Antimicrobial activity of torilin isolated from Torilis japonica fruit against Bacillus subtilis.

Torilis japonica fruit has been used in therapeutic antimicrobial treatments in Korea and China since ancient times, but there is still little information on the mechanism underlying this activity. We found that the ethanol extract of T. japonica fruit showed excellent antimicrobial activity against Bacillus subtilis ATCC 6633 spores and vegetative cells. The crude ethanol extract (75%) reduced the spore concentration by 3 log cycles and the vegetative cell concentration to lower than the detection level (reduction in spore concentration by more than 6 orders of magnitude) at a concentration of 1% (w/v). The ethanol extract of T. japonica fruit was fractionated into n-hexane (H) and a water layer. The active antimicrobial compound was isolated and purified from the hexane layer, and identified as torilin (5-[1-(acetyloxy)-1-methylethyl]-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octa-hydroazulen-6-yl(2E)-2-methylbut-2-enoate; C(22)H(32)O(5)). Torilin immediately reduced vegetative cells counts by 5 to 6 orders of magnitude, and reduced spores counts by 1 order of magnitude. The integrity of structures such as the inner, middle, and outer layers of the coat and the cortex, protoplast membrane, and core are vital to spores. Torilin functions as a surfactant with hydrophilic and hydrophobic properties related to denaturalization of various proteins. The distortion of coat proteins due to direct binding polar groups of spore coats with hydrophilic groups of torilin may be responsible for the observed rapid inactivation of bacterial spores.

Sphingomonas aquatilis sp. nov., Sphingomonas koreensis sp. nov., and Sphingomonas taejonensis sp. nov., yellow-pigmented bacteria isolated from natural mineral water.

The taxonomic status of four strains from several mineral water sources in Taejon, Korea, was examined. The DNA base compositions of these strains ranged from 62 to 66 mol %. All of the strains contained sphingolipids and ubiquinone 10 as the main respiratory quinone. The cellular fatty acids of these strains included octadecenoic acid, hexadecanoic acid and 2-hydroxymyristic acid, without 3-hydroxy fatty acids. On the basis of morphological, physiological and chemotaxonomic characteristics, together with 16S rDNA sequence comparison and DNA-DNA reassociation data, three new species of the genus Sphingomonas are proposed for the strains isolated from natural mineral water: Sphingomonas aquatilis sp. nov. for strain JSS7T (= KCTC 2881T = KCCM 41067T), Sphingomonas koreensis sp. nov. for strains JSS26T (= KCTC 2882T = KCCM 41069T) and JSS28 (= KCTC 2883 = KCCM 41066) and Sphingomonas taejonensis sp. nov. for strain JSS54T (= KCTC 2884T = KCCM 41068T).

Thermoanaerobacter yonseiensis sp. nov., a novel extremely thermophilic, xylose-utilizing bacterium that grows at up to 85 degrees C.

A novel strictly anaerobic, extremely thermophilic, spore-forming and xylose-utilizing bacterium, designated strain KB-1TP (type and patent strain), was isolated from a geothermal hot stream at Sileri on Java island, Indonesia. The cells were rod-shaped, motile and had terminal spores. The newly isolated strain stained gram-positive and the cells occurred singly or in pairs during the exponential growth phase. The temperature optimum for growth was 75 degrees C and growth occurred in the range 50-85 degrees C. The pH range for growth was 4.5-9.0, with an optimum at pH 6.5. Strain KB-1TP grew chemo-organotrophically by fermenting a wide range of substrates such as glucose, fructose, D-xylose, lactose, maltose, sucrose, mannose, galactose, cellobiose, pullulan and soluble starch. Arabinose, xylan, cellulose, olive oil and Tween 80 were not fermented. The predominant fermentation end products after growth on glucose were lactate, acetate, ethanol, CO2 and small amounts of isovaleric acid, butyric acid, propionic acid, 1-pentanol and 2-propanol. Thiosulfate was reduced to H2S. Strain KB-1TP was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, kanamycin, vancomycin and rifampicin at concentrations of 100 microg ml(-1). No effect was observed with chloramphenicol and neomycin at concentrations of 10 microg ml(-1). This indicates that strain KB-1TP belongs to the bacterial domain. The G+C content of the DNA was 37 mol%. The comparison of the 165 rDNA sequence to that of closely related strains revealed that strain KB-1TP belonged to clostridial cluster V, showing highest sequence identities (92.7%) to members of the genus Thermoanaerobacter. Taking into account the physiological and molecular properties of the new isolate, it is proposed that strain KB-1TP should be classified as a new species of the genus Thermoanaerobacter, designated Thermoanaerobacter yonseiensis. The type strain, KB-1TP, has been deposited in the Korean Federation of Culture Collections (KFCC 11116P) as a patent strain and in the Deutsche Sammlung von Mikroorganismen und Zellkulturen as a type strain (= DSM 13777T).

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae MNN9 gene.

A gene homologous to Saccharomyces cerevisiae MNN9 has been cloned and characterized in the methylotrophic yeast Hansenula polymorpha. This gene was cloned from a H. polymorpha genomic DNA library using the S. cerevisiae MNN9 gene as a probe. The H. polymorpha MNN9 homologue (HpMNN9) contained a 1062 bp open reading frame encoding a predicted protein of 354 amino acids. The deduced amino acid sequence showed 58% and 51% identity, respectively, with the S. cerevisiae and Candida albicans Mnn9 proteins. Disruption of HpMNN9 leads to phenotypic effects suggestive of cell wall defects, including detergent sensitivity and hygromycin B sensitivity. The hygromycin B sensitivity of S. cerevisiae mnn9 null mutant was complemented in the presence of the HpMNN9 gene. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession No. AF264786.

Membrane damage and enzyme inactivation of Lactobacillus plantarum by high pressure CO2 treatment.

Physiological changes of Lactobacillus plantarum (KFRI 815) by high pressure CO2 treatment were investigated to examine the relevance to microbial inactivation. Characteristic properties of the cells measured in this study included salt tolerance, release of UV-absorbing substances, Mg and K ions, proton permeability, glycolysis, H+-ATPase and constitutive enzymes, and dye uptake. The cells treated with high pressure CO2 of 7 MPa at 30 degrees C for 10 min showed the irreversible cellular membrane damages including loss of salt tolerance, leakage of UV-absorbing substances, release of intracellular ions, collapse of proton permeability and uptake of Phloxine B dye. L. plantarum cells after CO2 treatment also exhibited reduced glycolytic activity and inactivation of some constituent enzymes. However, H+-ATPase of the cell membrane maintained its initial specific activity of about 2.50 U/mg protein even though viability of the cells was reduced by several log cycles after high pressure CO2 treatment.

Cyclo(dehydroala-L-Leu), an alpha-glucosidase inhibitor from Penicillium sp. F70614.

A diketopiperazine (1) has been isolated from the culture broth of Penicillium sp. F70614 and its structure has been determined to be cyclo(dehydroala-L-Leu) by various spectroscopic analyses. This compound selectively inhibited yeast alpha-glucosidase and porcine intestinal alpha-glucosidase with IC50 values of 35 and 50 microg/ml, respectively. However, it did not show significant inhibitory effects against almond beta3-glucosidase, Aspergillus alpha-galactosidase, Escherichia coli beta-galactosidase and jack bean alpha-mannosidase.

Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae.

To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.

Antibacterial activity of xanthorrhizol from Curcuma xanthorrhiza against oral pathogens.

The antibacterial activity of xanthorrhizol, isolated from the methanol extract of Curcuma xanthorrhiza roots, was evaluated against oral microorganisms in comparison with chlorhexidine.

Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi.

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.

Cloning, sequencing, and expression of UDP-glucose pyrophosphorylase gene from Acetobacter xylinum BRC5.

A UDP-glucose pyrophosphorylase (UGPase) gene from Acetobacter xylinum BRC5 has been cloned, sequenced, and expressed in Escherichia coli. The gene consists of 867 nucleotides and encodes a polypeptide of 289 amino acid residues with a calculated molecular mass of 31,493 Da. The amino acid sequences of the enzyme showed an 85.8% identity to those of an enzyme from A. xilinum ATCC 23768. A polyhistidine-UGPase fusion enzyme was expressed and purified from the transformed E. coli. The enzyme showed a 35,620-Da single protein band on SDS/PAGE and an about 160,000-Da protein band on 8-16% pore-gradient polyacrylamide gel, indicating the enzyme may be a tetramer or pentamer composed of four or five identical subunits. Kinetic analysis of the enzyme showed a typical Michaelis-Menten substrate saturation pattern, from which Km and Vmax were calculated to be 3.22 mM and 175.4 micromol x min(-1) x mg(-1) for UDP-glucose and 0.24 mM and 69.4 micromol x min(-1) x mg(-1) for PPi, respectively, required Mg2+ for maximal activity, and was inhibited by free pyrophosphate. Computer-aided comparison of the Acetobacter enzyme sequence with those of other bacterial enzymes found significant similarities among them and predicted that Lys84 is a catalytically important residue. Lys84 in the enzyme, which was also conserved in other bacterial enzyme sequences, was replaced by arginine or leucine. The K84R mutant enzyme was successfully expressed in E. coli and showed enzyme activity (63% of the wild-type enzyme activity), but K84L was not isolated in stable form. These results suggest that Lys84 is significant in not only catalysis but also maintenance of the active structure.

alpha-Glycosidase inhibitory activity of hexagalloylglucose from the galls of Quercus infectoria.

Hexagalloylglucose (3-O-digalloyl-1,2,4,6-tetra-O-galloyl-beta-D- glucose), which was isolated from the methanol extract of the galls of Quercus infectoria, significantly inhibited alpha-glycosidases such as sucrase, maltase and isomaltase. Its inhibitory activity was comparable to acarbose being used as a hypoglycemic agent, while the inhibitory activity on alpha-amylase was approximately 10 times lower than that of acarbose. The results indicate that, when compared to acarbose, hexagalloylglucose might reduce the side effects by reducing inhibition of alpha-amylase.

Identification and partial characterization of lacticin BH5, a bacteriocin produced by Lactococcus lactis BH5 isolated from Kimchi.

Strain BH5 was isolated from naturally fermented Kimchi and identified as a bacteriocin producer that has bactericidal activity against Micrococcus flavus ATCC 10240. Strain BH5 was identified tentatively as Lactococcus lactis by API test. Lactococcus lactis BH5 showed a broad spectrum of activity against most of the nonpathogenic and pathogenic microorganisms tested by the modified deferred method. The activity of lacticin BH5, named tentatively as the bacteriocin produced by L. lactis BH5, was detected at the mid-log growth phase, reached its maximum during the early stationary phase, and decreased after the late stationary phase. Lacticin BH5 also showed a relatively broad spectrum of activity against nonpathogenic and pathogenic microorganisms as tested by the spot-on-lawn method. Its antimicrobial activity on sensitive indicator cells was completely destroyed by protease XIV. The inhibitory activities of lacticin BH5 were detected during treatments up to 100 degrees C for 30 min. Lacticin BH5 was very stable over a pH range of 2.0 to 9.0 and was stable with all the organic solvents examined. It demonstrated a typical bactericidal mode of inhibition against M. flavus ATCC 10240. The apparent molecular mass of lacticin BH5 was estimated to be in the region of 3 to 3.5 kDa, by the direct detection of bactericidal activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene.

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture.

Effects of pH and dissolved oxygen on cellulose production by Acetobacter xylinum BRC5 in agitated culture.

Acetobacter xylinum BRC5 was cultivated in a jar fermentor using glucose as the sole carbon source. Strain BRC5 oxidized almost all of the glucose to gluconic acid; thereafter, it biosynthesized cellulose by utilizing gluconic acid accumulated in the broth. The optimal pH for metabolizing glucose to gluconic acid was 4.0, while a pH of 5.5 was preferred for cell growth and cellulose production from the accumulated gluconic acid in the medium. Shifting the pH from 4.0 to 5.5 during the cellulose production phase in batch cultures improved cellulose production and reduced the total fermentation time, compared to batch cultures at constant pH. In constant fed-batch culture, 10 g/l of cellulose was obtained from 40 g/l of glucose, a yield which was approximately 2-fold higher than in batch culture with the same initial glucose concentration, even without control of the level of dissolved oxygen. The highest cellulose yield was obtained in fed-batch cultures in which the dissolved oxygen concentration was controlled at 10% saturation. Control of pH and dissolved oxygen to optimal levels was effective for improving the production rate and yield of cellulose, to achieve a high cellulose productivity of 0.3 g cellulose/l x h. Approximately 15 g/l of cellulose was considered to be the highest yield obtainable using conventional fermentors because the culture broth then became too viscous to allow satisfactory aeration.

Evidence that a beta-1,4-endoglucanase secreted by Acetobacter xylinum plays an essential role for the formation of cellulose fiber.

Cellulose-producing Acetobacter xylinum has been known to secrete a cellulose-hydrolyzing beta-1,4-endoglucanase (CMCax). When antibodies to recombinant CMCax were added to the culture medium, the formation of cellulose fiber was severely inhibited. Western blot analysis using the antibody showed that this enzyme was secreted into the medium even by a cellulose non-producing strain (Cel-). These results indicate that beta-1,4-endoglucanase in the medium plays a critical role in the formation of cellulose fiber by the microorganism.

Optimization of biphasic growth of Saccharomyces carlsbergensis in fed-batch culture.

The optimal glucose feeding policy for the fed-batch culture of Saccharomyces carlsbergensis is presented. The biphasic nature of growth results in a singular feed rate policy that is unique to this organism. When the operating cost is high, the reduction in operating time forces the cells to utilize both glucose and ethanol toward the end of fermentation time and results in a decreasing rate of glucose addition, unlike the normally observed increasing feed rate. The optimal feeding policy depends heavily on the initial conditions and is highly sensitive to changes in kinetic parameters. A semiempirical scheme for feedback optimization is suggested for the fed-batch yeast culture.

Adaptive optimization of a continuous culture with the use of a bilevel forgetting factor.

An adaptive optimization algorithm using a dynamic identification scheme with a bilevel forgetting factor (BFF) has been developed. The simulation results show superiority of this method to other methods when applied to maximize the cellular productivity of a continuous culture of baker's yeast, Saccharomyces cerievisiae. Within the limited ranges of tuning parameters tested the BFF algorithm is found to be superior in terms of initial optimization speed and accuracy and reoptimization speed and accuracy when there is an external change and long term stability (removal of "blowing up" phenomena). Algorithms tested include those based on a constant forgetting factor, an adaptive variable forgetting factor (VFF) and moving window (MW) identification.