Isolation and characterization of N-acylhomoserine lactonase from the thermophilic bacterium, Geobacillus caldoxylosilyticus YS-8.

Geobacillus caldoxylosilyticus YS-8, which was isolated from volcanic soil in Indonesia, was found to degrade various N-acylhomoserine lactones (AHLs) with different lengths and acyl side-chain substitutions over a wide temperature range of 30-70 °C. The purified AHL-degrading enzyme showed a single band of 32 kDa, and its N-terminal amino acid sequence was determined to be ANVIKARPKLYVMDN, tentatively suggesting that the AHL-degrading enzyme was AHL lactonase. The AHL-degrading activity of the purified enzyme was maximized at pH 7.5 and 50 °C, and it retained about 50% of its activity even after a heat treatment at 60 °C for 3 h, exhibiting properties consistent with a thermostable enzyme. The mass spectrometric analysis demonstrated that the AHL-degrading enzyme catalyzed lactone ring opening of N-3-oxohexanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone by hydrolyzing the lactones and working as an AHL lactonase.

Cloning, expression and characterization of xylose isomerase, XylA, from Caldanaerobacter subterraneus subsp. yonseiensis.

The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95 degrees C. The enzyme required divalent cations including Zn(2+) for its maximal activity and thermostability.

Enhanced production of gamma-aminobutyric acid using rice bran extracts by Lactobacillus sakei B2-16.

An efficient and simple fermentation process was developed for the production of gamma-amminobutyric acid (GABA) by Lactobacillus sakei B2-16. When the L. sakei B2-16 was cultivated in the rice bran extracts medium containing 4% sucrose, 1% yeast extract and 12% monosodium glutamate, the maximum GABA concentration reached 660.0 mM with 100% conversion yield, showing the 2.4-fold higher GABA concentration compared to the modified MRS medium without the rice bran extracts. The GABA production was scaled-up from a laboratory scale (5 L) to a pilot (300 L) and a plant scales (5,000 L) to investigate the application possibility of GABA production to industrial fields. The GABA production at the pilot and plant scales was similar to the laboratory scale using rice bran extracts medium which could be effective for the low-cost production of GABA.

Pulsed electric field (PEF) technology for microbial inactivation in low-alcohol red wine.

The decontamination of spoilage-related microbes in low-alcohol red wine was performed using a serial multiple electrode pulsed electric field (PEF) treatment system. The system consisted of seven electrodes connected in series, and it has been designed to produce square-wave high-voltage pulses of 1 µs duration at various electric field strengths and frequencies for decontamination. The initial counts of aerobic bacteria, yeast and lactic acid bacteria (spoilage-associated microbes) in the wine were 5.56, 5.61 and 5.22 log CFU/mL, respectively. The pattern of decontamination of the spoilage microorganisms followed first-order kinetics and the decontamination effect increased as the field strength and frequency increases. DHz and DPEF values were inversely related to the electric field strength of the PEF treatment. The yeast exhibited relatively low DPEF-value than the aerobic and lactic acid bacteria. The lowest ZPEF-value was observed for the lactic acid bacteria (24.6 kV/cm) among the spoilage microbes.

A sulfated fucan from the brown alga Laminaria cichorioides has mainly heparin cofactor II-dependent anticoagulant activity.

The major acidic polysaccharide from the brown alga Laminaria cichorioides is a complex and heterogeneous sulfated fucan. Its preponderant structure is a 2,3-disulfated, 4-linked alpha-fucose unit. The purified polysaccharide has a potent anticoagulant activity, as estimated by APTT assay ( approximately 40 IU/mg), which is mainly mediated by thrombin inhibition by heparin cofactor II. It also accelerates thrombin and factor Xa inhibition by antithrombin but at a lower potency. Sulfated fucan from L. cichorioides is a promising anticoagulant polysaccharide and a possible alternative for an antithrombotic compound due to its preferential heparin cofactor II-dependent activity.

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana.

Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t (1/2) = 43 days at 70 degrees C) in a continuous recycling mode at 70 degrees C produced 49 and 38 g D-tagatose/l from 180 and 90 g D-galactose/l, respectively, within 12 h.

Variations in protein glycosylation in Hansenula polymorpha depending on cell culture stage.

A simple way to prevent protein hyperglycosylation in Hansenula polymorpha was found. When glucose oxidase from Aspergillus niger and carboxymethyl cellulase from Bacillus subtilis were expressed under the control of an inducible methanol oxidase (MOX) promoter using methanol as a carbon source, hyperglycosylated forms occurred. In contrast, MOX-repressing carbon sources (e.g., glucose, sorbitol, and glycerol) greatly reduced the extent of hyperglycosylation. Carbon source starvation of the cells also reduced the level of glycosylation, which was reversed to hyperglycosylation by the resumption of cell growth. It was concluded that the proteins expressed under actively growing conditions are produced as hyperglycosylated forms, whereas those under slow or nongrowing conditions are as short-glycosylated forms. The prevention of hyperglycosylation in the Hansenula polymorpha expression system constitutes an additional advantage over the traditional Saccharomyces cerevisiae system in recombinant production of glycosylated proteins.

In vitro evolution of lipase B from Candida antarctica using surface display in hansenula polymorpha.

ALipase B from Candida antarctica (CalB) displayed on the cell surface of H. polymorpha has been functionally improved for catalytic activity by molecular evolution. CalB was displayed on the cell surface by fusing to a cell-wall anchor motif (CwpF). A library of CalB mutants was constructed by in vivo recombination in H. polymorpha. Several mutants with increased whole-cell CalB activity were acquired from screening seven thousand transformants. The two independent mutants CalB10 and CalB14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB 10 showed 6 times greater activity and CalB14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of Leu278Pro in CalB10 and Leu278Pro/Leu219Gln in CalB14. The substituted Pro278 in both mutants was located near the proline site of the alpha10 helix. This mutation was assumed to induce a conformational change in the alpha10 helix and increased the k(cat) value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ (Leu219Gln) was secreted into the culture supernatant at an amount of approximately 3 times more without an increase in the CalB transcript level, compared with the wild-type.

Enzymatic susceptibility of wheat gluten after subcritical water treatment.

Subcritical water (SCW) hydrolysis is an alternative to traditional methods of protein hydrolysis that uses water as a reaction medium. In this study, the effect of SCW treatment on heat-induced conformational changes in wheat gluten and its relation to enzymatic susceptibility were investigated. The degree of deamidation increased rapidly from 12.5 to 47.4% with increase in the temperature range of 160-220 °C. Protein solubility increased in a similar pattern with degree of deamidation and almost all protein was solubilized after treatment with SCW at 200 °C. SCW treatment in a particular time-temperature combination results in a significant decrease in enzymatic susceptibility. After SCW treatment at 220 °C for 20 min, enzymatic susceptibility of gluten protein was exceedingly decreased to nearly complete loss. Because of excess degradation and deamidation and small molecular size (less than 6500 Da) many hydrolysis sites disappear and are difficult to access by protease.

Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1.

Pediocin PA-1 is a representative class IIa bacteriocin which is small and heat-stable and has a consensus motif, -YGNGV-. The plasmid pQE40PED, encoding pediocin PA-1 fused with His-tagged mouse dihydrofolate reductase (DHFR), was constructed and introduced into Escherichia coli M15 strain. The fusion protein was overexpressed in the strain after induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active pediocin PA-1, the purified fusion protein was cleaved by Factor Xa protease and the liberated pediocin PA-1 was finally purified by ultrafiltration with a 75% yield. The molecular mass of the purified recombinant pediocin PA-1 was the same as that of native pediocin PA-1 on an electrophoresis gel.

Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from raw chicken meat and human stools in Korea.

Prevalence of Campylobacter in raw chicken meat and human stools and subsequent antibiotic resistance profiles of the pathogenic isolates obtained from 2000 through 2002 were investigated. Campylobacter jejuni and Campylobacter coli were isolated from 570 of the 923 raw chicken meat samples collected from traditional markets, large retail stores, or department stores in Korea, resulting in the isolation rate of 61.8%. A total of 579 Campylobacter isolates were obtained from raw chicken (36.3% for C. jejuni and 26.4% for C. coli) with the average population of 335.6 CFU/g. From 513 human stool samples, 15 isolates of Campylobacter were detected. Seasonal variation in the quantification of C. coli was not noticeable throughout the year, while the isolation rate of C. jejuni was the highest in September through October (840 CFU/g) followed by that of July through August and May through June in decreasing order, showing a significant seasonal effect (P < 0.05). Contamination of Campylobacter was more severe in raw chicken meat sold in traditional markets than in those sold in large retail stores and department stores. Prevalence of Campylobacter in raw chicken sold in traditional markets was significantly influenced by seasonal changes (P < 0.05), whereas the samples obtained from other places was less affected by the seasonal changes. Susceptibilities of the 594 chicken isolates to ciprofloxaxin, chloramphenicol, erythromycin, kanamycin, nalidixic acid, and tetracycline were determined by an E-test. Campylobacter isolates were the most resistant to nalidixic acid (91.4%) followed by ciprofloxaxin (87.9%), tetracycline (87.2%), kanamycin (30.6%), erythromycin (19.4%), and chloramphenicol (1.3%). Human isolates showed a similar resistance to the six antibiotics tested. The proportion of Campylobacter isolates with multidrug resistance to four or more antimicrobials obtained from 2000 through 2002 ranged from 28 to 43.5%, indicating that it could be a serious health-threatening factor. This study suggests that it is prudent to establish an effective National Monitoring Program in Korea for the prevention and control of Campylobacter spp.

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L-arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures.

To gain insight into the structural stability of homologous homo-tetrameric l-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DeltaGU) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn2+, those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (Tm) due to decreased heat capacity (DeltaGp). Moreover, the extent of difference in DeltaCp between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DeltaCp, may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.

Analysis of kimchi microflora using denaturing gradient gel electrophoresis.

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 degrees C or 20 degrees C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application of this culture-independent molecular technique determined that the lactic acid bacteria Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, and Lactobacillus curvatus were the main microorganisms responsible for kimchi fermentation.

Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: bioconversion of D-galactose to D-tagatose using the enzyme.

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.

Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi.

The influence of growth parameters on the fermentative production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi was studied. The bacteriocin production was greatly affected by carbon and nitrogen sources. Strain A164 produced at least 4-fold greater bacteriocin in M17 broth supplemented with lactose than other carbon sources. The amount of 3% yeast extract was found to be the optimal organic nitrogen source. While the maximum biomass was obtained at 37 degrees C, the optimal temperature for the bacteriocin production was 30 degrees C. The bacteriocin production was also affected by pH of the culture broth. The optimal pH for growth and bacteriocin production was 6.0. Although the cell growth at pH 6.0 was nearly the same level at pH 5.5 and 6.5, the greater bacteriocin activity was observed at pH 6.0. Exponential growth took place only during an initial period of the cultivation, and then linear growth was observed. Linear growth rates increased from 0.160 g(DCW) x l(-1) x h(-1) to 0.245 g(DCW) x l(-1) x h(-1) with increases in lactose concentrations from 0.5 to 3.0%. Maximum biomass was also increased from 1.88 g(DCW) x l(-1) to 4.29 g(DCW) x l(-1). However, increase in lactose concentration did not prolong the active growth phase. After 20 h cultivation, cell growth stopped regardless of lactose concentration. Production of the bacteriocin showed primary metabolic kinetics. However, bacteriocin yield based on cell mass increased greatly during the late growth phase. A maximum activity of 131x10(3) AU x ml(-1) was obtained at early stationary growth phase (20 h) during the batch fermentation in M17L broth (3.0% lactose) at 30 degrees C and pH 6.0.

The nontoxic mushroom Auricularia auricula contains a polysaccharide with anticoagulant activity mediated by antithrombin.

An acidic polysaccharide with anticoagulant activity was isolated from the edible mushroom Auricularia auricula using water, alkali or acid extracts. The alkali extract showed the highest anticoagulant activity and was thereby further purified using gel filtration chromatography. Specific anticoagulant activity of the purified polysaccharide was 2 IU/mg and its average mass was approximately 160 kDa. The polysaccharide from this species of mushroom contains mainly mannose, glucose, glucuronic acid and xylose but no sulfate esters. Its anticoagulant activity was due to catalysis of thrombin inhibition by antithrombin but not by heparin cofactor II. Inhibition of Factor Xa by antithrombin was not catalyzed by the polysaccharide. The glucuronic acid residues were essential for the anticoagulant action of the mushroom polysaccharide since the activity disappeared after reduction of its carboxyl groups. In ex vivo tests using rats orally fed with the polysaccharide, we observed an inhibitory effect on platelet aggregation as observed with aspirin, a well-known antiplatelet agent. The polysaccharides from these mushrooms may constitute a new source of compounds with action on coagulation, platelet aggregation and, perhaps, on thrombosis.

The medicinal plant Porana volubilis contains polysaccharides with anticoagulant activity mediated by heparin cofactor II.

We searched for polysaccharides with anticoagulant activity and inhibitory action on platelet aggregation induced by collagen in 59 species of medicinal plants. We then concentrated our studies on the polysaccharide from the species Porana volubilis, which showed the highest anticoagulant activity among the plants tested. The polysaccharide from this species has an average molecular mass of approximately 10 kDa, contains mainly galactose, galacturonic acid, and mannose but no sulfate esters. Its anticoagulant activity is mediated by the enhancement of thrombin inhibition that in turn is mediated by heparin cofactor II but not by antithrombin. The galacturonic acid residues are essential for activity since after reduction of its carboxyl groups the anticoagulant activity disappears. Our report is the first description of a natural nonsulfated polysaccharide from higher plants with anticoagulant activity, which may constitute a new source of compounds with action on coagulation and, perhaps, on thrombosis.

Cloning, expression, and characterization of thermostable DNA polymerase from Thermoanaerobacter yonseiensis.

A gene, coined tay, for a thermostable DNA polymerase from the novel, extremely thermophilic bacterium Thermoanaerobacter yonseiensis was cloned and expressed in E. coli. Using a DNA polymerase homologous PCR product as a hybridization probe, tay was isolated and sequenced to consist of 2,616 nucleotides that encode 872 amino acids. A database analysis showed that DNA polymerase, coined Tay, from T. yonseiensis shared a 39 percent to 47 percent identity in the amino acid sequence with those from other DNA polymerases. Tay was overexpressed in E. coli as a fusion protein with a poly-histidine tag at the Cterminus. It was purified by heat treatment, followed by a Ni(2+)-chelate column. The molecular weight of purified Tay was approximately 97 kDa, as shown by SDS PAGE, and it showed high DNA polymerase activity and thermostability. However, it had no 3'-->5' exonuclease activity

Melanocins A, B and C, new melanin synthesis inhibitors produced by Eupenicillium shearii. II. Physico-chemical properties and structure elucidation.

New melanin synthesis inhibitors, melanocins A, B and C, were isolated from the fermentation broth and extract of mycelium of Eupenicillium shearii F80695. The structures of melanocins were established by spectroscopic methods. They are formamide compounds. In particular, melanocin A has an isocyanide group.

Melanocins A, B and C, new melanin synthesis inhibitors produced by Eupenicillium shearii. I. Taxonomy, fermentation, isolation and biological properties.

New melanin synthesis inhibitors, melanocins A, B and C, were isolated from the fermentation broth and mycelium extract of Eupenicillium shearii F80695. Melanocin A, an isocyanide compound, inhibited mushroom tyrosinase and melanin biosynthesis of B16 melanoma cells with IC50 value of 9.0 nM and MIC value of 0.9 microM, respectively. Melanocin A also inhibited growth of Streptomyces bikiniensis. While, the structurally very related but non-isocyanide compounds melanocins B and C did not show inhibitory activity in these assays. Melanocins A, B and C showed potent antioxidant activity with scavenging activity of DPPH radical and superoxide anion radical.