous material at the out layer of cell wall. It was rod type bacterium $(0.5{\sim}0.8{\times}3{\sim}5{\mu}m)$ having biochemical characteristics such as gram staining(+), catalase(+), oxidase(-) and hydrolysis of esculin(+). The optimal medium compositions for production of antibacterial substance in the B. subtilis HS25 were 1% of soluble starch, 0.5% of yeast extract, 0.5% of peptone and 0.05% of MgCl$_2{\cdot}6H_{2}O$. The optimum temperature and pH of the growth of the B. subtilis HS25 was 35$^{\circ}C$ and pH 7.5, respectively. The antibacterial activity was more high in neutral to a little alkaline pH (6.5-10.5) than in acidic pH. The optimal shaking speed to grow and to produce antibacterial substance of the B. subtilis HS25 was 160${\sim}$200 rpm. The optimal culture time for antibacterial activities of the bacterium were shown to be in the range of 12-36 hr.

• Journal of Applied Biological Chemistry
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• v.59 no.4
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• pp.285-288
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• 2016

Proteolytic enzymes perform hydrolysis of the peptide bonds in the protein and most commonly use in the industry. Pseudoxanthomonas sp. WD12 and WD32 were previously isolated as protease producers from a rotten wood sample. Here, we report the secreted proteolytic enzymes. The optimum enzyme reaction temperature for the secreted crude enzyme from the strain WD12 and WD32 were $50^{\circ}C$ at pH 9.0 and $45^{\circ}C$ at pH 8.0, respectively. The enzyme activities of both strains were increased by addition of KCl, NaCl, $CaCl_2$ or $MnSO_4$, and decreased by addition of $AgNO_3$, $CuSO_4$, $FeCl_3$ or $AlCl_3$. Secreted enzymes of both strains were most strongly inhibited by addition of $FeCl_3$ or $CuSO_4$. Taken together these results, WD12 could be a candidate strain of industrial alkaline protease production.

• Journal of Life Science
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• v.23 no.12
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• pp.1486-1494
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• 2013

A high protease-producing strain was isolated and identified from the digestive tract of octopus vulgaris by detecting a hydrolysis circle of protease and its activity. The strain was identified by morphology observation, biochemical experiments, and 16S rRNA sequence analysis. The protease obtained from the strain was purified by a three-step process involving ammonium sulfate precipitation, carboxy methyl-cellulose (CM-52) cation-exchange chromatography, and DEAE-Sephadex A50 anion-exchange chromatography. The properties of protease were characterized as well. The strain Bacillus sp. QDV-3, which produced the highest activity of protease, was isolated. On the basis of the phenotypic and biochemical characterization and 16S rRNA gene-sequencing studies, the isolate was identified as follows: domain: Bacteria; phylum: Firmicutes; class: Bacilli; order: Bacillales; family: Bacillaceae; and genus: Bacillus. The isolate was shown to have a 99.2% similarity with Bacillus flexus. A high active protease designated as QDV-E, with a molecular weight of 61.6 kDa, was obtained. The enzyme was found to be active in the pH range of 9.0-9.5 and its optimum temperature was $40^{\circ}C$. The protease activity retained more than 96% at the temperature of $50^{\circ}C$ for 60 min. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme activity, thus confirming that this protease isolated from Bacillus sp. QDV-3 is an alkaline serine protease. Metal ions, $Mn^{2+}$ and $Mg^{2+}$, were determined to enhance the protease activity, whereas $Ba^{2+}$, $Zn^{2+}$, and $Cu^{2+}$ were found to inactivate the enzyme.

• Animal Bioscience
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• v.34 no.5
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• pp.867-879
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• 2021

Objective: Fibronectin 3 (FN3) and immunoglobulin like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researches have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase belonging to glycosyl hydorlase (GH) family 5 were determined. Methods: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete open reading frames (ORFs). The alkaline, thermostability, tertiary structure of deduced enzymes were predicted by AcalPred, TBI software, Phyre2 and Swiss models. Then, whole and truncated forms of a selected gene were expressed in Escherichia coli and purified by His-tag affinity column for assessment of FN3 ability to enhance enzyme activity, solubility and conformation. Results: From 297 complete ORFs coding for cellulases, 148 sequences containing FN3, Ig were identified. Mostly FN3 appeared in 90.9% beta-glucosidases belonging to glycosyl hydrolase family 3 (GH3) and situated downstream of catalytic domains. The Ig was found upstream of 100% endoglucanase GH9. Rarely FN3 was seen to be situated downstream of X domain and upstream of catalytic domain endoglucanase GH5. Whole enzyme (called XFN3GH5 based on modular structure) and truncate forms FN3, XFN3, FN3GH5, GH5 were cloned in pET22b (+) and pET22SUMO to be expressed in single and fusion forms with a small ubiquitin-related modifier partner (S). The FN3, SFN3 increased GH5 solubility in FN3GH5, SFN3GH5. The SFN3 partly served for GH5 conformation in SFN3GH5, increased modules interaction and enzyme-soluble substrate affinity to enhance SXFN3GH5, SFN3GH5 activities in mixtures. Both SFN3 and SXFN3 did not anchor enzyme on filter paper but exfoliate and separate cellulose chains on filter paper for enzyme hydrolysis. Conclusion: Based on these findings, the presence of FN3 module in certain cellulases was confirmed and it assisted for enzyme conformation and activity in both soluble and insoluble substrate.