Earticle

Optimal conditions for production of the carboxymethycellulase (CMCase) and filterpaperase (FPase) by E. coli pTDL-3 were investigated in a 7L bioreactor using an orthogonal array method. On basis of intuitive analysis and statistical calculations using data, significance of aeration rate on production of the CMCase by E. coli pTDL-3 was higher than that of agitation speed on production of the CMCase as well as for production of the FPase. Optimal agitation speed and aeration rate for the production of CMCase were found to be 200 rpm and 1.0 vvm in a 7 L bioreactor. Productivity of the CMCase by E. coli pTDL-3 from 5.0% (w/v) rice bran and 0.1% (w/v) tryptone under optimized conditions was 544.4 U/ml in a 100 L bioreactor with an inner pressure of 0.2 kgf/cm2.

Transglutaminase (TGase, EC 2.3.2.13) catalyses an acyl tansfer reaction between the primary grade amine and protein or γ-carboxyamide group of peptide bound glutamine residues. TGase is used as a material of many food processing stuffs jelling the protein foods after the formation of cross-links. The microbial TGase gene was cloned from Streptomyces platensis YK-2, which was newly isolated from a forest soil sample collected in Daegu. TGase gene (stgA) was obtained by PCR reaction, Southern blot, and Colony hybridization from Streptomyces platensis YK-2. To construct a high expression vector for stgA, a 1.5 kb fragment including the whole TGase gene was cloned into BamHΙ and XbaI-digested pSET152ET. The pSET152ET vector containing a stgA gene was introduced into S. platensis YK-2 as the host for TGase production by transconjugation using E. coli ET12567 (pUZ8002). Exconjugants were then confirmed by PCR and Southern hybridization. Also, the expression and activity of TGase were assayed by SDS-PAGE and N-terminal protein sequencing, and compared with the strain including only pSET152ET vector.

In order to enhance the production of psychrophilic chitinase, recombinant Escherichia coli Top 10 harboring chitinase gene from Antarctic microorganism KOPRI 22718 was cultivated using flask and jar fermentor. Expression of target protein was performed by Larabinose addition and confirmed by SDS-PAGE and activity test with p-nitrophenyl-N-acetyl-b-D-glucosaminide as a substrate.To increase the cell density and production of enzyme, modified R-medium was tested as a fermentation medium and effect of L-arabinose concentration was also observed. About 28 times higher enzyme production was obtained by using modified R-medium with lactose and 2.5g/L of L-arabinose in fermentor culture than that by using LB medium in flask culture.

Newcastle disease virus (NDV) has come into the spotlight because of its oncolytic property for cancer therapy and usefulness as a viral vector for vaccine development (1, 2). For the clinical use, the NDV production is performed in cell-based system rather than traditional egg-based system for compliance with GMP/GLP (4). In this study, the rapid detection and quantitation method for the NDV produced in cell-based system was developed. The SYBR Green I-based real-time RT-PCR was designed with conventional cheap RT-PCR kit by targeting F gene of the NDV LaSota strain. The developed method was validated by the contents of specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantitation (LOQ) and robustnesss (4-7). The validation results are satisfied with the predetermined acceptance criteria. The validated method was applied to quantitate the virus samples produced in cell-based production system. And the method was compared with other quantitating methods (3, 7). The comparison results showed that the real-time RT-PCR method would be well adequate to detect and quantitate the whole virus particles containing both infective and uninfective rapidly.

Propionibacterium acnes is a typically aerotolerant anaerobic Gram positive bacterium that can cause acne [1]. Many bacteria exhibited adaptation to various environmental stresses where pretreatment with sublethal level of stress induced resistance to subsequent lethal level of same or other stresses [2]. As there is little study on adaptation of P. acnes we examined whether P. acnes also exhibits adaptation phenomenon toward various stresses. A sublethal dose of ethanol (8%, vol/vol), acid (pH 5), H2O2 (0.01%, vol/vol), or NaCl (14%, wt/vol) was applied to a P. acnes KCTC 3314 culture, and the cells were allowed to grow for 0 to 1 h. The stress-adapted cells were then subjected to the following lethal level of stresses - NaCl (25%, wt/vol), ethanol (12%, vol/vol), H2O2 (0.1%, vol/vol), and acid (pH 3). Viability of P. acnes was measured by plate counting on BHI agar, and % survival rates were plotted. Adaptation to 14% NaCl significantly increased the resistance to NaCl, acid, ethanol, and H2O2 indicating cross-protection. Addition of chloramphenicol (translation inhibitor) during NaCl pretreatment prevented the development of resistance to lethal stresses suggesting that NaCl-induced crossprotection requires de novo protein synthesis.

Entomopathogenic fungi are a rich source of natural bioactive compounds. To confirm the productivity of secondary metabolites in entomopathogenic fungi and to select good genera as the producer, 47 typical entomopathogenic fungi were tested for their ability to produce antibiotic activity. The 38 strains (81%) and 30 strains (64%) of these fungi produced either anti-Bacillus compounds or anti-Staphylococcus compounds, respectively, indicating that the entomopathogenic fungi could be regarded as a rich source of natural bioactive compounds. To select suitable cultivation media which facilitate the production of bioactive compounds, nine species of the fungi were cultivated in four different liquid medium supplemented with or without insect-derived material, SANAGIKO. Six out of nine strains showed clear antibiotic activity against Bacillus sp. and Saccharomyces sp. only in the presence of SANAGIKO, suggesting that the production of antibacterial compounds by these fungi is triggered by the presence of the insect-derived material.

Aureobasidium pullulans has been known as microorganisms that produce a large amount of β-1,3-1,6-glucan (exopolysaccharides) used for health foods and cosmetics. To achieve production of higher EXPs (exopolysaccharides), we have optimized media for EXPs by using OFAT (one factor at a time) method and stastical analyses (Plackett-Burman design and Box-Behnken design). Basal medium were determinred in Czapek-Dox broth, Ashbya gossypii extract and amonium sulfate instead of sodium nitrate. Our study showed that sodium nitrate played an important role in cell growth and EXPs production. The relative importance of medium components for maximum glucan production was eavlauated by using Plackett-Burman design. It was showed that sucrose, amonium sulfate and A. gossypii extract were significant components for EXPs production. Also, on the basis of Plackett-Burman design analysis, Box-Behnken design was carried out to search for optimum medium of EPSs production. As a result of the box-behnken design and response surface methodology, EXPs production of the optimum medium was two or three times higher than one of the basal medium(5.34 g/L).

A xylanolytic bacterium, Cellulosimicrobium sp. HY-13, was isolated from the digestive tract of an earthworm, Eisenia fetida. The purified endo-β-1,4-xylanase (XylK) from strain HY-13 was an unique enzyme with the N-terminal amino acid sequence of APSTLEAAAE and a relative molecular mass of 36 kDa. It was most active at pH 6.0 and 55oC. The Vmax and Km values of XylK toward oat spelt xylan were determined to be 4,067 IU/mg and 2.78 mg/ml, respectively. The enzyme primarily degraded a xylan to a series of xylooligosaccharides of xylobiose to xylotetraose, but it could not further hydrolyze xylobiose to xylose. The present results suggest that the relatively highly active XylK lacking exo-xylanolytic activity is a promising candidate for the efficient production of non-digestible xylooligosaccharides that may have beneficial effects to gastrointestinal health by promoting the growth of probiotics.

The kinase cascade of the septation initiation network (SIN), first revealed in fission yeast, activates the contraction of the actomyosin ring, and plays an essential role in fungal septation. SmoAp is associated with SIN for septation in A. nidulans. In this study, SmoAinteracting proteins were isolated from the A. nidulans with SmoAFLAG affinity chromatography and mass spectrometry. As a result, 12 SmoA-interacting proteins were isolated and these proteins bypassed the requirement of the SIN kinases for septum formation and conidiation. These proteins also involved in chromatin remodeling process. Therefore, in A. nidulans, SmoA and SmoA-interacting proteins likely have an antagonistic interaction against the SIN pathway to regulate septation and conidiation.

The human cystathionine gamma-lyase (hCGL) is responsible for methionine-cysteine pathway. hCGL is also the enzyme that can catalyze the formation of hydrogen sulfide together with cystathionine beta synthase in human body. Recently, several mutations in hCGL have been described in patients with cystathioninuria, a rare but poorly understood genetic disease. Cystathioninuria can also be secondarily associated with a wide range of diseases including Diabetes insipidus, Down's syndrome, Neuoroblastoma, Hepatoblastoma, and Celiac disease. To characterize the enzymatic properties of the cgl gene overexpressed in E. coli, we investigated the various conditions for overexpression of pNIC28-Bas4 plasmid which has the T7 promoter under the control of lacO gene. Although the crystal structure was recently reported, the overexpression of hCGL is still important to pursue the studies of reaction mechanism and substrate specificities. Here we report that the optimum conditions such as low culturing temperature, very low aeration during the overexpression, and low concentration of IPTG for the induction are necessary for hCGL overexpression.

The use of sophorolipids (sophorose lipid), one of the main classes of bio-surfactants, has risen than chemical surfactants due to global environmental concerns and the awakening to the exhaustibility of our natural resources (1). Typically, sophorolipids from Candida sp. are mixture of lactone form that the carboxylic acid group of the fatty acid is covalently linked to the disaccharide ring and free acid form. And they have different characters of biosurfactant according to the types. Sophorolipids from Candida bombicola ATCC 22214 was cultured on standard medium in the jar fermenter. After the culture, sophorolipid was isolated from broth by extraction with ethyl acetate and evaporation (2). Isolated sophorolipids was analyzed by HPLC for observation of various forms in mixture (3).

Leuconostoc is a major lactic acid bacterium that produces D-lactate in fermented foods. Excessive dietary intake of D-lactate may cause metabolic stress in both infants and patients. Introduction of ldhL gene from Lb. plantarum into Leuconostoc citreum did not significantly shift D-lactate to L-lactate due to low L-LDH activity. To elevate the expression level of ldhL so that significantly alter D-/L-lactate ratio, nucleotides for pyruvate kinase promoter were fused to ldhL and cloned with pLeuCM42 to construct pLC42pkL. Introduction of pLC42pkL in L. mesenteroides subsp. mesenteroides ATCC8293 significantly improved both L-LDH activity and L-lactate productivity during MRS fermentation so that D-/L-lactate ratio was altered significantly. These results indicated that the enhanced L-LDH activity and L-lactate synthesis contributed to the active promoter of native pyruvate kinase and appropriate delivery vector of pLeuCM42. The resulting construct of actively expressing ldhL will further effectively applied in fermented foods. Furthermore, this work provides evidence that expression of other heterologous genes may facilitate the production of useful proteins in these economically important bacteria.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] can be tailored to achieve a variety of polymeric materials, from hard crystalline plastics to very elastic rubber by varying 4HB monomer composition. In general, 3HB is produced by feeding precursor carbon sources such as glucose, fructose, sucrose and plant oil, while 4HB is produced from precursors such as γ-butyrolactone, 1,4-butanediol and 4-hydroxybutyric acid. Plant oils are desirable feedstocks for PHA production because they are inexpensive carbon sources. The theoretical yield coefficients of PHA production from plant oils are as high as over 1.0 g-PHA per g-plant oils used, since they compose a much higher number of carbon atoms per weight. In this study, we attempted to produce P(3HB-co-4HB) copolymer by Ralstonia eutropha ATCC 17699 using a mixture of soybean oil and γ-butyrolactone. The effect of soybean oil and γ-butyrolactone on cell growth was investigated by varying the concentrations in flask cultures. The effects of culture volume on cell growth and P(3HB-co-4HB) production were also carried out. In addition, the 4HB precursor feeding strategies were studied to improve the copolymer production.

For efficient saccharification from alginate, a marine bacterium was isolated from seawater near the Korean south coast. Based on 16S rDNA sequence, the isolated strain was identified as Pseudoalteromonas agarovorans. Various environmental factors affecting saccharification of alginate using P. agarovorans CHO-12 have been investigated in flask cultures. The optimum concentration of sugar was obtained at 30 rpm and 29oC. Among various NaCl concentrations, when NaCl concentration was increased from 10g/l to 30g/l, the cell concentration sharply increased, while there is no increase at above 40g/l. The maximum sugar concentration was obtained at 13.8g/l when 30g/l of NaCl was used. Yeast extract and CSL were the best nitrogen source for efficient saccharification. Especially, the sugar concentration of 14.9 g/l was obtained after 3 days of culture using a mixture of 1.0 g/l of yeast extract and 1.5 g/l of peptone. Scale up was carried out at 50l of reactor for 3 days using P. agarovorans CHO-12 and Stenotrophomonas maltophilia sp. When S. maltophilia was used, cell concentration was about 2.0 fold higher than that of P. agarovorans CHO-12. On the other hand, when P. agarovorans CHO-12 was used, the maximum saccharification rate was obtained, 7.5 g/l/day after 2 days of culture, which was about 10.0 fold higher than that of S. maltophilia.

The uncharacterized abf gene encoding α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus DSM 8903, belonging to family 51 of the glycoside hydrolases, was cloned and expressed in Escherichia coli. The maximal activity of the enzyme was observed at pH 5.5 and 80°C. The half-lives of the enzyme at 65, 70, 75, 80, and 85°C were 419, 141, 58, 0.8, and 0.1 h, respectively. The enzyme showed activity for pNP-α-L-arabinofuranoside and pNP-α-Larabinopyranoside among pNP-glycosides. The enzyme released Larabinose from arabinan, debranched arabinan, and arabinoxyran. However, endoarabinanase activity was not detected. Thus, α-Larabinofuranosidase from C. saccharolyticus DSM 8903 was suggested as an exo-acting enzyme.

A recombinant endo-1,5-α-l-arabinanase from Caldicellulosiruptor saccharolyticus DSM 8903 with a specific activity of 12 U mg–1 was purified by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band on SDS-PAGE. Endo-1,5-α-larabinanase showed optimum activity at pH 6.5 and 75ºC for debranched arabinan. The half-lives of the enzyme at 65, 70 and 75ºC were 2440, 254 and 93 h, respectively. The addition of CoSO4 resulted in about 1.9 fold increase of the enzyme activity. However, the enzyme was relatively insensitive to other metal ions.

B-type natriuretic peptide is a cardiac derived peptide hormone. It is secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells. BNP levels are related to the severity of sings and symptoms of heart failure. In this respect, its concentrations have been able to accurately reflect diagnosis, prognosis and differentiate heart failure care pathway. It has emerged as a receptor of BNP, useful and cost-effective biomarkers, anti-BNP scFv antibody development is meaningful. But there is no research about anti-BNP scFv antibody, we try express anti-BNP scFv antibody through Escherichia coli expression. In an attempt to develop, we engineered anti-BNP scFv antibody in pET vector and it is possible to make folded and functional antibody fragments in excellent yield.

Streptococcus pneumoniae was cloned and expressed in E.coli Er2566. The recombinant RpiB was purified by HisTrap HP chromatography. The enzyme exhibited maximal activity at pH 7.5 and 35°C, and its activity was independent of metal ions. The molecular mass of native RpiB was estimated to be 98.2 kDa as a tetramer using Sephacryl S-300 gel filtration. The enzyme showed specificity for aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as L-lysose,L-talose, D-ribose, D-allose, Dgulose, D-xylose, and L-mannose. Especially, the RpiB exhibited high enzyme activity for the L-form aldoses such as L-lyxose and L-talose among various aldoses. The catalytic efficiency (kcat/Km) of RpiB for L-lyxose and L-talose were 3.86 and 1.2 mM-1s-1, respectively. Recently, L-form sugars have been increased the interest as a potential functional material in the food and pharmaceutical industry. Thus, it is suggested strongly that of RpiB from Streptococcus pneumonia is applied usefully for the production of L-form sugars.

The alginate lyase gene of Streptomyces sp. ALG-5 was cloned by using PCR, and the alginate lyase appears to be poly-guluronate lyase degrading poly-G block preferentially than poly-M block. The recombinant alginate lyase was purified and immobilized onto magnetic nanoparticles. The immobilized alginate lyase was used to hydrolyze alginic polymer, and oligosaccharides of di-, tri-, tetra- and pentasaccharides were produced. The immobilized alginate lyase was efficiently recovered and reused repeatedly by magnetic separation.

The rare ginsenosides (compound Y, Mc and K) were produced from reagent-grade ginsenosides (Rb1, Rb2 and Rc) by a thermostable β-glycosidase from Sulfolobus acidocaldarius. Optimum pH and temperature for producing rare ginsenosides were 5.5 and 85℃, respectively. Rare ginsenosides were produced via each pathway : Rb1à Rd à compound K, Rb2 à compound Y à compound K, or Rc à compound Mc. The product, were identified by LC/MS based on its molecular weight. At pH 5.5 and 85℃, 0.53 mg/ml compound K, 0.56 mg/ml compound Y and 0.75 mg/ml compound Mc was produced within 180 min from 1 mg/ml reagent-grade ginsenoside. Rb1, Rb2, and Rc, respectively.

Lactide that have been used as monomer for ring-opening polymerization of PLA (poly lactic acid) is important material for industrial production of PLA which is one of bio-plastics. However, enantioselective reaction from lactate to lactide has been not reported so in this study lactide was enantioselectively synthesized by esterification of lactate using lipase. The one of many lipases which was CAL-B only could synthesize lactide as highest conversion and showed R-specific enantioselectivity.[1],[2] Although lactide and lactate have less solublility than other ether solvent[3],[4], hexane is used as an appropriate solvent for reaction due to showing the higher activity than any other solvents. Also when the reaction temperature is changed from 5℃ to 60℃, the rate of consumption and conversion is gradually increased. However, the final amount of lactide is still same after 24 hours in spite of increasing temperature. The result of 2nd reaction showed that reaction was limited by absorption ability of molecular sieve about alcohol. Therefore, if a process for the effective removal of alcohol were developed, Enantiospecific lactide is produced by CAL-B with more efficiency.

The recombinant β-glycosidase from Sulfolobus acidocaldarius was purified by heat treatment and Hi-trap anion chromatography with a specific activity of 58.39 U mg-1 and identified as a single 54 kDa band on SDS-PAGE. The molecular mass of the native enzyme was 108 kDa as a dimer by gel filtration chromatograph. The maximum temperature and pH for β-glycosidase activity was 90ºC and 5.5 with pNP-glu, respectively. The half-lives of the enzyme at 70, 75, 80, 85, and 90ºC were 494, 453, 60, 9, and 0.2 h, respectively. The enzyme have broad substrate specificity to several aryl-glycosides of hydrolysis activity. The recombinant enzyme exhibited a higher transglycosylation activity for lactose as substrate than cellobiose, whereas higher hydrolysis activity for cellobiose.

The mpi gene encoding mannose-6-phosphate isomerase (GTMpi) from Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. GTMpi converted aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions, such as the D- and L-forms of ribose, lyxose, talose, mannose, and allose. D-Lyxose isomerization was optimal at pH 7.0 and 70°C with 1mM Co2+. The half-lives of the enzyme at 60°C, 65°C, and 70°C were 388 h, 73h, and 27 h, respectively. D-Lyxose was converted to D-xylulose by GTMpi with 38% conversion yields after about 3 h, whereas L-ribose was converted to L-ribulose with 29% conversion yields after about 3 h.

In this study, we utilized a catabolite repressorto improve the enzymatic activity of recombinantb-galactosidase inclusion bodies (IBs) produced inEscherichia coli under the araBAD promoter system.Specifically, we employed methyl a-D-glucopyranoside(a-MG) to lower the transcription rate of the b-galactosidasestructural gene. In deepwell microtiter plate and labscalefermentor culture systems, we demonstrated that theaddition of a-MG after induction improved the specificb-galactosidase production, even though b-galactosidasewas still produced as an IB. Particularly, the addition of0.0025% a-MG led to the most significant increase in thespecific activity of the bgalactosidase. Interestingly, theb-galactosidase IBs obtained in the presence of 0.0025%a-MG were more loosely packed, as determined by IBsolubilization in guanidine hydrochloride solution. Wepropose that the reduced gene transcription rate wasresponsible for the increased specific b-galactosidaseactivity and the loose packing that characterized the IBsproduced in the presence of a-MG. This principle could beapplied throughout the enzyme bioprocessing industry inorder to enhance the activity of aggregate-prone enzymeswithin IBs.

Human cystathionine gamma-lyase (hCGL) is a PLP dependent enzyme that catalyzes the cleavage reaction of L-cystathionine to Lcysteine and 2-ketobutyrate. hCGL also uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The hCGL/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we found a novel reaction of hCGL that directly converting seleno-L-methionine (SeMet) into methylselenol, 2-ketobutyrate, and NH3. The hCGL gene was cloned into a pET-28 vector which incorporating a TEV cleavable N-terminal His-tag fusion. hCGL was purified by a His-bind column chromatography followed by TEV protease cleavage. Authentic methylselenol was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The gaseous product of the enzymatic reaction with SeMet was determined by making the 2, 4-dinitrophenyl derivative, subjecting it to analysis by GC/MS spectrometry. GC/MS data was identical to that of authentic 2, 4-dinitrophenyl-methylselenol. 2-ketobutyrate was also detected by the MBTH method. These results suggested that hCGL is a critical enzyme charging SeMet metabolism.

The kanF and tobM1 are two glycosyltransferase (GTs) genes located in kanamycin and tobramycin biosynthetic gene clusters from Streptomyces kanamyceticus and Streptomyces tenebranius, respectively. The gene sequence analysis showed that kanF has 63%, 54%, 31% and 63% identities with tobM1, gtmG, rbmD and neo8 respectively whereas tobM1 expressed 59%, 64% and 68% identities with gtmG, neo8 and rbmD, respectively. Function of neo8 and rbmD has been characterized as UDP-N-acetlyglucosaminyltransferses from neomycin and ribostamycin producer, respectively. Thus, kanF and tobM1 are predicted to be responsible for the same enzymatic activities in paromamine biosynthesis and attempting to test the substract flexibility of these enzymes in vitro. So, we have successfully overexpressed these genes in Escherichia coli, got the soluble enzymes and further enzymatic reactions have to be carried out.

Effective immobilized techniques provide a great improvement on the stabilization of enzyme activity. In this study, biosilicification catalyzed by the silaffin R5 peptide from marine diatom Cylindrotheca fusiformis was investigated for the feasible evaluation of enzyme immobilization and stabilization. Glutathione S-transferase (GST) as the model enzyme was used, and GST and R5 tagged GST gene (GSTR5, R5-GST-R5) were amplified by PCR and inserted into the pET-28 a(+) vector for the expression in Escherichia coli BL21(DE3). Biosilicification was performed by adding tetramethoxysilane (TMOS) to R5 tagged proteins, which resulted in silica particle precipitation at ambient temperature and pressure in vitro. The silica nano-beads (about 200 nm in diameter) retained high GST activity. Loading efficiency (percent of enzyme with precipitated silica) and immobilization yield of R5 tagged GSTs (GST-R5, R5-GST-R5) increased as pH decreased. Maximum loading efficiency (95.48%) and immobilization yield (67.54%) with R5-GST-R5 at pH 6.0 were observed, which were higher than those with GST-R5.

It is of interest to develop a value-added chemical transformation of glycerol to facilitate the use of glycerol because large amount of glycerol is produced as the major byproduct in biodiesel production. We investigated lipase-catalyzed transesterification for synthesis glycerol carbonate from glycerol and dimethylcarbonate in the solvent-free system. Various lipases have also been tested for their abilities to synthesize glycerol carbonates. And immobilized lipase of Candida antartica was employed as the biocatalyst. To enhance miscibility of glycerol, glycerol was adsorbed onto silica gel and then suspended in dimethylcarbonate. The reaction conditions were optimized, and glycerol carbonate was successfully synthesized in solvent-free system.

In this study, the inactivation of Coprinus cinereus peroxidase (CiP) during the oxidation of various phenolic compounds originating from lignin was investigated. The CiP was significantly inactivated during the oxidation of phenolic compounds, such as vaniline, p-coumaric acid, 2, 6-dimethoxy phenol, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, p-cresol, m-cresol and phenol. Conversely, the CiP nearly maintained its initial activity for the oxidation of syringic acid, vanilic acid and ferulic acid. Hydrogen peroxide affected the CiP inactivation, while the polymerized reaction product hardly affected the CiP inactivation. The thermodynamic parameter(ΔΔG) and turnover capacity (ΔS/ΔE) were adapted to explain the CiP inactivation due to covalent bonding between the enzyme and phenolic compounds. In the cases of syringic acid, vanilic acid and ferulic acid, which maintained high residual CiP activities after reaction, the ΔΔG were more negative than the other values. This means that these compounds prefer to form a dimer rather than an enzyme-phenolics complex; hence, they might not cause CiP inactivation. The higher the turnover capacity of CiP the lower the ΔΔG° , indicating that some of the phenolic compounds require more CiP for their conversion due to the inactivation of CiP as a result of their formation of covalent bonding with CiP.

Aminoglycosides are a group of antibiotics that are used to treat certain bacterial infections. This group of antibiotics includes at least eight drugs, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, and tobramycin. UDP-kanosamine (UDP-3-amino-3-deoxyglucose) is a sugar moiety of kanamycin that was highly inhibitory to growth of plant-pathogenic oomycetes and moderately inhibitory to certain fungi and inhibited few bacterial species tested. Biosynthesis of UDP-kanosamine starts with UDPglucose via UDP-3-keto-glucose. Here we have characterized KanC as a UDP-glucose dehydrogenase to form UDP-3-keto-glucose, an intermediate of UDP-kanosamine. We have cloned KanC in expression vector pET32a+ and express the soluble protein. Enzymatic assay was carried out using UDP-glucose as a substrate and NADP+ as a cofactor. The product was analyzed by HPLC and ESI-MS.