Showing papers in "Journal of Microbiology and Biotechnology in 1999"

Direct Electrode Reaction of Fe(III)-Reducing Bacterium, Shewanella putrefaciens

Abstract: Anaerobically grown cells of an Fe(III)-reducing bacterium, Shewanella putrefaciens IR-1, were electrochemically active with an apparent reduction potential of about 0.15 V against a saturated calomel electrode in the cyclic voltammetry. The bacterium did not grow fermentatively on lactate, but grew in an anode compartment of a three-electrode electrochemical cell using lactate as an electron donor and the electrode as the electron acceptor. This property was shared by a large number of Fe(III)-reducing bacterial isolates. This is the first observation of a direct electrochemical reaction by an intact bacterial cell, which is believed to be possible due to the electron carrier(s) located at the cell surface involved in the reduction of the natural water insoluble electron acceptor, Fe(III).

A Microbial Fuel Cell Type Lactate Biosensor Using a Metal-Reducing Bacterium, Shewanella putrefaciens

Abstract: A fuel cell type biosensor for lactate was developed using a metal-reducing bacterium, Shewanella putrefaciens IR-1. Under the operational conditions, the bacterial cell suspension generated the current without an electrochemical mediator in the presence of lactate. The current was proportional to the lactate concentration up to 30 mM.

Novel SSF Process for Ethanol Production from Microcrystalline Cellulose Using the $\delta$-Integrated Recombinant Yeast, Saccharomyces cerevisiae L2612$\delta$GC

Abstract: A novel simultaneous saccharification and fermentation (SSF) process from the microcrystalline cellulose to ethanol was developed by using δ-integrated recombinant cellulolytic Saccharomyces cerevisiae L2612δGC, which can utilize cellulose as carbon and energy sources. The optimum amount of enzymes needed for the efficient conversion of cellulose to ethanol at 30°C was determined with commercial cellulolytic enzymes. By fed-batch cultivation, the heterologous cellulolytic enzymes were accumulated up to 42.67% of the total cellulase and 29% of the β-glucosidase needed for the efficient SSF process. When this δ-integrated recombinant yeast was applied to the successive SSF step for ethanol production, 20.35 g/l of ethanol was produced after 12 h from 50 g/l of microcrystalline cellulose. By using this novel SSF process, a considerable amount of commercial enzymes was reduced.

Isolation and characterization of a novel exopolysaccharide-producing Paenibacillus sp. WN9 KCTC 8951P

Abstract: A bacterial strain WN9, which produced a new type of extracellular polysaccharide, was isolated from soil samples. By morphological, physiological, biochemical, and phylogenetic studies, strain WN9 was identified as a Paenibacillus sp. and it was named as Paenibacillus sp. WN9, which produced a high molecular extracellular polysaccharide from glucose. The molecular weight of the exopolysaccharide (EPS-WN9) was estimated to be about 31.5 mega-Da. The FT-IR spectrum of EPS-WN9 revealed typical characteristics of polysaccharides. EPS- WN9 consisted of D-glucose and D-mannose with a molar ratio of 1:1.4 being identified as a neutral sugar component. The acidic component of EPS- WN9 was tyrosine. Rheological analysis of EPS- WN9 revealed that the pseudoplastic property and its apparent viscosity remained stable at various temperatures and pHs.

Mass production of poly(3-hydroxybutyrate) by fed-batch cultures of Ralstonia eutropha with nitrogen and phosphate limitation

Abstract: For mass production of poly(3-hydroxybutyrate) (PHB), high cell density cultures of Ralstonia eutropha were carried out in 2.5-1 and 60-1 fermentors by two fed-batch culture techniques of nitrogen and phosphate limitation. When the nitrogen limitation technique was employed using both an on-line glucose monitoring and control system, a high concentration level of PHB (121 g/l) was obtained in the small-scale fermentor of 2.51. However, the PHB concentration obtained in a large-scale fermentor of 601 only turned out to be 60 g/l. In contrast, when another fed-batch culture technique of the phosphate-limitation employing dissolved oxygen (DO) stat glucose feeding was used, a large amount of PHB was successfully produced in both 60-1 and 2.5-1 fermentors. In a 2.5-1 fermentor, concentrations of PHB and cells obtained in 58 h were 175 and 210 g/l, respectively, which corresponded to the PHB productivity level of 3.02 g/l/h. In a 60-1 fermentor, a final cell concentration of 221 g/l and a PHB concentration of 180 g/l with PHB productivity level of 3.75 g/l/h were obtained in 48 h. PHB content and yield from glucose were 81% and 0.38 g PHB/g glucose, respectively. These data suggest that the phosphate limitation technique is more effective compared to nitrogen limitation in the mass production of PHB by R. eutropha of a large scale.

Induction by carvone of the polychlorinated biphenyl (PCB)-degradative pathway in Alcaligenes eutrophus H850 and its molecular monitoring

Abstract: There is a possibility that carvone, a monoterpene from spearmint (Mentha spicata), could induce the bph degradative pathway and genes in Alcaligenes eutrophus H850, which is a known Gram-negative PCB degrader with a broad substrate specificity that was thoroughly investigated with Arthrobacter sp. B1B, a Gram-positive PCB degrader. The strains B1B and H850 were unable to utilize and grow on the plant terpene [(R)-(-)-carvone] (50 ppm) to be recognized as a sole carbon source. Nevertheless, the carvone did induce 2,3-dihydroxybiphenyl 1,2-dioxygenase (encoded by bphC) in the strain B1B, as observed by a resting cell assay that monitors accumulation of a yellow meta ring fission product from 4,4'-dichlorobiphenyl (DCBp). The monoterpene, however, did not appear to induce the meta cleavage pathway in the strain H850. Instead, an assumption was made that the strain might be using an alternative pathway, probably the ortho-cleavage pathway. A reverse transcription (RT)-PCR system, utilizing primers designed from a conserved region of the bphC gene of Arthrobacter sp. M5, was employed to verify the occurrence of the alternative pathway. A successful amplification (182bp) of mRNA transcribed from the N-terminal region of the bphC gene was accomplished in H850 cells induced by carvone (50 ppm) as well as in biphenyl-growth cells. It is, therefore, likely that H850 possesses a specific PCB degradation pathway and hence a different substrate specificity compared with B1B. This study will contribute to an elucidation of the dynamic aspects of PCB bioremediation in terms of roles played by PCB degraders and plant terpenes as natural inducer substrates that are ubiquitous and environmentally compatible.

Isolation of a phytase-producing Bacillus sp. KHU-10 and its phytase production

Abstract: A bacterial strain producing high level of an extracellular phytase was isolated from cooked rice and identified as a strain of Bacillus sp. and designated as Bacillus sp. KHU-10. Optimum culture conditions were investigated for the maximum productivity of phytase by Bacillus sp. KHU-10. 1.0% Maltose and 1.0% peptone with 0.5% beef extract were the best carbon source and nitrogen source, respectively. The addition of CaCl 2 stimulated the enzyme productivity with concentration between 0.01% and 0.2%, in the medium. Although sodium phosphate increased the cell mass, the enzyme activity decreased. Calcium phytate and wheat bran containing phytate did not enhance the enzyme production. Under the optimum medium, the production of the phytase reached the highest level of 0.2 unit/ml after 4 days of incubation.

Transgenic tobacco plants expressing the bacterial levansucrase gene show enhanced tolerance to osmotic stress

Abstract: Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plants. In addition, it has been suggested that, due to their solubility, they can play an important role in helping plants survive periods of osmotic stress. In order to study the effect of levan synthesis on plant growth, the coding region of the levansucrase gene, which was isolated from Zymomonas mobilis. was introduced into tobacco plants using Agrobacterium tumefaciens-mediated transformation. The presence of the levansucrase gene in transgenic plants was verified by genomic DNA gel blot analysis. RNA gel blot and immunoblot analyses showed an accumulation of the corresponding transcript and protein product of the bacterial levansucrase gene in transgenic plants. Furthermore, a thin layer chromatography analysis revealed that fructans were synthesized and deposited in transgenic tobacco plants. When T, seeds were germinated and grown under polyethylene glycol-mediated drought stress or cold stress, the transgenic seedlings displayed a substantially higher level of growth than that of untransformed plants. These results suggest that fructans may play a significant role in the tolerance of plants under osmotic stress.

Effect of fungal elicitor and heavy metals on the production of flavonol glycosides in cell cultures of Ginkgo biloba

Abstract: The effect of fungal elicitor and heavy metal salts on the production of flavonol glycosides in cell cultures of Ginkgo biloba was investigated. Among the fungi tested, Trichoderma longibrachiatum ATCC 52326 was found to be the most efficient in the production of flavonol glycosides. Kaempferol production from the elicited callus increased tenfold as compared to the unelicited callus, while quercetin concentration of elicited cells was nine-fold higher than that of unelicited cells in suspension cultures. The maximum quercetin concentration of 0.362 mg/l was obtained in 1.25 mg/l of the homogenate elicitor. Among the heavy metal salts tested, CuSO 4 showed a significant effect on quercetin accumulation, reaching to the concentration of 0.526 mg/l. Quercetin concentration increased to a maximum of 12-fold in response to CuSO 4 treatment as compared to that of untreated cells. The phenylalanine ammonia-lyase (PAL) activity and flavonol glycosides production simultaneously increased for 5 days of culture after fungal elicitor feeding, and their contents showed the same proportional patterns during the culture period. In contrast, PAL activity of cell cultures treated with CuSO 4 was almost constant during the culture period, although quercetin production increased remarkably.

Metabolic Analysis of Poly(3-Hydroxybutyrate) Production by Recombinant Escherichia coli

Abstract: Poly(3-hydroxybutyrate) (PHB) production by fermentation was examined under both restricted- and ample-oxygen supply conditions in a single fed-batch fermentation. Recombinant Escherichia coli transformed with the PHB production plasmid pSYL107 was grown to reach high cell density (227 g/l dry cell weight) with a high PHB content (78% of dry cell weight), using a glucose-based minimal medium. A simple flux model containing 12 fluxes was developed and applied to the fermentation data. A superior closure (95%) of the carbon mass balance was achieved. When the data were put into use, the results demonstrated a surprisingly large excretion of formate and lactate. Even though periods of severe oxygen limitation coincided with rapid acetate and lactate excretion, PHB productivity and carbon utilization efficiency were not significantly impaired. These results are very positive in reducing oxygen demand in an industrial PHA fermentation without sacrificing its PHA productivity, thereby reducing overall production costs.

Purification and Characterization of Biosurfactants Produced by Pseudomonas sp. SW1

Abstract: Pseudomonas sp. SWI grew and produced biosurfactants on 3% hexadecane as the energy and carbon source. As a result of biosurfactant synthesis, the surface tension of the medium was reduced from 72 dyne/cm to 30 dyne/cm. The properties of biosurfactants that were purified from Pseudomonas sp. SW1 were investigated. The purification procedure included acid precipitation from culture supernatant, silica gel G60 column chromatography, and Sephadex G-150 gel filtration. The biosurfactants were separated into two different types, viz., types I and II. Biosurfactant type I significantly reduced the surface tension of water from 72 to 27 dyne/cm at concentration levels above 30 mg/l. The surface tension of water was reduced to a minimum of approximately 30 dyne/cm by biosurfactant type II at concentration levels over 80 mg/l. The biosurfactants were effective in a wide range of pHs, at NaCl concentrations of up to 4%, at CaCl 2 concentration up to 100 mM, and at temperatures up to 200°C for 8 h.

Production of a fibrinolytic enzyme in bioreactor culture by Bacillus subtilis BK-17

Abstract: Bacillus subtilis BK-17 which produces a novel protease with fibrinolytic activity was isolated from soybean paste. Bioreactor production of the enzyme was studied in order to optimize fermentation conditions such as medium concentration, pH, agitation speed, and temperature. Under most cultural conditions, enzyme production initially began when the cell growth stopped. The onset of the enzyme production was indicated by rapid increase in both dissolved oxygen (DO) and pH. Two- to three-times more concentrated medium than the flask optimum medium yielded higher enzyme production in the bioreactor fermentation. When the medium pH was controlled constant, pH 6.5 exhibited the highest activity in the range of 6.0 to 7.5, but the activity was similar to the case when the pH was initially adjusted to 7.5 and subsequently maintained within a relatively wide range of 6.4 to 7.8. Agitation speed did not affect the enzyme production with an exception of DO reaching zero. Fermentation time was reduced when temperature increased within the range of 25C to 37°C. However, the highest activity, along with the slow decrease of the enzymatic activity after reaching the maximum value, was observed at 25C. By shifting the temperature from 37°C to 25°C immediately after DO reached the minimum level, the high enzyme production of 1,100 U/ml along with the short fermentation period of 13 h could be obtained.

Thelephoric acid and Kynapcin-9 in Mushroom Polyozellus multiflex Inhibit Prolyl Endopeptidase In Vitro

Abstract: Prolyl endopeptidase [PEP; EC 3.4.21.26], a serine protease which is known to cleave peptide bonds on the carboxy side of a proline residue, plays an important role in the degradation of proline-containing neuropeptides that have been suggested to participate in learning and memory processes. An abnormal increase in the level of PEP, which can lead to generation of Aβ, is also suggested to be involved in Alzheimer's type senile dementia. In the course of screening PEP inhibitors from Basidiomycetes, the mushroom Polyozellus multiplex exhibited a high inhibitory activity against PEP. Two active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification, using silica gel, Sephadex LH-20, and Lobar RP-18 chromatography. The chemical structures of these compounds were identified as thelephoric acid and 12-acetyl-2,3,7,8-tetrahydroxy-[12H]-12-hydroxymethylbenzobis[1.2b,3.4b'] benzofuran-11-one (kynapcin-9) by spectral data including UV, IR, MS, HR-MS, 'H-, 13 C-, and 2D-NMR. The IC 50 values of the thelephoric acid and kynapcin-9 were 0.157 ppm (446 nM) and 0.087 ppm (212 nM) and their inhibitor constants (K,) were 0.73 ppm (2.09 μM) and 0.060 ppm (146 nM), respectively. Furthermore, they were non-competitive with a substrate in Dixon plots.

Isolation of a Pseudomonas sp. Strain Exhibiting Unusual Behavior of Poly(3-hydroxyalkanoates) Biosynthesis and Characterization of Synthesized Polyesters

Abstract: A Pseudomonas sp. strain that is capable of utilizing dicarboxylic acids as a sole carbon source was isolated from activated sludge by using the enrichment culture technique. This organism accumulated polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units that depends on the carbon sources used. Polyhydroxybutyrate (PHB) homopolyester was synthesized from glucose or small C alkanoic acids, such as butyric acid and hexanoic acid. Accumulation of PHB homopolyester was also observed in the cells grown on C odd dicarboxylic acids, such as heptanedioic acid and nonanedioic acid as the sole carbon sources. In contrast, a copolyester consisting of 6 mol% 3-hydroxybutyrate (3HB) and 94 mol% 3-hydroxyvalerate (3HV) was produced with a PHA content of as much as 36% of the cellular dry matter. This strain produced PHAs consisting both of the short-chain-length (SCL) and the medium-chain-length (MCL) 3-hydroxyacid units when heptanoic acid to undecanoic acid were fed as the sole carbon sources. Most interestingly, polyester consisting of significant amount of relevant fractions, 3HB, 3HV, and 3-hydroxyheptanoate (3HHp), was accumulated from heptanoic acid. According to solvent fractionation experiments, the polymer produced from heptanoic acid was a blend of poly(3HHp) and of a copolyester of 3HB, 3HV, and 3HHp units. The hexane soluble fractions contained only 3HHp units while the hexane-insoluble fractions contained 3HB and 3HV units with a small amount of 3HHp unit. The copolyester was an elastomer with unusual mechanical properties. The maximum elongation ratio of the copolyester was 460% with an ultimate strength of 10 MPa, which was very different from those of poly(3HB-co-3HV) copolyesters having similar compositions produced from other microorganisms.

Facile Purification and Characterization of Dextransucrase from Leuconostoc mesenteroides B-512FMCM

Abstract: A simple sequence of membrane concentration and DEAE-Cellulose chromatography has been optimized to give a purified dextransucrase from Leuconostoc mesenteroides B-512FMCM with the highest specific activity (248.8 IU/mg protein) ever reported in high yield (overall 88.7%) for dextransucrase. When there was no sucrose in the dextransucrase and the dextran reaction digest, the dextransucrase hydrolyzed glucose from dextran. The glucose was transferred to the other glucoses from dextran and formed isomaltose and isomaltodextrin. The transglycosylation efficiency of glucose from dextran was much higher with acceptors. The dextransucrase can be used for the production of various kinds (or structures) of oligosaccharides using dextran and various acceptors with almost 100% theoretical yield.

Identification and characterization of bacteriocin-producing lactic acid bacteria isolated from Kimchi

Abstract: Lactic acid bacteria were isolated from Kimchi and screened for bacteriocin. A total of 99 strains showed antimicrobial activity when grown on solid media, yet only 10 showed antimicrobial activity in liquid media. Strain H-559, identified as Lactococcus lactis subsp. lactis, exhibited the strongest inhibitory activity and was active against pathogenic bacteria including Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus as well as other lactic acid bacteria. The antimicrobial substance produced by L. lactis subsp. lactis H-559 was confirmed to be a bacteriocin by the treatment of α-chymotrypsin, and protease type IX and XIV. The bacteriocin activity remained stable between pH 2.0 and pH 11.0 and during heating for 10 min at 100°C. The bacteriocin production started in the exponential phase and stopped in the stationary phase. L. lactis subsp. lactis H-559 showed the highest bacteriocin activity at a culture temperature of 25°C, and an inverse relationship between the bacterioci productivity and mean growth rate at different culture temperatures was observed. The mean growth rate and bacteriocin productivity of L. lactis subsp. lactis H-559 increased as the initial pH of the media increased.

Improvement of Bifidobacterium longum Stability Using Cell-Entrapment Technique

Abstract: A cell-entrapment technique using compressed air was applied to Bifidobacterium longum KCTC 3128 for the improvement of bifidobacteria viability. The main cell-entrapment matrix used was alginate, and viability improvement of the B. longum entrapped in alginate lattices was monitored along with the effects of other additional biopolymers. A prerequisite for acquiring consistent results was the uniformity of bead size and cell distribution which was achieved by using compressed air and mixing the cell suspension with sterilized alginate powder, respectively. The viability losses of the B. longum entrapped in alginate beads in the presence of three different substances logarithmically increased in relation to the reaction time, and proportionately decreased with an increased alginate concentration and bead diameter. The strongest improvement in B. longum viability was exhibited with a bead containing 3% alginate and 0.15% xanthan gum.

Examination of Cytopathic Effect and Apoptosis in Listeria monocytogenes-Infected Hybridoma B-Lymphocyte (Ped-2E9) Line In Vitro

Abstract: In our previous studies, we reported that hybridoma B-lymphocytes can be used to determine the virulence of Listeria species in an in vitro cytotoxicity assay. Here, we examined the cytopathic effect, i.e., membrane damage and the nature of cell death induced by Listeria monocytogenes on murine hybridoma B-lymphocytes (Ped-2E9). Membrane damage was assessed by microscopic analyses and by measuring the release of intracellular alkaline phosphatase (AP) and lactate dehydrogenase (LDH). Cell death was determined by DNA fragmentation analyses using agarose gel electrophoresis. Infection by listeriolysin O (LLO)-producing L. monocytogenes strains induced substantial amounts of AP and LDH release from Ped-2E9 hybridoma B-cells, suggesting severe membrane damage in these cells, while an LLO-negative L. monocytogenes mutant strain had no effect. An LLO-producing recombinant L. innocua (prfA + hly + ) strain also induced high AP and LDH release and cytopathic changes in Ped-2E9 cells. Light or scanning electron microscopic examination revealed L. monocytogenes mediated membrane destabilization, pore formation, intense cytoplasmic granulation, bleb formation, and lysis of Ped-2E9 cells. LLO-producing L. monocytogenes and L. innocua (prfA + hly + ) also induced ladder-like DNA fragmentation in Ped-2E9 cells. Collectively, these results suggest that L. monocytogenes, specifically LLO-producing strains, can induce a severe cytopathic effect leading to apoptosis in hybridoma B-lymphocytes (Ped-2E9).

Enhancement of Succinate Production by Organic Solvents, Detergents, and Vegetable Oils

Abstract: Bioconversion of fumarate to succinate by Enterococcus sp. RKY1 was enhanced when Tween surfactant, organic solvent, and vegetable oil were added to the fermentation medium. The maximum amount of succinate produced was 80.4 g/l after a 24 h incubation when Tween 80 was added to the culture to a final concentration of 0.1 g/l. Triton X-100 was observed to damage the enzymes and inhibit the formation of succinate. The addition of 10 ml/l acetone increased the production of succinate by 110%. Vegetable oils used were found to be effective for succinate production as well as for the cell growth. Similar productivity increases were obtained with corn oil and Tween 80 plus biotin with the total productivity being 3.6 g/l/h, and 3.5 g/l/h, respectively, which was approximately 25% greater than that of the control. Therefore, these results indicate that corn oil can be considered the most appropriate agent for the production of succinate where succinic acid was primarily used in the production of food, medicine, and cosmetics.

Effect of ammonium phosphate on mycelial growth and exopolysaccharides production of Ganoderma lucidum in an air-lift fermenter

Abstract: It was discovered that ammonium phosphate in the medium played an important role in both growing mycelium and producing exopolysaccharides (EPS) from G. lucidum. In lower concentration levels of ammonium phosphate (0-3 g/l), an improved mycelial growth was observed by maintaining more filamentous morphology than in high concentrations (5-11 g/l). In addition, it was confirmed by comparing the factual dimension and frequency of the area regarding the mycelial pellets. This must be attributed to limitations of nutrient transfer by maintaining filamentous mycelium during the cultivation in a low ammonium phosphate containing medium. On the other hand, the best EPS production was observed in medium with the absence or low concentration of ammonium phosphate. The shear stress of the culture broth was greatly affected by the shear rate, as compared with that of the culture broth with high ammonium phosphate concentration. The rheological characteristics of the fermentation broth and filtrate worked well according to the Herschel-Bulkley model. It was also found that the morphological changes of the mycelium resulting from the ammonium phosphate concentration directly affected the rheological characteristics of the system and resulted in reversely affecting the EPS production levels. Based on these results, it can be concluded that delicate regulation of the ammonium phosphate concentration in the culture media should be provided in order to obtain optimal mycelial growth and/or EPS production.

Molecular characterization and bitter taste formation of tryptic hydrolysis of 11S glycinin

Abstract: The molecular size reduction and the formation of bitterness during a tryptic hydrolysis of soybean 11S glycinin were determined by using quantitative analysis and organoleptic evaluation. The 11S glycinin of 90% purity was prepared by cryoprecipitation and Con A Sepharose 4B affinity chromatography, and hydrolyzed with trypsin in a pH-stat reactor for 4h. Bitterness was formed within 1 h of hydrolysis, and then slowly increased up to 3.5×10 -5 M quinine-HC1 equivalent. The extent of hydrolysis (DH) was 7% at 1 h and increased up to 12% by the end of the reaction. The α-amino nitrogen content increased from an initial 0.7 mM to 7 mM at the end of the period. The SDS-PAGE analysis showed that the acidic subunit of 11S glycinin was mostly hydrolyzed. The GP-HPLC analysis indicated that the bitterness was mainly contributed by the peptide fractions of molecular weights of 360-2,100 Da.

Characterization of a novel carbohydrase from Lipomyces starkeyi KSM 22 for dental application

Abstract: The combined activities of dextranase and amylase (DXAMase) from Lipomyces starkeyi KSM 22 produced from starch fermentation inhibited or prevented dental plaque formation. The activities were stable in commercial mouthwash products; DXAMase activity retained over 93% of original activity after 6 months at 23°C. We examined the effects of enzyme inhibitors and active ingredients in mouthwash on DXAMase activity. The DXAMase was stable with 0.29% (w/v) EDTA, 20% (v/v) ethanol, 0.05% (w/v) fluoride, and 0.05% (w/v) SDS. Among the active ingredients of mouthwash, sodium benzoate (up to 1%, w/v) had no inhibitory effect on either dextranase or amylase activity. In the case of cetylpyridinium chloride, the addition of 0.05% (w/v) inhibited 6% of dextranase activity and 13% of amylase activity. Propylene glycol (up to 1%, w/v) showed no inhibitory effect on either enzyme activity. DXAMase (5 IU/ml) in mouthwash could remove pre-formed films of glucan-bound S. mutans cells. The addition of 0.1 IU/ml DXAMase in mouthwash prevented the formation of insoluble-glucan. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.

Characterization of the β-cyclodextrin glucanotransferase gene of Bacillus firmus var. alkalophilus and its expression in E. coli

Abstract: The β-CGTase gene of alkalophilic Bacillus firmus var. alkalophilus was cloned into E. coli using pZErO-2 as a vector. The cloned gene encoded a total of 710 amino acid residues consisting of 674 amino acids of the matured protein and 36 amino acids of the signal peptide, including 20 amino acids from the lacZ gene in the vector. Although the cloned β-CGTase gene did not contain the promoter and start codons, it was expressed by the lac promoter and lacZ start codon in the pZErO-2 vector. A comparison was made with the amino acid sequence and ten other CGTases from Bacillus sp. Also, ten highly conserved regions, which are important amino acid residues in catalysis of CGTase, were identified. The lac promoter used for expression of the β-CGTase gene was induced constitutively in recombinant E. coli even without IPTG possibly because of a lack of the lacI gene in both host and vector, repressing the lacZ gene in the lac operon. Its expression was catabolically repressed by glucose, however, its repression was reduced by soluble starch, mainly because of the extremely high increase of the cAMP level. β-CGTase can be overproduced in the recombinant E. coli by maintaining intracellular cAMP levels mostly through the intermittent feeding of glucose during cultivation.

Control of both foam and dissolved oxygen in the presence of a surfactant for production of β-carotene in Blakeslea trispora

Abstract: A production of β-carotene was attempted in a fed-batch culture of Blakeslea trispora by controlling both foam and dissolved oxygen in the presence of surfactant. Span 20. Results obtained from the shake flask cultures indicated that a high concentration of dissolved oxygen was needed for both cell growth and β-carotene synthesis, and the optimal concentration of glucose was found to be in the range of 50-100 g/l. In order to maintain the dissolved oxygen concentration level at higher than 50% of air saturation, pure oxygen was automatically sparged into the medium with air. Foam was controlled by bypassing air from the submerged aeration to the headspace in response to the foam that was caused by Span 20. High agitation speed was found to be detrimental to the cell growth due to shear damage, even though it provided sufficient dissolved oxygen. On the other hand, a low aeration speed caused stagnant regions in the fermentor because of improper mixing. Thus, for the fed-batch operation, agitation speed was increased gradually from 300 to 700 rpm to prevent cell damage at the initial stage of fermentation and to give efficient mixing for a viscous culture broth as the culture proceeded. By controlling dissolved oxygen and foam, a high concentration of β-carotene (1,190 mg/l) was obtained in 6 days of the fed-batch culture of B. trispora with 2.5% of the dry cell weight, which was approximately 5 times higher than that of the batch cultures.

Production of poly(3-hydroxybutyrate) [P(3HB)] with high P(3HB) content by recombinant Escherichia coli harboring the Alcaligenes latus P(3HB) biosynthesis genes and the E-coli ftsZ gene

Abstract: Filamentation-suppressed recombinant Escherichia coli strain harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes and the E. coli ftsZ gene was constructed and cultivated for the production of poly(3-hydroxybutyrate) [P(3HB)] with high concentration and high content. By the pH-stat fed-batch culture of this recombinant E. coli strain XL1-Blue(pJC5), the final cell concentration and P(3HB) concentration obtained in 44.25 h were 172.2 g cell dry weight/l and 141.9 g P(3HB)/l, respectively, resulting in productivity of 3.21 g P(3HB)/l-h. More importantly, the P(3HB) content obtained was 82.4 wt %, which was significantly higher than that obtained with the recombinant E. coli harboring only the PHA biosynthesis genes.

Increased production of recombinant protein by Escherichia coli deficient in acetic acid formation

Abstract: The effect of acetic acid formation deficiency on recombinant E. coli fermentation was investigated using a mutant strain deficient in acetic acid formation. A mutant strain which does not grow under anaerobic conditions was isolated. The acetic acid production in this strain was negligible in aerobic batch fermentation. The cloned-gene expression in the mutant strain was higher than the wild-type strain. Fed-batch fermentations with controlled specific growth rates were carried out in order to compare the cloned-gene expression between the wild-type and the mutant strains. The expression decreased along with the specific growth rate in both strains. The cloned-gene expression in the mutant strain was 60% higher than in the wild-type strain at the same specific growth rate.

Improvement of Kimchi Fermentation by Using Acid-Tolerant Mutant of Leuconostoc mesenteroides and Aromatic Yeast Saccharomyces fermentati as Starters

Abstract: Saccharomyces fermentati and Leuconostoc mesenteroides were isolated from a traditional kimchi, and then the Leu, mesenteroides was mutated to the acid-tolerant mutant Leu. mesenteroides M-100. In the result of growth properties in MRS broth with various pHs adjusted with HCl and acid solution (latic acid:acetic acid = 1:2), an acid-tolerant mutant Leu. mesenteroides M-100 showed more increased ability for growth than its wild strain at various temperatures. The strains were used as starters for the fermentation of kimchi. The experiments were performed with classified experimental groups (Group I, control kimchi; Group II, addition of YK-19 only; Group III, addition of M-100 only; Group IV, addition of mixture of M-100 and YK-19), and their pH, total acidity, reducing sugars content, organic acid productivity, organoleptic tests, and microfloral changes were compared. As a result, in pH and acidity, the optimal ripening period of Group IV was about 13.5 days (i.e. from the 8.5 to 22 days of fermentation). This result indicates that the optimal ripening period of Group IV was about 1.5 times longer than that of Group I. Furthermore, Group IV was edible to 28 days of fermentation. In addition, high contents of succinc acid was observed in Group IV. Group IV was also highly ranked on the organoleptic test. During the fermentation of kimchi, the number of Leuconostoc sp. in group I reduced after 7 days; however, the number of Leuconostoc sp. in Group II, III, and IV decresed after 14 days of fermentation. An especially high number of Leu. sp. was observed in Group IV, and this gave better flavor of kimchi than any other during the whole fermentation period. Citric acid, tartaric acid, succinic acid, fumaric acid, and lactic acid were detected in the kimchi, and a significant increase in the concentration of lactic acid during fermentation was observed in the all experimental groups.

Purification and characterization of metalloproteases from Pleurotus sajor-caju

Abstract: Fibrinolytic protease activity was detected in the fruit body of Pleurotus sajor-caju using a fibrin plate method. Two fibrinolytic activities (FPI and Ⅱ) were found at the regions of 14.5 and 86.0 kDa by using gel-filtration column chromatography. FPⅡ was identified as an alkaline protease, whereas FPⅠ was a neutral protease. Both were inhibited by phenanthrolin and EDTA, suggesting that they are metalloprotease. Inactivated enzyme activities were restored by adding Co/sup 2+/ or Zn/sup 2+/. Iodoacetate inhibited FPⅠ, but not FPⅡ. Both enzymes cleaved B/sub β/ and γ chains of the human fibrinogen. FPⅡ showed a preference to hydrophobic and bulky residues of nitroanilidine compounds as substrates, whereas FPⅠ preferred positively charged residues.

Effect of gluconic acid on the production of cellulose in Acetobacter xylinum BRC5

Abstract: Four mutants of Acetobacter xylinum BRC5 defective in gluconic acid production were isolated from UV-irradiated cells. The gluconic acid-negative mutants did not show glucose oxidase activity. The mutants were also defective in cellulose production. A randomly selected mutant grown in the Hestrin-Schramm medium (pH 6.0) supplemented with gluconic acid, however, was found to synthesize cellulose. The mutant grown in Hestrin-Schramm medium whose pH was adjusted to 5.0 with HC1 and contained no gluconic acid also produced cellulose. Wild-type cells grown under the same condition synthesized cellulose more rapidly than those grown in the pH 6.0 medium.