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Applied Microbiology and Biotechnology (v.65, #3)
Current studies on biological tagatose production using l-arabinose isomerase: a review and future perspective by Pil Kim (pp. 243-249).
d-Tagatose is a hexoketose monosaccharide sweetener, which is an isomer of d-galactose and is rarely found in nature. Recently, there has been industrial interest in d-tagatose as a low-calorie sugar-substituting sweetener. This article describes the properties and metabolism of tagatose as well as its commercial importance. The comparison between the biological tagatose production and the chemical production was reviewed based on the example of the glucose isomerization into fructose. The industrial problems facing its commercial application is described and evolving potential solutions are suggested.
Strategies for bioremediation of polychlorinated biphenyls by Yoshiyuki Ohtsubo; Toshiaki Kudo; Masataka Tsuda; Yuji Nagata (pp. 250-258).
Polychlorinated biphenyls (PCBs) are serious environmental pollutants that threaten both the natural ecosystem and human health. For remediation of environments contaminated with PCBs, several approaches that exploit the potential of microbes to degrade PCBs have been developed. These approaches include improvement of PCB solubilization and entry into the cell, pathway and enzyme engineering, and control of enzyme expression. In this mini-review, we briefly summarize these strategies and provide potentially useful knowledge for the further improvement of the bacterial breakdown of PCBs.
Production of antithrombotic hirudin in GAL1-disrupted Saccharomyces cerevisiae by M.-D. Kim; T.-H. Lee; H.-K. Lim; J.-H. Seo (pp. 259-262).
A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h−1 produced a maximum hirudin concentration of 62.1 mg l−1, which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.
Batch production of deacetyl 7-aminocephalosporanic acid by immobilized cephalosporin-C deacetylase by Akio Takimoto; Tomoaki Takakura; Hiroyoshi Tani; Shigeo Yagi; Kenji Mitsushima (pp. 263-267).
Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5–10) was broader than that for free enzyme. The K m app value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis–Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics.
Evaluation of agar diffusion bioassay for nisin quantification by T. Pongtharangkul; A. Demirci (pp. 268-272).
The agar diffusion bioassay is the most widely used method for the quantification of nisin, due to its high sensitivity, simplicity, and cost-effectiveness. This method is based on the measurement of the inhibition zone produced in nisin-sensitive microorganisms. The size of the zone is affected by many factors, such as nisin-sensitive strain, amount of added agar and surfactant, and pre-diffusion step. This research aims to evaluate the effects of nisin-sensitive strains and pre-diffusion on the accuracy and precision of nisin quantification. Three strains of nisin-sensitive microorganisms (Micrococcus luteus, Lactobacillus sakei, Brochothrix thermosphacta) were tested along with three different incubation processes. The best combination was the method using L. sakei as an indicator strain with pre-diffusion at 4 °C for 24 h. Compared with M. luteus and B. thermosphacta, L. sakei gave more accurate and reproducible results. Moreover, the pre-diffusion step resulted in larger inhibition zones and more precise results. Finally, the best combination was validated and compared with the method that is usually used and the result showed that the method using L. sakei with pre-diffusion gave more accurate and precise results.
Isolation and characterization of a novel intracellular glucosyltransferase from the acarbose producer Actinoplanes sp. CKD485-16 by B. T. Choi; C. S. Shin (pp. 273-280).
A novel intracellular glucosyltransferase (GTase) was isolated from cells of Actinoplanes sp. CKD485-16—acarbose-producing cells. The enzyme was purified by DEAE-cellulose and G75-40 Sephadex chromatography. The molecular mass of the enzyme was estimated to be 62 kDa by SDS-polyacrylamide gel electrophoresis, and its isoelectric point (pI) was pH 4.3. The N-terminal sequence of the GTase consisted of NH2-Ser-Val-Pro-Leu-Ser-Leu-Pro-Ala-Glu-Trp. The optimum pH and temperature were 7.5 and 30°C. The enzyme was stable in a pH range of 5.5–9.0 and below 40°C. Enzymatic reactions were performed by incubating the GTase with various substrates. The GTase converted acarbose into component C, maltose into trehalose, and maltooligosaccharides into maltooligosyl trehaloses. The reactions were reversible. Various acarbose analogs were tested as inhibitors against the GTase as a means to suppress component C formation. Valienamine was the most potent, with an IC50 value of 2.4×10−3 mM and showed a competitive inhibition mode.
Purification, substrate specificity, and N-terminal amino acid sequence analysis of a β-lactamase-free penicillin amidase from Alcaligenes sp. by S. Das; J. R. Gayen; A. Pal; K. Ghosh; J. P. N. Rosazza; T. B. Samanta (pp. 281-286).
A β-lactamase-free penicillin amidase from Alcaligenes sp. active against various β-lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.
Production and induction of manganese peroxidase isozymes in a white-rot fungus Pleurotus ostreatus by H. Kamitsuji; Y. Honda; T. Watanabe; M. Kuwahara (pp. 287-294).
The production of MnP by Pleurotus ostreatus in different liquid cultures was investigated. The highest level of activity was observed after 8 days of culture in peptone-glucose-yeast extract medium (PGY), whereas maximal activity was achieved after 30 days in glucose-yeast extract medium (GY). MnP was purified to homogeneity from PGY (designated MnP-PGY) and GY (MnP-GY). The isoelectric points of MnP-PGY and MnP-GY were 3.77 and 4.06, respectively. The molecular mass of both enzymes was 42 kDa. Analysis of the N-terminal amino acid sequence of purified MnPs and nucleotide sequence of cloned mnp indicated that MnP-GY has VTCATGQTTANE at the N-terminus, whereas MnP-PGY has ATCADGRTTANA. A putative exposed tryptophan residue (W170) was found in MnP-GY. Both isozymes oxidized veratryl alcohol, although the K m of MnP-GY was lower than that of MnP-PGY. Thus, the presence of peptone in the medium affected the production of MnP isozymes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the synthesis of MnP isozymes is controlled by culture conditions at the transcriptional level.
Orally administrable enterotoxigenic Escherichia coli vaccine encapsulated by ethylcellulose powder dispersion by C.-W. Liao; S.-H. Lin; P.-Y. Lin; H.-Y. Chiou; W.-F. Chang; C. N. Weng (pp. 295-300).
To overcome the limitations of injection administration to vaccinate neonatal piglets against diarrheal disease, an oral vaccine needs to be developed. Enteric microspheres of oral vaccines were developed by a co-spray drying process based on formalin-inactivated enterotoxigenic Escherichia coli antigens with various encapsulating materials. The encapsulating efficiencies of ECN7m, ECN14m and ECN22m (vaccine microsphere formulations) tested by extraction procedure are high, more than 85%. To assess enteric characteristics, an in vitro dissolution test was performed with microspheres. Formulations with ethylcellulose ECN14m and ECN22m allow controlled release in a neutral or basic environment and resisted acid damage. In all cases, 95% of the E. coli protein was released within 2 h at pH 6.8–7, but there was no release at pH 1.5–2. However, ECN7m was less acid-resistant and had lower release at low pH. In animal immunization tests, oral immunization with microspheres of formulations ECN14 and ECN22m effectively evoked both systemic IgG and mucosal IgA responses against E. coli whole cell antigens in mice. In the mice challenge test, orally administrable ECNm14 (12 mg) or ECN22m (12.6 mg) vaccine (i.e., encapsulating 3.0×109 cfu inactive bacterial mass) provided good protection from infection in animals.
Enantioselective transesterification using immobilized Aspergillus oryzae overexpressing lipase by M. Kaieda; M. Nagayoshi; S. Hama; A. Kondo; H. Fukuda (pp. 301-305).
In the present study, we used gene manipulation to construct a recombinant Aspergillus oryzae strain overexpressing lipase and investigated its application to the optical resolution of chiral compounds. A. oryzae niaD300, which was derived from the wild-type strain RIB40, was used as the host strain. The tglA gene, which encodes a triacylglycerol lipase, was cloned from the A. oryzae niaD300 chromosomal genome, then reintroduced, with and without a secretion-signal sequence, into the genome and expressed under the control of the improved glaA promoter of plasmid pNGA142. The resulting recombinant strain overexpressing A. oryzae lipase was immobilized within biomass-support particles and used as a whole-cell biocatalyst. The immobilized lipase-overexpressing strain with secretion-signal sequence showed high activity and was used to selectively synthesize (R)-1-phenylethyl acetate from (RS)-1-phenylethanol and vinyl acetate. After 48 h reaction at 30°C with molecular sieve 4A, the yield and enantiomeric excess (%ee) of (R)-1-phenylethyl acetate reached approximately 90 and 95%ee, respectively. The whole-cell biocatalyst for optical resolution of chiral compounds produced in this study maintained its activity over 25 batch-reaction cycles.
Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain by T. Salusjärvi; M. Povelainen; N. Hvorslev; E. V. Eneyskaya; A. A. Kulminskaya; K. A. Shabalin; K. N. Neustroev; N. Kalkkinen; A. N. Miasnikov (pp. 306-314).
A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.
Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of Acetobacter xylinum BPR 2001 by S. O. Bae; Y. Sugano; K. Ohi; M. Shoda (pp. 315-322).
The diguanylate cyclase 1 (DGC1) (dgc1) gene in Acetobacter xylinum BPR 2001—a bacterial cellulose (BC) producer—was cloned and sequenced, and a DGC1 gene-disrupted mutant, strain DD, was constructed. The production and structural characteristics of the BC formed by DD were compared with those of the parental strain BPR 2001. BC production by DD was almost the same as that by BPR 2001 in static cultivation and in shake flask cultivation. However, in a jar fermentor DD produced about 36% more BC than the parental strain. DD produced suspended particle materials that cannot aggregate owing to their random structural characteristics in static cultivation; more uniformly dispersed BC pellicles and smaller BC pellets are produced on average in a jar fermentor, as reflected by the higher BC production by DD than by the parental strain in a jar fermentor. Micrographs of BC produced by DD revealed that the width of cellulose ribbons assemblies decreased as a result of differences in the ultrastructure and mechanism of formation of BC between the two strains. These results reveal that disruption of the dgc1 gene, which catalyzes synthesis of c-di-GMP (an effector of BC synthase), is not fatal for BC synthesis, although it affects BC structure.
Characterization of Pseudomonas sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by H.-W. Chang; H.-Y. Kahng; S.-I. Kim; J.-W. Chun; K.-H. Oh (pp. 323-329).
The cellular responses of Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have been extensively analyzed in this study. The stress shock proteins, which might contribute to enhancing the cellular resistance to the cytotoxic effect of RDX, were induced at different concentrations of RDX used as a substrate for cell culture of Pseudomonas sp. HK-6. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using the anti-DnaK and anti-GroEL monoclonal antibodies. The stress shock proteins induced by RDX were found to increase in proportion to the RDX concentration used for this work. Analysis of membrane fatty acids of strain HK-6 following exposure to RDX showed that the amounts of dominant lipids 16:1 ω7c/15:0 iso 2OH, 16:0 and 18:1 ω7c/ω9t/ω12t decreased substantially or were not detected in the cells exposed to RDX, while amounts of lipids 10:0 iso, 14:1 ω5c/ω5t and 16:10 methyl increased dramatically. Scanning electron microcopy analyses revealed the presence of perforations and irregular rod shapes with wrinkled surfaces for cells treated with 0.135 mM RDX for 12 h, suggesting that RDX has a substantial cytotoxic impact on cells of strain HK-6.
Reductive transformation of methyl parathion by the cyanobacterium Anabaena sp. strain PCC7120 by J. W. Barton; T. Kuritz; L. E. O’Connor; C. Y. Ma; M. P. Maskarinec; B. H. Davison (pp. 330-335).
Organophosphorus compounds are toxic chemicals that are applied worldwide as household pesticides and for crop protection, and they are stockpiled for chemical warfare. As a result, they are routinely detected in air and water. Methods and routes of biodegradation of these compounds are being sought. We report that under aerobic, photosynthetic conditions, the cyanobacterium Anabaena sp. transformed methyl parathion first to o,o-dimethyl o-p-nitrosophenyl thiophosphate and then to o,o-dimethyl o-p-aminophenyl thiophosphate by reducing the nitro group. The process of methyl parathion transformation occurred in the light, but not in the dark. Methyl parathion was toxic to cyanobacteria in the dark but did not affect their viability in the light. Methyl parathion transformation was not affected by mutations in the genes involved in nitrate reduction in cyanobacteria.
Combined effects of pH and biosurfactant addition on solubilization and biodegradation of phenanthrene by K.-H. Shin; K.-W. Kim; E. A. Seagren (pp. 336-343).
Phenanthrene solubilization and biodegradation with a biosurfactant (rhamnolipid) solution were investigated as a function of pH. Batch phenanthrene solubilization experiments were performed in the pH range 4–8 and the highest solubilities with the biosurfactant were detected around a pH of 4.5–5.5. The apparent solubility at pH 5.5 was 3.8 times greater than at pH 7 in the presence of 240 ppm rhamnolipid, probably due to the rhamnolipid—an anionic surfactant—forming different pH-dependent structures. Biodegradation experiments using Pseudomonas putida CRE 7 were performed in the absence and the presence of the rhamnolipid solution. Without the biosurfactant, the specific growth rate (μ) at pH 6 was higher than at other pH values, and analysis for the total phenanthrene loss confirmed the trends in μ, with the greatest phenanthrene removal at pH 6. In presence of the rhamnolipid, the maximum μ value shifted to around pH 5, which showed maximum enhancement of solubility in the abiotic experiment. Although there was an increase in the observed specific growth rate with the biosurfactant, this increase was not as great as the increase in solubilization. For example, the 1.44 times increase in the μ value at pH 5 was lower than the 3.8 times enhancement in the solubility at the same pH. Thus, as observed by others, not all of the solubilized phenanthrene was bioavailable to the microorganisms. Interestingly, the results of a size distribution experiment showed that a large portion of the phenanthrene-rhamnolipid aggregates existed at a molecular weight of >300,000. Furthermore, this fraction appeared to be the most available for biodegradation, although not all the phenanthrene was bioavailable.
Treatment of the yeast Rhodotorula glutinis with AlCl3 leads to adaptive acquirement of heritable aluminum resistance by A. Tani; D. Zhang; J. A. Duine; F. Kawai (pp. 344-348).
When aluminum (Al) was added to a culture, growth of Rhodotorula glutinis IFO1125 was temporarily arrested, showing longer lag phases, depending on the Al concentrations (50–300 μM) added, but the growth rates were not affected at all. Resistant strains obtained by one round of plate treatment containing Al reverted the resistance level to the wild-type level when cultivated without Al. Repeated Al treatments, however, induced heritable and stable Al resistance, the level of which was increased up to 4,000 μM by stepwise increments in Al concentrations. Thus, the heritable Al resistance adaptively acquired was due neither to adaptation nor to mutation, but to a mechanism which has yet to be studied. Heritable Al resistance seemed to release the Al inhibition of magnesium uptake.
Styrene degradation by Pseudomonas sp. SR-5 in biofilters with organic and inorganic packing materials by J. H. Jang; M. Hirai; M. Shoda (pp. 349-355).
Pseudomonas sp. SR-5 was isolated as a styrene-degrading bacterium. In liquid culture containing 1% (v/v) styrene, more than 90% styrene was degraded in 53 h and the doubling time of SR-5 was 2 h. The removal of styrene gas was investigated in biofilters for 31 days using an organic packing material of peat and an inorganic packing material of ceramic inoculated with SR-5. The maximum-styrene-elimination capacities for peat and ceramic packing materials were 236 and 81 g m−3 h−1, respectively. The percentage of styrene converted to low molecular weight compounds including CO2 in the peat and ceramic biofilters during a 10-day operation were estimated to be 90.4 and 36.7%, respectively. As the pressure drop in the peat bioflter at the end of experiment was significantly higher than that in ceramic biofilter, a biofilter using a mixture of peat and ceramic was tested. We determined that the maximum elimination capacity was 170 g m−3 h−1 and the production of low molecular weight compounds was 95% at a low pressure drop for this mixed packing material filter.
Coprecipitation of Th4+ and the purified extracellular polysaccharide produced by bacterium Bradyrhizobium (Chamaecytisus) BGA-1 by Ana R. Díaz-Marrero; Mónica Santamaría; Jairo Hernández; Javier Corzo (pp. 356-362).
The soil bacterium Bradyrhizobium (Chamaecytisus) strain BGA-1 produces an extracellular polymeric substance (EPS) that, in the presence of Fe3+, Al3+ or Th4+ solutions, forms a gel-like precipitate composed of polysaccharide, protein, lipopolysaccharide and the metal. Precipitation of the main component of the EPS, the extracellular polysaccharide, and thorium was studied. The precipitate was stable, but redissolved at pH values below 3.0 or in the presence of 10 mM EDTA. In the precipitate, the ratio thorium/basic repeating unit of the polysaccharide ranged from 0.4 to 0.8 mol/mol. Soluble polysaccharide–thorium complexes were not found, and larger polysaccharide molecules were precipitated in preference to smaller ones. Kinetic studies showed a non-linear dependence of the precipitate on the concentrations of both thorium and polysaccharide. The behaviors of the purified polysaccharide and of whole EPS with the thorium-containing precipitate were compared. The results suggested that EPS components other than polysaccharide are able to modify the precipitating ability of the polysaccharide. Thus, whole EPS is a better substrate than the purified polysaccharide for the removal of thorium from its solutions.