Earticle

The formation of β-carotene detergent micelles and their conversion into retinal by recombinant human β,β-carotene 15,15’-monooxygenase was optimized under aqueous conditions. Toluene was the most hydrophobic among the organic solvents tested; thus, it was used to dissolve β-carotene, which is a hydrophobic compound. Tween 80 was selected as the detergent because it supported the highest level of retinal production among all of the detergents tested. The maximum production of retinal was achieved in detergent micelles containing 200 mg/L β-carotene and 2.4% (w/v) Tween 80. Under these conditions, the recombinant enzyme produced 98 mg/L retinal within 20 h with a conversion yield of 49% (w/w). The amount of retinal produced, which is the highest ever reported, is a result of the ability of our system to dissolve large amounts of β- carotene.

Two-component oxygenases catalyze a wide variety of important oxidation reactions. Recently, we characterized a novel arylamine N-oxygenase (PrnD), a new member of the two-component oxygenase family.(1) Although arylamine N-oxygenases are widespread in nature, aminopyrrolnitrin N-oxygenase (PrnD)represents the only biochemically and mechanistically characterized arylamine N-oxygenase to date.(2) Here we report the use of bioinformatic and biochemical tools to identify and characterize the reductase component (prnF) involved in the PrnD- catalyzed unusual arylamine oxidation. The prnF gene was identified via sequence analysis of the whole genome of Pseudomonas fluorescens and subsequently cloned and overexpressed in E. coli. The purified PrnF protein catalyzes reduction of FAD by NADH with kcat of 65 s-1 (Km = 3.2mM for FAD and 43.1mM for NADH) and supplies reduced FAD to the PrnD oxygenase component. Unlike other known reductases in two-component oxygenase systems, PrnF strictly requires NADH as an electron donor to reduce FAD, and requires unusual protein-protein interaction with the PrnD component for the efficient transfer of reduced FAD. This PrnF enzyme represents the first cloned and characterized flavin reductase component in a novel two-component arylamine oxygenase system.

Pancreatic or tissue carboxypeptidase B (CPB; EC 3.4.17.2) is a key enzyme involved in insulin conversion and highly specific for excising C-terminal Lys and Arg resiudes from peptides and proteins. Also, CPB is naturally synthesized in form of zymogen with an 11 kDa N-terminal prodomain that covers the catalytic pocket of the enzyme.1) A cDNA containing the human pancreatic pro-CPB was fused to three mutants of exoinulinase secretion signal (m1, m2, and m3) from Kluyveromyces marxianus. We used these plasmids in order to further improve the secretion efficiency of proCPB. The recombinant plasmids, pYInum1-hproCPB, pYInum2-hproCPB, and pYInum3-hproCPB, were transformed into Saccharomyces cerevisiae 2805 strain. Yeast transformants were selected on the synthetic defined media lacking uracil. The recombinant hproCPBs were successfully expressed in S. cerevisiae after induction of galactose, and could be secreted into the supernatant of the culture. The secretion efficiency of proCPBs from each vectors were measured and analyzed. Each the human proCPB protein secreted by three mutants of exoinulinase signal sequence was purified and the enzymatic properties were examined.

토양시료로부터 얻은 바실러스를 배양하여 카탈라제 활성이 높은 3가지 균주를 선별하고, TSB 복합배지를 영양원으로 하여 30 ℃, 200 rpm의 조건으로 대수증식기까지 배양하여 원심분리하고, 얻어진 상등액을 카탈라제 활성 측정에 이용하였다. 각 시료에 혈청알부민, 글리세롤, 이노시톨, 폴리에틸렌글리콜, 자일리톨 등을0~10%의 농도로 처리하여 30 ℃에서 24시간 반응시켜 카탈라제 활성의 변화를 측정하였다. 각각의 균주 배양액에 몇 가지 첨가물질을 넣은 시료를 온도와 pH를 변화시켜 일정 시간 반응시킨 후 카탈라제 활성에 미치는 영향을 조사하였다. 본 연구에서 얻은 카탈라제에 첨가물질을 넣었을 때 효소 활성이 대체로 증가하였다. 또한 pH 7 이상에서 대부분 높은 효소 활성을 나타냈다. 온도 변화실험에서는 BKS 14와 39로부터 얻은 효소는 글리세롤을 첨가하여 65℃에서 반응시킨 경우에 서 최대의 활성을 보였고 , BKS 15로부터 얻은 카탈라제는 글리세롤을 첨가하여 30 ℃에서 반응시킨 경우 높은 카탈라제 활성이 관찰되었다.

In this work, the production of catalase was investigated. Several soil microorganisms were cultivated in shaking flask with TSB(tryptic soy broth) complex medium at 30 , 200 rpm. The additive compounds ℃ included bovine serum albumine, glycerol, inositol, polyetylene glycol and xylitol. Concentration of additives was of 0~10%. Catalase activity was determined by measuring with spectrophotometric method at 525 nm. pH profile of catalase activity in the presence of additives was investigated in the range of pH 7 ~ 13. The effect of the temperature on the activity of native and additives treated catalase was studied in the temperature range of 30 ~ 65 ℃. Almost catalase from soil Bacilli were higher activity over the pH 7.0 and 40 ℃.

Streptococcus mutans, the major oral pathogen, produces glucan that has α-(1→3), α-(1→6), and α-(1→4)-glycosidic linkages and this glucan forms dental plaque. In this study, a hybrid enzyme having both dextranase and maltohexaose-producing amylase activities was constructed to apply for the removal of dental plaque or retarding formation of it. Dextranase (Dex2) and maltohexaose-producing amylase (G6-Amy) gene were cloned from Arthrobacter oxydanas and Klebsiella pneumoniae, for respectively and then ligated with each other. The open reading frame of the constructed dex2-G6Amy hybrid gene consists of 3,759 bp encoding 1,253 amino acid residues. The gene was ligated with pRSET expression vector and transformed to Escherichia coli BL21 (DE3) pLysS for enzyme expression. Expressed enzymes were purified by application to Ni Sepharose High Performance column. The purified dex2-G6Amy enzyme has the dextranolytic and amylolytic bifunctional activities.

Chitopearl beads were used as immobilization supports for D-tagatose production by L-arabinose isomerase from Thermotoga neapolitana because chitopearl beads were more stable than alginate beads at temperatures above 60°C. The optimal pH and temperature for the isomerization of D-galactose were 7.5 and 90ºC, respectively. The half-life of immobilized L-arabinose isomerase was 11 times higher than that of free enzyme at 90°C. The pH of a mixture containing partially purified enzyme and galactose decreased as the reaction time, galactose concentration, and temperature increased, resulting in decreased tagatose production. When the pH was maintained at 7.5 in a stirred tank reactor containing immobilized enzyme, 138 g/L tagatose was produced at 70°C from 300 g/L galactose, while only 70 g/L was produced without pH control.

본 연구에서는 효모가수분해액을 BC 생산배양의 질소원으로 첨가하여 BC 생산성을 제고시켰다. 효모가수분해액 15%을 첨가하여 BC 생산배지의 총 질소원의 농도(T-N)가 2.60 g/L 일때 BC 농도는 3.4 g/L로 가장 높은 생산성을 보였다. 그리고 50 L 공기순환배양기에서 당화액배지(환원당 농도 70 g/L)에 효모가수분해액을 15% 첨가하고, 공기 공급 시 순산소를 함께 주입하고, 한천분말 0.6%를 첨가하였 을 때, BC 농도는 8.84 g/L에 도달하였다. 이는 질소원을 첨가하지 않고 환원당 농도를 40 g/L로, agar의 농도를 0.4%, 공기만을 주입하여 얻은 BC 농도 6.79 g/L 보다 BC생산성이 30.2% 향상되었다.

To improve bacterial cellulose productivity, yeast hydrolyzed by culture broth of Bacillus alcalophilus, a protease production strain, was added to BC production medium. BC concentration was increased to 3.4 g/L at 100 mL flask when 15% of hydrolyzed yeast was added to BC production medium. At 50 L air circulation bioreactor, BC concentration was increased to 8.84 g/L when 15% of hydrolyzed yeast, 0.6% of agar and pure oxygen were supplemented to BC production medium. As a result, 30.2% of BC productivity was improved comparing to that of our previous study.

Daidzein is diphenolic phytoestrogen compound found in numerous plants and soybean .Hydroxylated products have been reported to have potent antioxid ant properties that contributes to their cholesterol lowering effects,cardiovascular protection ,antitumor effects and anticarcinogenic properties .Compounds with the ortho-dihydroxy groups are known to exhibit anti-inflamatory and anti-allergic activity and to express anticarcinogenic properties due to the inhibition of protein tyrosin kinase to a potent tyrosinase inhibitor of lipoxygenases .Hydroxylation of the aromatic compounds by using chemical synthesis method is difficult and involves diverse reaction steps.Since the conversion of a non activated carbon -hydrogen bond by the microbial enzyme is of increasing interest .Hydroxylation of daidzein by recombinant streptomyces avermitilis was examined.Strucuture analyisis of the reactive products was determined using gas chromatography mass spectrometry(GC-MASS).Recombinant streptomyces avermitilis is highy regioselective to give 7,3',4'-trihydroxyisoflavone.Quantification analysis of the 3'-hydroxylated daidzein were performed by HPLC.

본 연구에서는 섬유소분해효소를 연속배양 하고자 먼저 refill하는 멘델배지의 농도를 0.5%로, 새로운 배지의 주입시간을 12시간으로 결정하였다. Flask 레벨에서는 fill-and-draw 방법으로 12 시간 단위로 연속배양한 결과, amylase 활성은 300시간까지 1.0 U/mL 내외로 유지되었으며, FPase 활성은 156시간까지 0.40 U/mL 이상으로 유지되었다. 이때의 효소생산성은 amylase 3.5 U/L․hr, FPase 1.0 U/L․hr 이었다. 10 L에서는 batch, fed-batch, fill-and-draw 방법으로 효소를 생산한 결과 batch에서 가장 높은 효소생산성을 나 타내었으며, 그다음은 fed-batch 이었다. Batch에서의 효소생산성은 amylase 42.3 U/L․hr, FPase 5.6 L․hr, fed-batch에서는 각각 23.0, 2.8 U/L․hr 이었다.

For continuous culture of cellulolytic enzymes, refill concentration of Mandel's medium for continuous culture was 0.5%, and refill intervals were determined to 12 hours by analysis of COD and total nitrogen concentration after 4 days batch culture in flask level. As a result, amylase and FPase activities were 3.5 and 1.0 U/L․hr, respectively. In 10 L bioreactor, the batch culture mode was compared with fed-batch, fill-and-draw for continuous production of cellulolytic enzyme. Enzyme productivities were most high at batch culture and followed by fed-batch culture. Amylase and FPase activities were 42.3 and 5.6 U/L․hr at batch culture, and 23.0, 2.8 U/L․hr at fed-batch culture, respectively.

Aerobic biodegradation was carried out using original fish-meal wastewater (FMW), and successfully carried out in 100-L bioreactor for 3 days. During biodegradation, ORP was maintained at a range of positive values, and this caused a pleasant smell in the end. Eight-folds dilution of the final biodegraded FMW broth gave no phytotoxicity claerly. The amino-acids composition increased from 3.13% to 5.22% by the biodegradation. The total amount of amino acids in final biodegraded FMW was comparable to that of a commercial fertilizer. The concentrations of N, P and K in biodegraded FMW were in a range of 3.8-6.0%, 2.8-3.0% and 2.1-3.5%, respectively, and the concentrations of all noxious components were much lower than the standard.

Racemase is novel enzyme which has been biochemically classified as a subgroup of the isomerases. Although many report of sulfur containing amino acids transformation have been studied, homocysteine and sulfur containing amino acid racemase was not clearly identified. We here report the identification of free homocysteine isomers without making any derivatization. The separation of homocysteine isomers were performed on a chiralpacR MA(+) column by isocratic elution with 2mM copper sulfate-acetonitrile (85:15, v/v) as mobile phase. Authentic amino acid, D or L-homocysteine was incubated with crude extracts of Pseudomonas putida and diminuta under the neutral conditions at 37 ℃ for 3 hours. The change of proportion for D and L- isomers were detected and analyzed by UV-visible detector at 240nm within 30 minutes. Our new detection method for homocysteine isomers demonstrates the high sensitivity and rapid separation of each isomer and enabled us to detect the putative homocysteine racemase. The Pseudomonas putida exhibits a more great potential for preparative bio-transformations than diminuta. [Supported by grant No. R01-2006-000-10232-0 from MOST]

Polyaniline nanofibers were used as immobilization media for glucose oxidase. Since polyaniline is conducting polymer, immobilized glucose oxidase can be used for the glucose biosensor and other applications. 10 mg of glucose oxidase was immobilized onto 2 mg of polyaniline nanofiber and enzyme loading capacity of polyaniline was 0.267 mg/mg. Developed immobilized glucose oxidase was easily recovered from the reaction mixture. The stability of immobilized enzyme was high and 60 % of its initial activity was maintained after 60 days of incubation at room temperature and under rigorous shaking condition.

Activity is an important character of enzyme because industry requires efficient catalysts. In this study, bacteriophage T4 lysozyme was selected as a model enzyme and the flexibility of mutants was analyzed to understand mechanism of activity enhancement. Mutants data were selected in Mutant database and mutants having activity data were chosen. The group having enhanced activity showed a tendency in residue; the changed residues were 7 Glu, 3 Asp, and 1 Pro and 6 of them were located in helix edge. The charge-charge interaction was negligible between the mutated residues and substrate from the point on the distance. The flexibility is related with the enzyme motion and this factor can affect the catalytic activity of the enzyme. The standardized B-factor of Cα can be a measure of residual flexibility. Compared with the wild type, the standardized B-factor change of mutation site in edge region showed a positive value whilst in helix, a negative value. The mutants having increased activity had more rigid helix body and more flexible helix edge than the wild type. This can be a clue to obtain mutant having enhanced activity.

Ketoprofen is one of non-steroidal anti-inflammatory drug. It is generally alleged that the (S)-ketoprofen has the higher pharmacological effect compare with the racemic mixture of profens.1) Because of their abundance and great versatility in mediated reactions, esterases have recently been considered as a possible candidate for the chiral resolution of ketoprofen. Esterase-mediated chiral resolution of racemic compound has been ubiquitously found in a variety of biomolecules and synthetic chemicals.2) Est25, novel esterase from a metagenomic library, efficiently hydrolyzed (R,S)- ketoprofen ethyl ester which was significantly improved by the addition of emulsifier surfactants, but showed no significant enantioselectivity.3) In the process of (R,S)-ketoprofen ethyl ester hydrolysis enantioselectivity of Est 25 toward S-ketoprofen ethyl ester was remarkably increased when (R,S)-ketoprofen ethyl ester disolved in ethanol then surfactant such as triton X-100 was added separately, instead (R,S)-ketoprofen ethyl ester was disolved in the buffer containg surfactant. We are studying the effect of other organic solvents on enantioselectivity and results will be discussed.

Regiospecific hydroxylation of isoflavonoids using cells of Bacteria, Actinomyce was examined. Current researches reported when isoflavones have more hydroxy group on the aromatic ring, the anticancer, antitumor, and antioxidant effects of hydroxy isoflavone showed higher. Among 27 P450s, nfa33880 showed highest activity for daidzein and using this system daidzein was hydroxylated to produce 6,7,4’-trihydroxyisoflavone and 7,8,4’-trihydroxyisoflavone. Also we conducted substrate and P450 docking modeling and the result was well consistant with the experimental result of which nfa33880 P450 enzyme had bi-functional activity for daidzein.We determined the regiospecific hydroxylation position of daidzein and the turning point of the highest convertion of hydroxyisoflavone and we identified P450s participated in the hydroxylation reaction of the Nocardia farcinica IFM10152 and co-expressed candidate P450s and redox partner in E. coli. And the hydroxylated and O-methylated products were determined using high performance liquid chromatograph (HPLC), gas chrmomatograph / mass spectrometry (GC/MS).

We purified recombinant galactose 6-phosphate isomerase (LacAB) from Lactococcus lactis using HiTrap Q HP and Phenyl-Sepharose columns. The purified LacAB had a final specific activity of 1.79 units/mg to produce D-allose. The molecular mass of native galactose 6-phosphate isomerase was estimated at 135.5 kDa using Sephacryl S-300 gel filtration, and the enzyme exists as a hetero-octamer of LacA and LacB subunits. The activity of galactose 6-phosphate isomerase was maximal at pH 7.0 and 30°C, and enzyme activity was independent of metal ions. When 100 g/L of D-psicose was used as the substrate, 25 g/L of D-allose and 13 g/L of D-altrose were simultaneously produced at pH 7.0 and 30°C after 12 h of incubation. The enzyme had broad specificity for various aldoses and ketoses. The interconversion of sugars with the same configuration except at the C2 position was driven by using a large amount of enzyme in extended reactions. The interconversion occurred via two isomerization reactions, i.e., the interconversion of D-allose↔D-psicose↔Daltrose, and D-allose to D-psicose reaction was faster than D-altrose to D-psicose reaction.

Yeast, Saccharomyces cerevisiae, produces hydrogen sulfide (H2S) as an undesirable by-product during alcoholic fermentation. Very low levels of H2S can be detected, so it can have a profound effect on final product quality. It is highly desirable to have yeast strains available for various fermented production that will not produce and release H2S. To confirm H2S producing proteins in Saccharomyces cerevisiae, we assayed the beta-replacement reaction activity with beta-mercaptoethanol and L-cysteine, which will form S-hydroxyethyl-L-cysteine and H2S. Formed H2S can easily be detected by the incubation with Pb-acetate that will make Pb-sulfide precipitation. When the crude extract of S. cerevisiae was incubated with beta-mercaptoethanol, L-cysteine and Pb-acetate, two proteins which made H2S on the Native-PAGE staining were detected. Because CYS4 gene deleted crude extracts gave only one H2S producing protein in the Native gel, cystathionine beta-synthase is one of the H2S producing protein. The other protein was identified as O-acetylhomoserine sulfhydrylase by MALDI-TOF mass analysis. We could produce CYS4 and Met 17 deficient yeast strain.

flavobacterium sp.로부터 재조합 시킨 Escherichia coli로부터 생산되는 OPH는 자석으로 쉽게 분리 및 회수가 가능한 DEAE, CM에 고정화시켜 활성도가 2일에 50%이상 떨어지는 free enzyme에 비해 32일까지 남아있었다. 생물적 환경 정화를 위한process 중 하나로 Paraoxon의 연속적으로 분해할 수 있는 packed-bed reactor에 고정화를 시켜 채류시간 10.8초에 기질인 paraoxon 0.1 mM이 44.3% 분해효율을유지되어 연속적인분해를 안정적으로 수행하였다.

Electrospun polymer nanofibers and carboxy methyl cellulose nanofibers providelarge surface area for enzyme coating and easily recoverable from solution for iterative use. The enzyme stability of crosslinked organophosphorus hydrolase (OPH) coating on nanofibers was greatly improved than that of free enzyme. The half life of immobilized enzyme was 32 days and that of free enzyme was 2 days. OPH coated nanofibers were used for the bioremediation of paraoxon which is one of organophosphrus toxic compounds. When the retention time of paraoxon was 4 h, 68 % of paraoxon was removed from the solution. Packed bed enzyme reactor with enzyme coated carboxy methyl cellulose nanofiber was developed for the bioremediation of paraoxon. About 44 % of paraoxon was degraded with 10.8 sec retention time.

현재 심각한 공해를 유발시키는 디젤유에 대한 청정 대체연료인 바이오디젤은 동, 식물성 기름(triacylglyceride)이 methanol과 에스테르 반응 후 생성되는 메틸에스테르 (methyl ester)이다. 기존의 석유계 경유와 섞어서 사용할 경우 대기오염 주범인 동차 공해를 획기적으로 줄일 수 있어 대체에너지로 최근 그 수요가 늘고 있다. 환경오염을 유발시키는 화학적 촉매방법 대신에 lipase를 이용한 연구가 늘고 있지만 고가의 효소 가격으로 인한 경제성이 문제가 되어왔다. 본 실험에서 는 생화학적 촉매로 이용되는 lipase를 자석으로 쉽게 분리가 가능한 MP에 고정화하여 열에 따른 효소의 불활성 및 효소의 재이용성의 단점을 보완시키고, 식물유지(콩기름, 포도씨유)를 이용하여 바이오디젤 생산을 시도하였다. 본 실험에서는 유지 0.5g과 enzyme 0.1mg을 24시간 37℃(회분식 반응) 반응하여 free 65%, 고정화 효소는 56% 전환되었고 재사용 전환율을 처음 사용의 100%였다.

In this work, the immobilized PAMP-LP was found to be very stable for the transesterification of vegetable oil. This enzyme was found to be much more stable than the free enzyme system and the half life of PAMP-LP was 110 days which was greatly improved from that of free enzyme. It could be used repeatedly for the different batches with simple magnetic recovery process. The produced biodiesel composition using soybean oil was similar to that of grapestone oil. The activity of recycled immobilized PAMP-LP after transesterification was similar to that of first time transesterification of soybean oil.

A recombinant β-galactosidase from Sulfolobus solfataricus produced galactooligosaccharides (GOS) from lactose by transgalactosylation. The enzyme activity for GOS production was maximal at pH 6.0 and 85°C. In thermal stability experiments, the half-lives of the enzyme at 70, 75, 80, 85, and 90°C were 700, 111, 72, 43, and 2.4 h, respectively. The optimal amount of enzyme for effective GOS production was 3.6 units ml-1 of enzyme. GOS production increased with increasing lactose concentration, whereas the yield of GOS from lactose was almost constant. Under the conditions of pH 6.0, 80°C, 600 g/L of lactose, and 3.6 units ml-1 of enzyme, GOS production was 310 g/L for 56 h, with a conversion yield of GOS from lactose of 52%. The final concentration of GOS and GOS yield from lactose used by β-galactosidases from S. solfataricus was the highest among thermostable β-galactosidases.

One of the most attractive features of cell-free protein synthesis is reproducible gene expression in an artificial environment designed to produce specific proteins.1) We report here a system of bacterial translation machinery entrapped in the silica sol-gel matrix and the cell-free protein synthesis in the system. The translation machinery of E.coli was encapsulated within a silica sol-gel matrix by mixing the cell extract with alkoxysilane sol solution prior to gel formation. Translation factors survived the harsh conditions of sol-gel transition and were able to decode the co-immobilized DNA into functional protein molecules. In addition, protein expression was affected by additives incorporated during the gel formation. Study of scanning electron microscopy informs that the different expression level was caused by the alteration of structure and pore size in the gel matrix. To the best of our knowledge, this is the first report describing the convergence of organic translation machinery and inorganic sol-gel material. We expect that this technology will offer various applications including high-throughput protein expression and the developments of protein chip and biosensors.

Amphiphilic molecules self-assemble into various structures depending on the molecular architecture, hydrophilic/hydrophobic ratio and assembly methodologies.1)-4) Here, we report the structural transition of self-aggregates of a series of Poly(2-hydroxyethyl aspartamide) (PHEA) graftedwith dodecyl chains (PHEA-g-C12) with various degree of substitution (DS). Amphiphilic graft copolymers, PHEA-g-C12 with the different DS of C12, were simply prepared by aminolysis of the precursor polymer poly(succinimide) (PSI) with dodecylamine and ethanolamine. Synthesis and DS values were verified with 1H NMR spectra. Stable self-aggregates in aqueous media were successfully prepared by direct dissolution and characterized mainly in terms of size and structure by light scattering methods and visualized by transmission electron microscopy (TEM). Spherical micelles, wormlike micelles, tubule networks, and vesicles were observed.The structure is changed as a function of DS of hydrophobic dodecyl chain and can be explained in terms of packing parameter and interface energy. The tubular network system shows unique and interesting viscose behavior.

Several glycosidases were reported to produce 2-deoxyglycoside conjugates from glycals and alcoholic or phenolic compound. α-Glucosidase [EC 3.2.1.20, α-D-glucoside glucohydrolase] is a group of typical exo-type carbohydrases, which catalyze the split of α-glucosyl linkage to liberate α-glucose from the non-reducing terminal of substrate. α-Glucosidases belonging to glycoside hydrolase (GH) 13 cannot hydrate D-glucal, but α-glucosidases belonging to GH 31, such as Aspergillus niger, pig serum, rice, buckwheat and sugar beet α-glucosidases, are able to catalyze the hydration of the double bond in D-glucal and produce the α-anomer of 2-deoxyglucose [1]. α-Glucosidase from Aspergillus niger (ANGase) was found to synthesize high yield of the alkyl α-D-2-deoxyglucoside (A2DG) from D-glucal and alkyl alcohols [2, 3]. In this study, the synthetic method of A2DG derivatives was investigated and the structure of product was analyzed. ANGase was much more stable than the tested three enzymes, S. pombe α-glucosidase, pig serum α-glucosidase, and buckwheat α-glucosidase. ANGase maintained the high residual activity was considered to be an enzyme suitable for synthesizing of A2DG in alcoholic solvents. Using ANGase, the optimum reaction conditions in synthesis of methyl α-D-2-deoxyglucoside (M2DG) were investigated with pH (4.0-4.5), temperature (35-35 oC), methanol concentration (70%, v/v), and D-glucal concentration. The 93% of D-glucal was converted into M2DG under the optimum reaction conditions The 14 kinds of A2DG were synthesized and isolated by silica gel column chromatography or liquid/liquid extraction. The yields of ethyl and propyl α-D-2-deoxyglucosides by ANGase in 90% (v/v) ethanol and 1-propanol were 90% and 88%, respectively, and those from alcohols having ling alkyl chain were low yield (83-45%). The structures of 14 kinds of A2DGs were determined to be a-D-2-deoxyglucosides having each alkyl group by MS and NMR analysis, all of which were novel compounds [2, 3]. For high yield production of 2-deoxyglucoside derivatives, the used organic solvents have the two kinds of advantage. During the formation of addition product, alcohols had two kinds function. One is the suppressor of hydration by reduced water molecule; the other function is acted as acceptors by itself in the reaction. It was found that ANGase had four benefits: (1) the simplicity of one step reaction, (2) the high stability in alcohols, (3) its strong addition activity, and (4) the wide acceptability of various alcohols.

We have found that the maltol and pulegone were presence in large amounts in Red Ginseng and Schizonepeta tenuifolia Briquet, respectively. We prepared large scale purification of maltol and pulegone by various column chromatographies. The purity of each phytochemical compound was determined by liquid and gas chromatography. The aim of our study is to evaluate supramolecular complex which containing phytonutrients. We could detect the UV-visible absorption change before and after mixing the purified maltol with β- and γ-cyclodextrin, which suggested the complex formation between maltol and cyclodextrins. The rates of complex formation were variable by incubation time, temperature, pH and UV light exposure. The complex formation between pulegone and cyclodextrins were also detected. 1H nuclear magnetic resonance (NMR) spectroscopy data demonstrates the pulegone signal in D2O solution. UV-visible spectrum and infra-red spectroscopy data of cyclodextrin with pulegone also gave the evidences for complex formation. The data of thermo gravimetric analyzer (TGA) and dynamic light scattering (DLS) also exhibited the complex formation. [Supported by grant No.KB-05-1-012 from KOTEF]

Self-assembeld nanostructures from amphiphilic molecules have been extensively studied due to their unique characteristics and the variety of applications in many areas.1)-4) Here, we report the synthesis of novel amphiphilic biodegradable polymer and its self-aggregation behavior in aqueous solution. A series of amphiphilic copolymer, poly(2-hydroxyethyl aspartamide) grafted with fatty acids from palm oil (PHEA-g-palm FA) with various degree of substitution (DS, defined as grafted mole percent), was synthesized. Synthetic, hydrophilic poly(amino acid) polymer, PHEA, can be simply prepared by aminolysis of precursor polymer poly(succinimide) (PSI) withethanolamine. Fatty acids were grafted onto the backbone polymer PHEA through DCC/DMAP mediated ester bond (-COO-) formation between PHEA (-OH) and fatty acids (-COOH). Synthesis and DS values were verified with 1H NMR spectra. Stable self-aggregates in aqueous media were successfully prepared by direct dissolution/sonication method. The effect of DS on the self-aggregation was investigated by characterizing the size and structure of self-aggregates with light scattering methods and transmission electron microscopy (TEM).

NAD(P)-dependant oxidoreductases catalyze valuable reactions for synthesis of chiral compounds that chemical catalysts fail. Due to the economical infeasibility of stoichiometric use of cofactors, regeneration of nicotinamide cofactors such as NAD(P) and NAD(P)H is a key object for the application of cell-free biocatalytic redox reactions catalyzed by versitile classes of oxidoreductases. Among many approaches under development, electrochemical method gains the upper hand over other approaches like enzymatic method due to its cheaper and cleaner properties. However, this electrochemical method suffers from its low efficiency arising from the short-range electron transfer that occurs only between electrode surface and target molecules. The low rate of electrochemical reduction of NADH was greatly enhanced by inducing conductive materials such as vanadium pentaoxide and Pt nanoparticles. The increased reduction rate was dependant on the concentration of conductive material and the stirring rate of reaction medium. Enzymatic synthesis of glutamate coupled with the electrochemical regeneration of NADH was successfully made up resulting complete ocontinuous conversion of substrate.

Nitrilase is one of the enzymes which has been studied from the beginning of the biotechnology era. Because of the simple and mild reaction condition and useful functionality of carboxylic acid group, its ability to convert cyanide group to carboxylic group was of interest to industry, and as a result of such industrial interest, many results were reported and actually applied to industry like polyacrylamide synthesis.(1,2). Nowadays, nitrilase, especially arylacetonitrilase, is coming into spotlight again as a promising biotool to synthesize optically pure alpha-hydroxy acid. Alpha-hydroxy acid is industrially very promising compound. Because, its structure is similiar with natural amino acid structure. To find nitrilase activity, enrichment culture system has been mainly used as a screening method using cyanide compounds of interest as a sole N-source (6, 7, 8). However, because there are always some limitations of enrichment process, like instability of cyanide compounds or the limited species , like alcaligense, pseudomonas found, finding new enzyme using this old-fashioned method doesn't seem to be promising. Faced with this obstacle, other screening tools were investigated as a alternative, and metagenome (9) , bioinformatics (10,11) has showed results and possibility so far. We report the new enzyme screened from NCBI DB via bioinformatics tools. Using profile analysis based on nitrilase sequence database, one of putative nitrilase was given the most probability, and over-expressed in E.coli heterologously.and purified using N-terminal His-tag separation. Recombinant enzyme shows good activity toward mandelonitrile, which means it belongs to arylacetonitrilase subgroup, and proves to be good partner for synthesis of stereospecific mandelic acid combined with stereospecific oxynitrilase.

The Full gene of Paenibacillus sp. dextranase was open reading frame comprises of 5,091 bp encoding 1,696 amino acids. The primary structure of PDEX has 30% identity with CITase from Bacillus circulans and shows 10-15% identities with Streptococcus dextranases. PDEX has a catalytic domain and three-repeated cell surface layer homology (SLH) domains at the C-terminus. GH family 66 enzymes have the nine conserved regions, which may be important for their enzyme activities. CITase or PDEX has a long insertion part, especially the insertion of PDEX is very long. These amino acids may contribute to the synthesis of CI. SLH domains might be responsible for binding of cell wall. PDEX has proline rich region between catalytic domain and SLH domains, which seems to be a linker. PDEX produces CIs as well as isomaltooligosaccharides from dextran in the initial stage of enzyme reaction. The cyclic structure of CIs is determined by 1H-NMR and 13C-NMR. ESI-MS analysis confirms that CIs are composed of 7-14 glucosyl units.

The herbal extracts of Salvia miltiorrhiza Bunge was widely used as traditional Chinese preparation for the treatment of cerebrovascular diseases. We analyzed methanol and ethyl acetate extracted fractions from Salvia miltiorrhiza Bunge. We could identify rosemarinic acid and salvianolic acid B using reversed-phase high performance liquid chromatographic (HPLC) method. Both compounds were detected within 40min by linear gradient elution with acetonitrile and water. The elution fractions of rosemarinic acid and salvianolic acid B were further analyzed and compared with authentic samples using UV-visible absorbance, fluorescence spectrum, and mass spectroscopy. Absorbance and fluorescence spectra demonstrated the completely same patterns between elution fractions and authentic compounds. Rosemarinic acid and salvianolic acid B from HPLC elution fractions were also detected by ESI (+) MS and MS/MS spectrometer. Our data suggested that S. miltiorrhiza Bunge is a good source of rosemarinic acid and salvianolic acid B. The extraction yields were 0.12% for rosemarinic acid and 0.53% for salvianolic acid B. [Supported by grant No.KB-05-1-012 from KOTEF]

연교를 메탄올로 1차 추출한 물질의 물, 부탄올, 에틸아세테이트 분획에서 물과 부탄올 분획은 한약재 추출물을 넣지 않은 대조군과 O.D. 값의 차이가 크지 않았으나 에틸아세테이트 분획물의 경우에는 대조군과 비교하여 O.D. 값이 70% 감소하였다. 이것으로 보아 연교의 에틸아세테이트 분획물 중에 urease 활성억제 물질이 포함되어 있음을 확인 할 수 있다. 연교로부터 분리된 betulinic acid와 oleanolic acid 모두 기존 항생제에 비하여 강한 urease 활성억제 효과가 있으며, 특히 butulinic acid가 oleanolic acid보다 강한 활성억제 효과가 있었다.

Betulinic and oleanolic acids isolated from Forsythia suspensa Vahl. Showed stronger urease inhibition activity compared with popular antibiotics such as amoxicilin, clarithromycin, metronidazole, and tetracycline. Betulinic and oleanolic acids lowered urease activity by 20% and 25%, respectively compared with the control experiments without the addition of the two acids.