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The sweet-tasting protein brazzein offers considerable potential as a functional sweetener with antioxidant, anti-inflammatory, and anti-allergic properties. Here, we optimized a chemically defined medium to produce secretory recombinant brazzein in Kluyveromyces lactis, with applications in mass production. Compositions of defined media were investigated for two phases of fermentation: the first phase for cell growth, and the second for maximum brazzein secretory production. Secretory brazzein expressed in the optimized defined medium exhibited higher purity than in the complex medium; purification was by ultrafiltration using a molecular weight cutoff, yielding approximately 107 mg L−1. Moreover, the total media cost in this defined medium system was approximately 11% of that in the optimized complex medium to generate equal amounts of brazzein. Therefore, the K. lactis expression system is useful for mass-producing recombinant brazzein with high purity and yield at low production cost and indicates a promising potential for applications in the food industry.

Obesity is a global chronic disease linked to various diseases. Increased consumption of added sugars, especially in beverages, is a key contributor to the obesity epidemic. It is essential to reduce or replace sugar intake with low-calorie sweeteners. Here, a natural sweet protein, 3M-brazzein, was investigated as a possible sugar substitute. Mice were exposed to 3M-brazzein or 10% sucrose of equivalent sweetness, in drinking water to mimic human obesity development over 15 weeks. Consumption of 3M-brazzein in liquid form did not cause adiposity hypertrophy, resulting in 33.1 ± 0.4 g body weight and 0.90 ± 0.2 mm fat accumulation, which were 35.9 ± 0.7 g (p = 0.0094) and 1.53 ± 0.067 mm (p = 0.0031), respectively, for sucrose supplement. Additionally, 3M-brazzein did not disrupt glucose homeostasis or affect insulin resistance and inflammation. Due to its naturally low-calorie content, 3M-brazzein could also be a potential sugar substitute that reduces adiposity.

The sweet-tasting protein, brazzein, has potential as a low-calorie sugar substitute owing to its high sweetness, stability, and water solubility. In this study, the synthetic brazzein gene was expressed in the tobacco plant, Nicotiana tabacum. Three types of expression cassettes containing the brazzein gene were constructed to examine the expression and purification efficiency of the brazzein: pBI-BZ1 containing a signal sequence and His-tag, pBI-BZ2 containing a signal sequence, and pBI-BZ3 containing only the brazzein gene. Brazzein expression confirmed by ELISA was purified using ammonium sulfate precipitation, heat treatment, and CM-sepharose chromatography. The purity and conformational state of the brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the brazzein was confirmed by N-terminal amino acid analysis, ESI-MS/MS, and sweetness analysis. We successfully generated brazzein overexpression tobacco plants, suggesting that this method could be used as a brazzein production platform to provide an alternative to currently produced sweeteners.

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast Kluyveromices lactis and purification method for maximum yield. Transformed K. lactis was cultured in YPGlu (pH 7.0) at 25 °C and induced by adding glucose:galactose at a weight ratio of 1:2 (%/%) during the stationary phase, which increased brazzein expression 2.5 fold compared to the previous conditions. Cultures were subjected to heat treatment at 80 °C for 1 h, and brazzein containing supernatant was purified using carboxymethyl-sepharose cation exchange chromatography using 50 mM NaCl in 50 mM sodium acetate buffer (pH 4.0) as a wash buffer and 400 mM NaCl (pH 7.0) for elution. The yield of purified brazzein under these conditions was 2.0-fold higher than that from previous purification methods. We also determined that the NanoOrange assay was a suitable method for quantifying tryptophan-deficient brazzein. Thus, it is possible to obtain pure recombinant brazzein with high yield in K. lactis using our optimized expression, purification, and quantification protocols, which has potential applications in the food industry.

Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase. Seven histidine mutants in the two copper-binding sites were generated, and catalytic activities were characterised. The tyrosine hydroxylase activities of the CuA site mutants were approximately 50% lower than those of the wild-type tyrosinase, while the dopa oxidation activities of the mutants were not significantly different from that of wild-type tyrosinase. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylation and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetic and pharmaceutical industries.

Rapid and simple spectrophotometric methods are required to detect various oligosaccharides produced by agar-hydrolysing enzymes. Herein, we present a quantitative agarose-iodine assay for agarase activity determination via the detection of the extent of agarose degradation. The agarose-iodine complex becomes reddish orange upon the addition of Lugol solution, and the enzymatic activity can be detected with ultraviolet-visible spectroscopy at 600 nm. The main advantages of this modified Lugol assay are high sensitivity, simple detection, and cost effectiveness. A novel definition of the unit to measure and compare the activities of agarases is also suggested.

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6 mM isopropyl β-D-1-thiogalactopyranoside for 9 h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ∼10 mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu

To identify critical residues responsible for sweetness in brazzein and elucidate the interaction mechanisms of brazzein with the sweet taste receptor, three mutants of Lys5 residue in N-terminal β-strand I of brazzein were constructed by site-directed mutagenesis. Mutations of Lys to Asp or Glu at position 5 of brazzein significantly decreased its sweetness, while mutation of Lys5 to Arg resulted in a molecule with slightly decreased sweetness to des-pE1M-brazzein. From these results, it is suggested that the positive charge of Lys5 in β-strand I of brazzein is essential for its function and necessary for structural integrity. Practical Applications Brazzein is a sweet-tasting protein which has been isolated from the fruit of the West African plant Pentadiplandra brazzeana Baillon and is 500 to 2,000 times sweeter than sucrose. Brazzein is attractive as a candidate sweetener for control of obesity, because of its potential sweetness, sugar-like taste and good stability. The present study offers information on the precise interaction mechanism of brazzein with human sweet taste receptor responsible for the sweetness of brazzein, and can be of great value in future design of sweeter brazzein variants.

BACKGROUND The sweetness of brazzein, one of the known sweet proteins, is dependent on charges and/or structures of its specific amino acid side chains. As the residues in the C-terminus of brazzein are known to play a critical role in sweetness, the currently unknown function of Glu53 requires further study. RESULTS To identify important residues responsible for the sweetness of the protein brazzein, four mutants of the Glu53 residue in the C-terminal region of des-pE1M-brazzein, which lacks the N-terminal pyroglutamate, were constructed using site-directed mutagenesis. Mutations of Glu53 substitution to Ala or Asp significantly decreased the sweetness. On the other hand, a Lys mutation resulted in a molecule with sweetness similar to that of des-pE1M-brazzein. Mutation of Glu53 to Arg resulted in a molecule significantly sweeter than des-pE1M-brazzein, which agrees with previous findings showing that mutation with positively charged residues results in a sweeter protein. CONCLUSION Our results suggest that the residue at position 53 is crucial for the sweetness of brazzein, which may be interacting with the sweet-taste receptor. © 2015 Society of Chemical Industry

Sweet-tasting proteins may be useful as low-calorie sugar substitutes in foods, beverages, and medicines. Brazzein is an attractive sweetener because of its high sweetness, sugar-like taste, and good stability at high temperature and wide pH ranges. To investigate the bioactivities of brazzein, the antibacterial, antifungal, antioxidant, anti-inflammatory, and anti-allergic activities were determined in vitro. Brazzein showed no antibacterial and antifungal activities, although it showed approximately 45% or greater similarity to defensin, which has antimicrobial effects, and drosomycin, which is used as an antifungal agent. However, brazzein exhibited strong antioxidant effects, showing ABTS radical scavenging activity (IC50=12.55μM) and DPPH activity (IC50>30μM). Brazzein also showed anti-inflammatory activity and anti-allergic activity in a β-hexosaminidase assay (IC50>15μM) and cyclooxygenase-2 inhibition assay (IC50=12.62μM), respectively. These results suggest that brazzein has antioxidant, anti-inflammatory, and anti-allergic activities and considerable potential as a functional sweetener.

E-mail: khkong@cau.ac.krReceived May 14, 2014, Accepted July 19, 2014Key Words : Brazzein, Model building, Peptide, Sweetness loop, Sweet-tasting proteinHumans recognize five tastes, namely, sweet, bitter, sour,salty, and umami, through taste buds on the tongue. Thesefive tastes give organism essential information with which toevaluate the nutritional components of food, as well asprevent the intake of toxic substances. The desirable tastesof umami and sweetness promote consumption of nutritivefood. On the other hand, bitterness and sourness alertorganisms to toxins and promote rejection of harmful foods.Identifying sweet-tasting foods is particularly important as itprovides organisms with a means to seek out carbohydrateswith high nutritive value.

To identify the critical residue for the catalytic activity of rice glutathione S -transferases (GSTs), the evolutionarily conserved serine residues in the N-terminal domain of rice phi class GST F5 ( Os GST F5) were replaced with Ala by site-directed mutagenesis. Replacement of Ser9 with Ala significantly affected the catalytic activity and kinetic parameters of the enzyme, whereas substitutions of Ser34 and Ser65 with Ala showed little effect. The S9A mutant showed a decrease of approximately 90–99% in the specific activities and k cat values. From the plot of pH against log( k cat / K m ), the p K a values for glutathione (GSH) in the wild-type enzyme-GSH complex and the S9A-GSH complex were estimated to be 6.8 and 8.6, respectively. From these results, we suggest that the evolutionarily conserved Ser9 residue in Os GST F5 plays a major role in the catalytic mechanism of rice GST by lowering the enzyme's p K a and enhancing the nucleophilicity of the GSH thiol in the active site.

To elucidate the role of the evolutionarily conserved Tyr8 residue in rice Phi-class GSTF3, this amino acid was replaced with alanine and phenylalanine by site-directed mutagenesis, respectively. The replacement of Tyr8 with Ala significantly affected the catalytic activity and the kinetic parameters, whereas the substitutions of Tyr8 with Phe had almost no effect. The Y8A mutant resulted in approximately 90-100% decrease of the specific activity. Moreover, the Y8A mutant resulted approximately in 2-fold increase of Km, approximately 60-80% decrease of kcat, and approximately 6.5-fold decrease in kcat/Km. From the pH/log kcat/Km plot, pKa values of the GSH in the wild-type enzyme-GSH complex, Y8A-GSH complex and Y8F-GSH complex were estimated to be approximately 6.8, 8.5 and 6.9, respectively. From these results, we suggest that the evolutionarily conserved Tyr8 residue in OsGSTF3 seems to influence the structural stability of the active site of OsGSTF3 rather than directly its catalytic activity.

Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis . The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomyces cerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96 h at 30 °C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS–PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry.

We have previously identified critical residues important for sweetness of the sweet protein brazzein by site-directed mutagenesis ( Yoon, Kong, Jo, & Kong, 2011 ). In order to elucidate the interaction mechanisms of brazzein with the sweet taste receptor, we made multiple mutations of three residues (His31 in loop 30–33, Glu36 in β-strand III, and Glu41 in loop 40–43). We found that all double mutations (H31R/E36D, H31R/E41A and E36D/E41A) made the molecules sweeter than des-pE1M-brazzein and three single mutants. Moreover, the triple mutation (H31R/E36D/E41A) made the molecule significantly sweeter than three double mutants. These results strongly support the hypothesis that brazzein binds to the multisite surface of the sweet taste receptor. Our findings also suggest that mutations reducing the overall negative charge and/or increasing the positive charge favour sweet-tasting protein potency.

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that catalyze the formation of conjugates between reduced glutathione (GSH) and a variety of carcinogenic, mutagenic, toxic and pharmacologically active compounds. 1,2 GSTs have also been shown to participate in other biological process. Certain GSTs can detoxify lipid and DNA hydroperoxide by their intrinsic peroxidase activity, while others can catalyze the isomerization of certain steroids. Mammalian cytosolic GSTs, which exist as homo- or heterodimers, are grouped into at least seven distinct classes alpha, mu, pi, sigma, theta, sigma, kappa and zeta - according to their physical, chemical, immunological and structural properties. 1,3,4 Human pi-class hGST P1-1 contains eight arginine residues. Among them, Arg13 is the only residue conserved in all the known Pi class GSTs and in most of the Alpha class GSTs. The highly conserved residues are expected to be important to the enzyme's structure and function. hGST P1-1 is particularly interesting as it can be used as a reliable preneoplastic or neoplastic marker because it is expressed at raised levels by a number of human tumors. 5,6 It has also

Brazzein is an intensely sweet protein with high stability over a wide range of pH values and temperatures, due to its four disulfide bridges. Recombinant brazzein production through secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect disulfide formation, which is crucial for achieving the final protein structure and stability. Protein disulfide bond formation requires protein disulfide isomerase (PDI) and Ero1p. Here, we overexpressed KlPDI in K. lactis or treated the cells with dithiothreitol to overexpress KlERO1 and improve brazzein secretion. KlPDI and KlERO1 overexpression independently increased brazzein secretion in K. lactis by 1.7-2.2- and 1.3-1.6-fold, respectively. Simultaneous overexpression of KlPDI and KlERO1 accelerated des-pE1M-brazzein secretion by approximately 2.6-fold compared to the previous system. Moreover, intracellular misfolded/unfolded recombinant des-pE1M-brazzein was significantly decreased. In conclusion, increased KlPDI and KlERO1 expression favors brazzein secretion, suggesting that correct protein folding may be crucial to brazzein secretion in K. lactis.

Department of Fine Chemistry, Seoul National University of Science and Technology, Seoul 139-743, KoreaReceived May 17, 2012, Accepted June 2, 2012Key Words : Human tyrosinase, Deletion mutants, Biochemical properties, Expression in Escherichia coliTyrosinase (monophenol, dihydroxyphenylalanine: oxygenoxidoreductase, EC 1.14.18.1) is an essential enzyme in thebiosynthesis of melanin pigment, which has a protectiveeffect against injurious ultraviolet radiation.

The only human antimicrobial peptide, LL-37, was overexpressed in soluble form using Escherichia coli-based expression system. Recombinant LL-37 production in E. coli was optimized for use in large quantities for studying antimicrobial activity against Helicobacter pylori strains. We previously reported a method to express and purify LL-37 using Glutathione S-transferase (GST) fusion system. Herein presents method suitable for producing recombinant LL-37 from the recombinant GST-LL-37 fusion protein by recovering from both soluble protein fractions and inclusion bodies. Compared to the yield reported previously, the yield of recombinant GST-LL-37 protein was much improved to 10 mg/L of culture by affinity chromatography using GSTrap FF (1 mL) affinity chromatography column. These results suggest that the production method used in present study is useful in obtaining large quantity of recombinant LL-37 peptide. The optimized recovery protocol from inclusion bodies was highly contributable in raising yield of GST-LL-37 fusion protein. When treated with Factor Xa, GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies released an approximate 4.5-kDa protein, which was the expected size of LL-37; GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies was also confirmed by enzymatic digestion with thrombin, which produced LL-37 peptide containing six extra amino acid residues, Gly-Ile-Ile-Glu-Gly-Arg, on Nterminus of LL-37. Purified recombinant LL-37 showed nearly identical antimicrobial activities against H. pylori strains as that of synthetic LL-37, suggesting its application to functional study with therapeutic potential on gastric pathogen.

To identify critical residues, important for sweetness, of the sweet protein brazzein, 11 mutants of the residues in three loops of brazzein were constructed by site-directed mutagenesis. We found that mutations of Glu41 to Ala, Lys, or Arg at position 41 in loop 40–43 made the molecules significantly sweeter than brazzein, while mutations at two distant residues (changing Arg43 to Lys or Glu) decreased sweetness. A similar pattern occurred at loop 30–33, where mutation of the His31 to Arg significantly increased sweetness, while mutations at positions 30 or 33 in the immediate vicinity of this region significantly decreased sweetness. In addition, a Gln17 residue in the loop 9–19 was necessary for structural integrity. From these results, we suggest that the loops containing His31 and Glu41 are critical regions of the molecule for eliciting sweetness, and the charge and/or structure of the side chain of these residues play an important role in the multi-point interactions between brazzein and the sweet-taste receptor.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are enzymes that detoxify endobiotic and xenobiotic compounds by covalently linking glutathione (GSH) to a hydrophobic substrate, forming a less reactive and more polar glutathione S-conjugate. 1 In plants, GSTs detoxify herbicides, organic pollutants, and natural toxins; they also protect cells from a wide range of biotic and abiotic stresses, including pathogen attack, xenobiotic and heavy-metal toxicity, and oxidative stress. 2,3 Plant GSTs are grouped into five classes (phi, zeta, tau, theta and lambda) based on sequence identity, gene organization, and active site residues in the proteins. A further GST-like class, DHAR (proteins with dehydroascorbate reductase activity), was recently reported with members in Arabidopsis, rice and soybean. 2 The function of these plant-specific GSTs is detoxification of herbicides in both crops and weeds. Despite all available research, both the biochemical properties and functions of plant GSTs remain to be elucidated. Rice is the most important crop for human consumption with more than a half of the world’s population utilizing it as an energy source. The entire rice genomes for the japonica and indica subspecies have been sequenced. 4 From these genome sequences, a GST gene homolog (GenBank Accession No. AF309379) was identified from Oryza sativa by homolog searches in the NCBI database. As yet, there have been no reports concerning this gene product. Therefore, we cloned this gene and expressed it in Escherichia coli. The mRNA from Oryza sativa L. cv. Yamahousi was isolated from the cell culture and transcribed into cDNA. Primers for PCR, using the cDNA as a template, were derived from a published cDNA sequence from Oryza sativa L. cv. Japonica. 5 Subsequently, a 702-bp fragment of Oryza sativa cDNA was amplified by PCR, subcloned into the expression vector pET-26b(+) and transformed into the E. coli strain BL21 (DE3). Both DNA strands were sequenced with the final amino acid sequence bearing a difference between the PCR-identified sequence and the earlier cDNAderived sequence (GenBank Accession No. AF309379). The gene whereby guanine was substituted with thymine at position of nucleotide 166 gave an Asn 55 residue instead of Lys 55. The cloned gene was composed of 702-bp encoding for the 233 amino acids and designated OsGSTU3. A multiple alignment of the OsGSTU3 protein with other GSTs is shown in Figure 1. Sequence aliment was performed using the T-Coffee and Boxshade programe (http:// www.ch.embnet.org). GST isoenzymes belonging to the same class showed more than approximately 60% identity in their primary structure, whereas enzymes belonging to phi and theta classes generally had less than 2% sequence identity. 2 Zeta class enzymes also bore less than 24% sequence identity. Based on its gene sequence, OsGSTU3 was presumed to belong to the plant-specific tau class GST. The E. coli BL21 (pET-OsGSTU3) clone was tested for production of recombinant OsGSTU3 protein. The expressed OsGSTU3 protein was purified by S-hexylGSH affinity

The DNA sequence of the synthesized brazzein gene en-coding 53 amino acids that combined with the pelB leader sequence and amino acid sequence of the resulted recombinant brazzein. The sequence ends with two TAG and TAA termination codons. Gray box( ) indicates the sequence of the pelB leader sequence.

Arg13 is a conserved active-site residue in all known Pi class glutathione S-transferases (GSTs) and in most Alpha class GSTs. To evaluate its contribution to substrate binding and catalysis of this residue, three mutants (R13A, R13K, and R13L) were expressed in Escherichia coli and purified by GSH affinity chromatography. The substitutions of Arg13 significantly affected GSH-conjugation activity, while scarcely affecting glutathione peroxidase or steroid isomerase activities. Mutation of Arg13 into Ala largely reduced the GSH-conjugation activity by approximately 85 - 95%, whereas substitutions by Lys and Leu barely affected activity. These results suggest that, in the GSH-conjugation activity of hGST P1-1, the contribution of Arg13 toward catalytic activity is highly dependent on substrate specificities and the size of the side chain at position 13. From the kinetic parameters, introduction of larger side chains at position 13 results in stronger affinity (Leu > Lys, Arg > Ala) towards GSH. The substitutions of Arg13 with alanine and leucine significantly affected k cat , whereas substitution with Lys was similar to that of the wild type, indicating the significance of a positively charged residue at position 13. From the plots of log (k cat /K m CDNH ) against pH, the pK a values of the thiol group of GSH bound in R13A, R13K, and R 13 L were estimated to be 1.8,1.4, and 1.8 pK units higher than the pK a value of the wild-type enzyme, demonstrating the contribution of the Arg13 guanidinium group to the electrostatic field in the active site. From these results, we suggest that contribution of Arg13 in substrate binding is highly dependent on the nature of the electrophilic substrates, while in the catalytic mechanism, it stabilizes the GSH thiolate through hydrogen bonding.

The human tyrosinase ectodomain has been expressed in Escherichia coli as a soluble form and purified by immobilized metal affinity column chromatography. The ectodomain exhibited tyrosinase activities toward the hydroxylation and oxidation reactions. Biochemical properties of the ectodomain appeared to be distinct from those of the human tyrosinase, although common features were retained.

Glutathione S-transferase is known to play a crucial role in detoxification in many cases. To develop a herbicide detection biosensor, we in this study attempted to immobilize glutathione S-transferase enzyme on solid supports, polystyrene and agarose, and Na-alginate. These matrixes were attractive materials for the construction of biosensors and might also have utility for the production of immobilized enzyme bioreactors. We also compared the activities of glutathione-S-transferase immobilized OsGSTF3 and free OsGSTF3. The specific activity of the free enzyme in solution was 3.3 higher than the immobilized enzyme. These results suggest that 50% of the enzyme was bound with the catalytic site in polystyrene-alkylamine bead and immobilized enzymes showed 80% remaining activity until 3 times reuse.

A gene (PH0311) encoding a hypothetical protein from the genome sequence data of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and over-expressed in Escherichia coli. The purified recombinant protein was found to possess FAD-dependent NADH oxidase activity, although it lacked sequence homology to any other known general NADH oxidase family. The product of the PH0311 gene was thus designated PhNOX (NADH oxidase from Pyrococcus horikoshii), with an estimated molecular weight of 84 kDa by gel filtration and 22 kDa by SDS-PAGE, indicating it to be a homotetramer of 22 kDa subunits. PhNOX catalyzed the oxidation of reduced β-NADH with subsequent formation of H2O2 in the presence of FAD as a cofactor, but not α-NADH, α-NADPH, or β-NADPH. PhNOX

Comparison of amino acid sequences of ApAAT and other AATs. Sequence alignment was performed by T-Coffee and Boxshadesoftware (http://www.ch.embnet.org). Dashes indicate gaps, and consensus identical (black) or similar (gray) amino acids are shaded. Thefollowing sequences were aligned: ApAAT, AAT from

Alcohol oxidase catalyzes the oxidation of short lines alcohol to aldehyde. In this study, alcohol oxidase from Hansenula polymorpha (HpAOD) was induced by addition of 0.5% methanol as the carbon source and purified to electrophoretic homogeneity by column chromatographies. The purified HpAOD was immobilized with DEAE-cellulose particles and its biochemical properties were compared with those of free enzyme. The substrate specificity and the optimum pH of immobilized enzyme were similar to those of free enzyme. On the other hand, the Km values of free and immobilized enzymes for ethanol were 6.66 and 14.65 mM, respectively. The optimum temperature for free enzyme was 50°C, whereas that for immobilized enzyme was 65°C. Immobilized enzyme showed high stability against long storage. Immobilized enzyme was also tested for the enzymatic determination of ethanol by the colorimetric method. We detected 1 mg/liter ethanol (1×10 -4 % ethanol) by 2,6- dichloroindophenol system. Therefore, the present study demonstrated that immobilized HpAOD has high substrate specificity toward ethanol and storage stability, which may be of considerable interest for alcohol biosensor and industrial application.

In human, the telomer-ase plays an important role in cancer. Most somatic cellslack telomerase activity and correspondingly lose telomericDNA, thus are limited their proliferative capacity. Cancercells can overcome this proliferative block through theillegitimate transcriptional up-regulation of the gene encod-ing the catalytic subunit of the enzyme.

To gain further insight into herbicide detoxification, we studied the herbicide activity and specificity toward glutathione S-transferases from human and rice. In this study, the genes of the plant specific phi and tau class GST enzymes from Oryza sativa (OsGST) and human pi class GST enzyme (hGSTP1-1) were cloned and expressed in Escherichia coli with the pET and pKK vector systems, respectively. The gene products were purified to homogeneity by GSH Sepharose affinity column chromatography. The herbicide specificity of the enzymes was investigated by enzyme-catalyzed conjugation of GSH with chloroacetanilide, diphenylether and chloro-s-triazine herbicides. The hGSTP1-1 showed very high specific activity toward atrazine. On the other hand, the phi class OsGST enzymes showed high specific activity toward chloroacetanilide herbicides, acetochlor, alachlor and metolachlor. The tau class GST enzymes displayed remarkable activity toward the diphenylether herbicide, fluorodifen. From these results, we conclude that the phi and the tau class GST enzymes show herbicide specificities and also they play an important role in the detoxification reaction of plant toward herbicides.

The GST cDNA was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the Os GSTU5 (GenBank Accession No. AF309377) sequence. The cDNA was composed of a 687-bp open reading frame encoding for 228 amino acids. The deduced amino acid sequence of this gene shared over 60% sequence identity with the sequences of the tau class Zm GSTU6 and Zm GSTU19. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH–Sepharose affinity column chromatography. The expressed Os GSTU5 formed a homo-dimer composed of 25 kDa subunit and its p I value was approximately 7.5. The Os GSTU5 displays high activities towards 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-( p -nitrophenoxy)propane. The activity of the Os GSTU5 was significantly inhibited by hematin and ethacrynic acid. The Os GSTU5 shows the highest activity towards chloro- s -triazine and acetanilide herbicides.

To gain further insight on the relationship between structure and functions of glutathione S-transferase (GST), the three tyrosine 108 mutants, Y108A, Y108F, and Y108W, of human GST P1-1 were expressed in Escherichia coliand purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitution of Tyr 108 with alanine resulted in significant decrease of the GSH-conjugation activity and the GSH peroxidase activity, but approximately 63% increase of steroid isomerase activity toward Δ 5 -androstene 3,17-dione. On the other hand, the substitution of Tyr 108 with phenylalanine resulted in decreases of k c a t and k c a t /K m E P N P by 2 orders of magnitude, suggesting that Tyr 108 residue of hGSTP1-1 are considered to be important for the catalysis and the binding of the epoxide substrates. The substitution of Tyr 108 with tryptophan resulted in significant decreases of the specific activities toward EPNP, cumene hydroperoxide and Δ 5 -androstene 3,17-dione, but approximately 2-fold increase on the enzyme-catalyzed addition of GSH to DCNB. We conclude from these results that Tyr 108 in hGST P1-1 plays very different roles depending upon the nature of the electrophilic substrates.

A lectin that exhibits hemagglutination activity and cytotoxicity against human cancer cell lines has been purified from the legume Maackia fauriei. This lectin, designated M. fauriei agglutinin (MFA), is a tetramer of 115.6 kDa consisting of 30 kDa subunits with a pI of 4.9. The hemagglutination activity of MFA was inhibited by N-acetylneuraminic acid, Neu5Acα2–3Galβ1–4GlcNAc, and sialoglycoproteins. MFA was stable at pH values from 4.0 to 8.5, and at temperatures below 50 °C, and its activity was affected by demetalization with EDTA. MFA has a high homology with lectins from M. amurensis—which is the only legume source of lectins that bind to specific carbohydrate chains containing sialic acid—in its N-terminal 20 amino acid sequence.

A lectin gene homolog of Oryza sativa was successfully cloned and expressed in Escherichia coli. The deduced amino acid sequence of the protein product showed a significant similarity with known chitin-binding lectins. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 8 M urea in Tris/HCl buffer, pH 7.0 and renaturation by 10-fold dilution in the same buffer. The recombinant lectin with His-tag was simply purified to homogeneity by the process of affinity chromatography and was obtained with a yield of 6-8 mg/L culture. The recombinant lectin was a homo-dimer composed of 22 kDa. The hemagglutination activity of the recombinant lectin was optimal at pH 4.0-7.0 and it was very sensitive to inhibition by N-acetylneuraminic acid and thyroglobulin.

In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. Km GSH , Km DCNB and Km EPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I50 values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.

An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. The native enzyme was found to be a homo-dimer of 43-kDa subunits. The pI of the enzyme was determined to be 6.2, while its optimum pH is 10.0. The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol, n-propanol and crotyl alcohol. The highest reaction rate was observed when ethanol was used as substrate and the K(m) value of the enzyme for ethanol was 24.2 mM. Pyrazole notably inhibited the enzymatic activity. The enzyme had the optimal temperature of 70 degrees C and was highly stable against high temperature.

Determining the in vivo gustatory thresholds of sweet-tasting molecules is more difficult and laborious than other sensory analyses. In this study, we refined the single-interval adjustment matrix (SIAM) yes-no task to produce threshold estimates accurately and efficiently. Experimental procedures were optimized such that tasters provided bias-free results and achieved maximum performance. We found that the refined SIAM yes-no task accurately estimated the threshold values of the sweet-tasting protein brazzein within the sensitive threshold range, which was confirmed using the two-alternative forced-choice task. Threshold values of various artificial sweeteners were also investigated using the SIAM yes-no task. Our results suggest that the refined SIAM yes-no task is advantageous for obtaining thresholds and comparing the biological activity of various sweet-tasting molecules. Practical applications The refined SIAM yes-no task suggested in this study would enable researchers to accurately obtain thresholds of various sweet-tasting molecules, in order to develop potential sugar substitutes and elucidate interactions with their corresponding sweet-taste receptors T1R2-T1R3 for sweet-taste mechanism studies.

E-mail: khkong@cau.ac.krReceived September 17, 2011, Accepted September 26, 2011Key Words : Brazzein, Sweet protein, Site-directed mutagenesis, Sweetness determinant, Sweetness evalua-tionThere is a high demand for non-calorigenic, protein-basedsweeteners with favorable taste properties. The optimaldesign of such sweeteners requires knowledge of structure-function relationships and identification of chemical entitiesthat trigger the sweetness response. Brazzein is an intenselysweet-tasting plant protein with good stability at hightemperatures and over a wide pH range. Brazzein, with amolecular mass of 6.5 kDa, is the smallest naturally occurr-ing, sweet-tasting protein described to date.

Palladium acetate complexes have been incorporated into polycarbonate (PC) films as a dispersion medium using tetrahydrofuran (THF). The resulting light brown-coloured homogeneous films of 10–30 wt.% Pd metal complex concentration are found to be thermally stable up to 150°C, and the relatively electron rich oxygen atoms of ketone groups in polycarbonate molecules are assumed to interact with Pd complexes on the basis of their infrared spectroscopic and thermal analyses. The reaction chemistry of Pd in PC has been investigated by reacting Pd sites with small gaseous molecules such as CO, H 2 , O 2 , NO, and olefins in the temperature range of 25 to 150°C, and various Pd-carbonyls, -hydride, -nitrosyl, and -ethylene species formed in PC are characterized. The treatment of Pd-containing PC film with H 2 gas at 120°C produces small Pd metal particles of ca. 40–330 A in diameter in PC, which show various catalytic reactivities in CO oxidation, NO reduction, dimethyl carbonate formation and hydrogenation of styrene-monomer.

A glutathione S -transferase (GST) from Oryza sativa was purified to electrophoretic homogeneity approximately 742-fold with a 16% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be ≈23 000 by SDS-polyacrylamide gel electrophoresis and 48 000 by gel chromatography, indicating a homodimeric structure. The p I value of the enzyme was 6.4 by chromatofocusing using a Mono P column and the optimum pH was 7.5. The enzyme was retained on GSH affinity column and its K m value for GSH was 0.36 mM. The activity of the enzyme was significantly inhibited by S -hexyl-GSH and S -(2,4-dinitrophenyl)glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards 4-nitrophenethyl bromide and 1,2-epoxy-3-( p -nitrophenoxy) propane, a marker substrate for the theta-class GSTs. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide.

The mutational replacement of Arg182 with threonine markedly decreased the specific activities for GSH-conjugation reaction. The Kcat of R182T for GSH-[1-chloro-2,4-dinitrobenzene] conjugation reaction was about 100-fold smaller than that of the wild type. On the other hand, the affinity for GSH of R182T was not significantly affected. The pKa of the thiol group of GSH bound in R182T was approximately 0.9 pK units higher than those in the wild type, but the Kcat/Km1-chloro-2,4-dinitrobenzene values at high pH were not so much lower than those of the wild type. The thermostability of R182T was significantly lower than that of the wild type. Therefore, Arg182 seems to be important for the construction of the active enzyme structure.

Determination of inorganic anions by capillary zone electrophoresis is reported in this study. An anions mixture of bromide, chloride, fluoride, nitrite, sulfite, sulfate, perchlorate, chlorate and chlorite was separated by a capillary column and detected by indirect UV method. The running buffer contained 5mM ammonium dichromate, 10mM ammonium acetate, 20mM diethylenetriamine, and in 10% methanol solution at pH 9.3. A potential of 15kV at the cathode(reversed polarity) was utilized for the separation of inorganic anions. A complete separation of anions was achieved in less than 10 min and the applicabilities of the method were demonstrated for forensic evidences. We performed analysis of inorganic anions in toluene inhaled human's urine and in postmortem bloods by capillary zone electrophoresis and ion chromatography

Tyrosinase (EC 1.14.18.1) is a copper-containing monooxygenase catalyzing the o-hydroxylation of monophenols to the corresponding catechols (monophenolase or cresolase activity), and the oxidation of monophenols to the corresponding o-quinones (diphenolase or catecholase activity). These functions of tyrosinase play an important role in the formation of melanin pigments during melanogenesis. Melanin production is principally responsible for skin color and plays an important role in prevention of sun-induced skin injury. However, abnormal accumulation of melanin products is responsible for hyperpigmentations including melasma, freckles, and senile lentigines, which could be a serious aesthetic problem. Recently, a global market demand has developed for skinwhitening agents because some dark-skinned individuals in many countries and races are prefer lighter skin color. Several tyrosinase inhibitors have been used in the cosmetic industry as skin-whitening agents. Unfortunately, many of these inhibitors have not been demonstrated yet to be clinically efficacious when critically analyzed in carefully controlled studies. Thus, many efforts are still devoted to screening recognized and putative skin-whitening agents. A correct assay method is essential for the development of effective inhibitors of tyrosinase. Until now, the screening and evaluation methods of these skin-whitening agents have been adopted dopa oxidation test by mushroom tyrosinase because of its commercial availability, not human tyrosinase. Moreover, a comparative study on the inhibitory effects of skin-whitening agents by human tyrosinase and mushroom tyrosinase is still undone. In this study, we investigated inhibitory effects of skinwhitening agents on L-dopa oxidation by human tyrosinase and mushroom tyrosinase, and compared their effects. According to our study described here, L-dopa oxidation activities of human tyrosinase and mushroom tyrosinase show very different inhibitory effects depending upon the nature of skin-whitening agents.

Glutathione S-transferase (GST) was purified from Escherichia coli K-12, and its N-terminal sequence was determined to be MKLFYKPGAXSLAS. The gene encoding this sequence was cloned and mapped at 1731-1732 kilobases on the E. coli gene map. It encoded a polypeptide of 201 amino acid residues with a calculated molecular weight of 22,860. The overexpressed product of the gene was confirmed to have GST activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid and GSH-dependent peroxidase activity toward cumene hydroperoxide. The relative molecular mass of the gene product was determined to be 40,000 by gel chromatography and 25,000 by SDS-polyacrylamide gel electrophoresis, indicating a homodimeric structure. The deduced amino acid sequence was 54% identical with that of Proteus mirabilis GST. Although the homologies between the GSTs from E. coli and mammals were low, many of the residues assigned to be important for the enzymatic function or structure in mammalian cytosolic GSTs were found to be conserved in E. coli GST. Therefore, E. coli GST is considered to have diverged from the same ancestor with other cytosolic GSTs. A specific tyrosyl residue in the vicinity of the N terminus is conserved in all of the known cytosolic GSTs and has been shown to function as a catalytic residue in alpha, mu, and pi class GSTs from mammals. Although Tyr5 in E. coli GST appeared to be the counterpart of the catalytic residue, its replacement with phenylalanine did not significantly affect the enzymatic activity. Therefore, this apparently conserved tyrosyl residue is not essential for catalytic activity in E. coli GST.

The gene APE0743 encoding the superoxide dismutase (ApSOD) of a hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and over-expressed as a GST fusion protein at a high level in Escherichia coli. The expressed protein was simply purified by the process of glutathione affinity chromatography and thrombin treatment. The ApSOD was a homodimer of 25 kDa subunits and a cambialistic SOD which was active with either Fe(II) or Mn(II) as a cofactor. The ApSOD was highly stable against high temperature. This thermostable ApSOD is expected to be applicable as a useful biocatalyst for medicine and bio-industrial processes.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GSTπ) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GSTπ, which is conserved in all cytosolic GSTs, with phenylalanine by sitedirected mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GSTπ.