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Applied Microbiology and Biotechnology (v.66, #3)
Glutathione: a review on biotechnological production by Yin Li; Gongyuan Wei; Jian Chen (pp. 233-242).
This Mini-Review summarizes the historic developments and technological achievements in the biotechnological production of glutathione in the past 30 years. Glutathione is the most abundant non-protein thiol compound present in living organisms. It is used as a pharmaceutical compound and can be used in food additives and the cosmetic industries. Glutathione can be produced using enzymatic methods in the presence of ATP and its three precursor amino acids (l-glutamic acid, l-cysteine, glycine). Alternatively, glutathione can be produced by direct fermentative methods using sugar as a starting material. In the latter method, Saccharomyces cerevisiae and Candida utilis are currently used to produce glutathione on an industrial scale. At the molecular level, the genes gshA and gshB, which encode the enzymes γ-glutamylcysteine synthetase and glutathione synthetase, respectively, have been cloned from Escherichia coli and over-expressed in E. coli, S. cerevisiae, and Lactococcus lactis. It is anticipated that, with the design and/or discovery of novel producers, the biotechnological production of glutathione will be further improved to expand the application range of this physiologically and medically important tripeptide.
Improvement of arachidonic acid production by mutants with lower n-3 desaturation activity derived from Mortierella alpina 1S-4 by Eiji Sakuradani; Yuriko Hirano; Nozomu Kamada; Masutoshi Nojiri; Jun Ogawa; Sakayu Shimizu (pp. 243-248).
Five mutants were obtained, Y11, Y135, Y164, Y180 and Y61, capable of accumulating higher amounts of arachidonic acid (AA) than Mortierella alpina 1S-4, an industrial strain for the production of AA-rich triacylglycerol (TG). This is thought to be due to low or no activity of n-3 desaturation with conversion of AA to eicosapentaenoic acid, which functions at a cultural temperature below 20°C. In small-scale cultivation under optimum conditions, Y11 and Y61 respectively accumulated 4.97 mg/ml and 4.11 mg/ml of AA, using a high concentration of glucose at 20°C, compared with 3.74 mg/ml for M. alpina 1S-4. In a 5-l jar fermentor, the AA content in Y11 and Y61 kept increasing during cultivation, with consumption of the glucose in the medium; and this reached 1.48 mg/ml and 1.77 mg/ml (118 mg/g, 120 mg/g of dry mycelia) at day 10, respectively, compared with 0.95 mg/ml (86 mg/g of dry mycelia) for M. alpina 1S-4. From the results of lipid analysis, the TG contents of Y11 and Y61 in the major lipids were significantly higher than that of M. alpina 1S-4; and the AA percentages in TG of Y11 and Y61 were also higher. Both Y11 and Y61 are potential producers of TG rich in AA.
Astaxanthin hyperproduction by Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) with raw coconut milk as sole source of energy by A. R. Domínguez-Bocanegra; J. A. Torres-Muñoz (pp. 249-252).
Natural carbon sources, such as those present in cane sugar molasses and grape juice, promote the synthesis of astaxanthin in different Phaffia rhodozyma yeasts. One of these, coconut milk, has a very rich nutrient composition. The aim of this work was to investigate the utility of coconut milk as sole source of energy for astaxanthin pigment production by P. rhodozyma strains. Currently, coconut pulp is widely used in industrial processes in Mexico for the production of shampoos, candies, food, etc. However, coconut milk is a waste product. We show that coconut milk enhances astaxanthin production. The fermentation yielded 850 μg/g yeast with the NRRL-10921 wild-type strain and 1,850 μg/g yeast with the mutated R1 strain. Production was better than reported results employing other natural carbon sources.
d-Hexosaminate production by oxidative fermentation by D. Moonmangmee; O. Adachi; H. Toyama; K. Matsushita (pp. 253-258).
Microbial production of d-hexosaminate was examined by means of oxidative fermentation with acetic acid bacteria. In most strains of acetic acid bacteria, membrane-bound d-glucosamine dehydrogenase (synonymous with an alternative d-glucose dehydrogenase distinct from quinoprotein d-glucose dehydrogenase) oxidized d-hexosamines to the corresponding d-hexosaminates in a stoichiometric manner. Conversion of d-hexosamines to the corresponding d-hexosaminates was observed with growing cells of acetic acid bacteria, and d-hexosaminate was stably accumulated in the culture medium even though d-hexosamine was exhausted. Since the enzyme responsible is located on the outer surface of the cytoplasmic membrane, and the enzyme activity is linked to the respiratory chain of the organisms, resting cells, dried cells, and immobilized cells of acetic acid bacteria were effective catalysts for d-hexosaminate production. d-Mannosaminate and d-galactosaminate were also prepared for the first time by means of oxidative fermentation, and three different d-hexosaminates were isolated from unreacted substrate by a chromatographic separation. In this paper, d-hexosaminate production by oxidative fermentation carried out mainly with Gluconobacter frateurii IFO 3264 is exemplified as a typical example.
Use of activated carbon as a support medium for H2S biofiltration and effect of bacterial immobilization on available pore surface by Y. L. Ng; R. Yan; X. G. Chen; A. L. Geng; W. D. Gould; D. T. Liang; L. C. C. Koe (pp. 259-265).
The use of support media for the immobilization of microorganisms is widely known to provide a surface for microbial growth and a shelter that protects the microorganisms from inhibitory compounds. In this study, activated carbon is used as a support medium for the immobilization of microorganisms enriched from municipal sewage activated sludge to remove gas-phase hydrogen sulfide (H2S), a major odorous component of waste gas from sewage treatment plants. A series of designed experiments is used to examine the effect on bacteria-immobilized activated carbon (termed “biocarbon”) due to physical adsorption, chemical reaction, and microbial degradation in the overall removal of H2S. H2S breakthrough tests are conducted with various samples, including microbe-immobilized carbon and Teflon discs, salts-medium-washed carbon, and ultra-pure water-washed carbon. The results show a higher removal capacity for the microbe-immobilized activated carbon compared with the activated carbon control in a batch biofilter column. The increase in removal capacity is attributed to the role played by the immobilized microorganisms in metabolizing adsorbed sulfur and sulfur compounds on the biocarbon, hence releasing the adsorption sites for further H2S uptake. The advantage for activated carbon serving as the support medium is to adsorb a high initial concentration of substrate and progressively release this for microbial degradation, hence acting as a buffer for the microorganisms. Results obtained from surface area and pore size distribution analyses of the biocarbon show a correlation between the available surface area and pore volume with the extent of microbial immobilization and H2S uptake. The depletion of surface area and pore volume is seen as one of the factors which cause the onset of column breakthrough. Microbial growth retardation is due to the accumulation of metabolic products (i.e., sulfuric acid); and a lack of water and nutrient salts in the batch biofilter are other possible causes of column breakthrough.
Cloning, expression, and characterization of a glycoside hydrolase family 86 β-agarase from a deep-sea Microbulbifer-like isolate by Yukari Ohta; Yuji Hatada; Yuichi Nogi; Zhijun Li; Susumu Ito; Koki Horikoshi (pp. 266-275).
The gene for a novel β-agarase from a deep-sea Microbulbifer-like isolate was cloned and sequenced. It encoded a mature protein of 126,921 Da (1,146 amino acids), which was a modular protein including two tandem carbohydrate-binding module (CBM)-like sequences and a catalytic module. The catalytic module resembled a glycoside hydrolase family 86 β-agarase, AgrA, from Pseudoalteromonas atlantica T6c with 31% amino acid identity. Its recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host and purified to homogeneity. The activity and stability were strongly enhanced by CaCl2. The maximal enzyme activity was observed at 45°C and pH 7.5 in the presence of 10 mM CaCl2. The enzyme was an endo-type β-agarase and degraded agarose and agarose oligosaccharides more polymerized than hexamers to yield neoagarohexaose as the main product. This is the first glycoside hydrolase family 86 enzyme to be homogeneously purified and characterized.
β-Galactosidase from Bifidobacterium adolescentis DSM20083 prefers β(1,4)-galactosides over lactose by Sandra W. A. Hinz; Lambertus A. M. van den Broek; Gerrit Beldman; Jean-Paul Vincken; Alphons G. J. Voragen (pp. 276-284).
A β-galactosidase gene (β-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (−) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. β-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that β-Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl-β-d-galactopyranoside as a substrate. The Km and Vmax for Gal(β1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant β-Gal II was highly active towards Gal(β1–4)Gal and Gal(β1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(β1–3)Gal, lactose, and Gal(β1–3)GalOMe. No activity was found towards Gal(β1–6)Gal, Gal(β1–4)Man, Gal(α1–4)Gal, Gal(α1–3)Gal(β1–4)Gal, cellobiose, maltose and sucrose. β-Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(β1–4)]2Gal peak area ratio was 9:1.
Carbon isotope fractionation during cis–trans isomerization of unsaturated fatty acids in Pseudomonas putida by Hermann J. Heipieper; Grit Neumann; Nadja Kabelitz; Matthias Kastner; Hans Hermann Richnow (pp. 285-290).
The molecular mechanism of the unique cis to trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12 was studied. For this purpose, the carbon isotope fractionation of the cis–trans isomerase was estimated. In resting cell experiments, addition of 3-nitrotoluene for activation of the cis–trans isomerase resulted in the conversion of the cis-unsaturated fatty acids into the corresponding trans isomers. For the conversion of C16:1 cis to its corresponding trans isomer, a significant fractionation was measured. The intensity of this fractionation strongly depended on the rate of cis–trans isomerization and the added concentration of 3-nitrotoluene, respectively. The presence of a significant fractionation provides additional indication for a transition from the sp2 carbon linkage of the cis-double bond to an intermediate sp3 within an enzyme–substrate complex. The sp2 linkage is reconstituted after rotation to the trans configuration has occurred. As cytochrome c plays a major role in the catabolism of Cti polypeptide, these findings favour a mechanism for the enzyme in which electrophilic iron (Fe3+), provided by a heme domain, removes an electron of the cis double bond thereby transferring the sp2 linkage into sp3.
Translation efficiency mediated by the 5′ untranslated region greatly affects protein production in Aspergillus oryzae by Akio Koda; Toshitaka Minetoki; Kenji Ozeki; Masato Hirotsune (pp. 291-296).
We demonstrate that the 5′ untranslated region (5′UTR) plays an important role in determining translation efficiency in Aspergillus oryzae, using a model β-glucuronidase (GUS) expression system. Alterations in the 5′UTR resulted in an increase in GUS activity of up to eight-fold, without affecting mRNA levels. Moreover, using the most effective 5′UTR construct, we could achieve remarkable intracellular overproduction of GUS protein; and the GUS level reached more than 50% of the total soluble protein. This is the first experimental evidence indicating the feasibility of improving recombinant protein yield by promoting translation initiation in filamentous fungi.
Fed-batch production of d-ribose from sugar mixtures by transketolase-deficient Bacillus subtilis SPK1 by Yong-Cheol Park; Sung-Gun Kim; Kyungmoon Park; Kelvin H. Lee; Jin-Ho Seo (pp. 297-302).
d-Ribose, a five-carbon sugar, is used as a key intermediate for the production of various biomaterials, such as riboflavin and inosine monophosphate. A high d-ribose-producing Bacillus subtilis SPK1 strain was constructed by the chemical mutation of the transketolase-deficient strain, B. subtilis JY1. Batch fermentation of B. subtilis SPK1 with 20 g l−1 xylose and 20 g l−1 glucose resulted in 4.78 g l−1 dry cell mass, 23.0 g l−1d-ribose concentration, and 0.72 g l−1 h−1 productivity, corresponding to a 1.5- to 1.7-fold increase when compared with values for the parental strain. A late-exponential phase was chosen as the best point for switching to a fed-batch process. Optimized fed-batch fermentation of B. subtilis SPK1, feeding a mixture of 200 g l−1 xylose and 50 g l−1 glucose after the late-exponential phase reduced the residual xylose and glucose concentrations to less than 7.0 g l−1 and gave the best results of 46.6 g l−1d-ribose concentration and 0.88 g l−1 h−1 productivity which were 2.0- and 1.2-fold higher than the corresponding values in a simple batch fermentation.
Deficiency in the glycerol channel Fps1p confers increased freeze tolerance to yeast cells: application of the fps1Δ mutant to frozen dough technology by Shingo Izawa; Kayo Ikeda; Kazuhiro Maeta; Yoshiharu Inoue (pp. 303-305).
Intracellular glycerol content affects the freeze-thaw stress tolerance of Saccharomyces cerevisiae. We have recently reported that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability even in dough after frozen storage [Izawa et al. (2004) Appl Microbiol Biotechnol http://dx.doi.org/10.1007/s00253-004-1624-4]. A deletion mutant of the FPS1 gene, which encodes a glycerol channel, accumulates glycerol inside the cell without an exogenous supply of glycerol into the medium. We found that the fps1Δ cells acquired tolerance to freeze stress and retained high leavening ability in dough after frozen storage for 7 days. These results suggest that the fps1Δ mutant is a useful strain for developing better frozen-dough with a commercial advantage.
Biosurfactant from Lactococcus lactis 53 inhibits microbial adhesion on silicone rubber by Lígia Rodrigues; Henny van der Mei; José António Teixeira; Rosário Oliveira (pp. 306-311).
The ability of biosurfactant obtained from the probiotic bacterium Lactococcus lactis 53 to inhibit adhesion of four bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed biosurfactant layer was investigated in a parallel-plate flow chamber. The microbial cell surfaces and the silicone rubber with and without an adsorbed biosurfactant layer were characterized using contact-angle measurements. Water contact angles indicated that the silicone-rubber surface with adsorbed biosurfactant was more hydrophilic (48°) than bare silicone rubber (109°). The results showed that the biosurfactant was effective in decreasing the initial deposition rates of Staphylococcus epidermidis GB 9/6 from 2,100 to 220 microorganisms cm−2 s−1, Streptococcus salivarius GB 24/9 from 1,560 to 137 microorganisms cm−2 s−1, and Staphylococcus aureus GB 2/1 from 1,255 to 135 microorganisms cm−2 s−1, allowing for a 90% reduction of the deposition rates. The deposition rates of Rothia dentocariosa GBJ 52/2B, Candida albicans GBJ 13/4A, and Candida tropicalis GB 9/9 were far less reduced in the presence of the biosurfactant as compared with the other strains. This study constitutes a step ahead in developing strategies to prevent microbial colonization of silicone-rubber voice prostheses.
Degradation of 4-nitrophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium by Hiroshi Teramoto; Hiroo Tanaka; Hiroyuki Wariishi (pp. 312-317).
The fungal metabolism of 4-nitrophenol (4-NP) was investigated using the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Despite its phenolic feature, 4-NP was not oxidized by extracellular ligninolytic peroxidases. However, 4-NP was converted to 1,2-dimethoxy-4-nitrobenzene via intermediate formation of 4-nitroanisole by the fungus only under ligninolytic conditions. The metabolism proceeded via hydroxylation of the aromatic ring and methylation of phenolic hydroxyl groups. Although the involvement of nitroreductase in the metabolism of 2,4-dinitrotoluene by many aerobic and anaerobic microorganisms including P. chrysosporium has been reported, no formation of 4-aminophenol was observed during 4-NP metabolism. The formation of 1,2-dimethoxy-4-nitrobenzene was effectively inhibited by exogenously added piperonyl butoxide, a cytochrome P450 inhibitor, suggesting that cytochrome P450 is involved in the hydroxylation reaction. Thus, P. chrysosporium seems to utilize hydroxylation and methylation reactions to produce a more susceptible structure for an oxidative metabolic system.
Effect of hyperbaric stress on yeast morphology: study by automated image analysis by M. A. Z. Coelho; I. Belo; R. Pinheiro; A. L. Amaral; M. Mota; J. A. P. Coutinho; E. C. Ferreira (pp. 318-324).
The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO2.
Enrichment and molecular characterization of chloropicrin- and metam-sodium-degrading microbial communities by A. Mark Ibekwe; Sharon K. Papiernik; Ching-Hong Yang (pp. 325-332).
Chloropicrin (CP) and metam sodium are commonly used as fumigants in agricultural soils in order to provide effective control of nematodes, soil-borne pathogens, and weeds in preparation for planting of high-value cash crops. Repeated application of these compounds to agricultural soils for many years may result in the enrichment of microorganisms capable of degrading them. In this study, a microcosm-enrichment approach was used to investigate bacterial populations that may be components of metam-sodium- and CP-degrading microorganisms in compost-amended soils. After 6 months incubation, with repeated application of metam sodium and CP, degradation was ≥70% faster in compost-manure-amended (CM) soil compared to ≤50% in the unamended soils. The accelerated fumigant degradation may have been due to the addition of compost or to the development of new microbial populations with enhanced degradation capacity. Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified regions of 16S rRNA genes were used to identify dominant bacterial populations responsible for the accelerated fumigant degradation. The DGGE results indicated that specific bacterial types had been enriched and these were similar to strains isolated from basal minimal media. Fragments from DGGE bands and colonies were cloned, sequenced, and compared with published 16S rRNA sequences. Cloned sequences were dominated by Pseudomonas, Bacillus, Arthrobacter, Mycobacterium and uncultured bacterial species. The addition of organic amendment to soil during fumigation practices has the potential to increase the diversity of different microbial species, thereby accelerating fumigant degradation and reducing atmospheric emissions.
Biological nitrogen and organic matter removal from tannery wastewater in pilot plant operations in Ethiopia by S. Leta; F. Assefa; L. Gumaelius; G. Dalhammar (pp. 333-339).
The objective of this study was to set-up a pilot plant and to evaluate its effectiveness for biological nitrogen and organic matter removal from tannery wastewater in Ethiopia. A pilot wastewater treatment plant consisting of a predenitrification-nitrification process was constructed and operated for 6 months. This was fed with a raw tannery wastewater obtained from the Modjo Tannery located 70 km south of the capital, Addis Ababa. Up to 98% total nitrogen and chemical oxygen demand, and 95% ammonium nitrogen removal efficiencies were achieved in the system. The average effluent ammonium nitrogen ranged from 8.4 mg l−1 to 86.0 mg l−1, whereas the average effluent for nitrate nitrogen ranged from 2.9 mg l−1 to 4.4 mg l−1. The average values of denitrification and nitrification rates determined by nitrate and ammonium uptake rates (NUR and AUR) were 8.0 mg NO3-N [g volatile suspended solids (VSS)]−1 h−1 and 5.4 mg NH4-N (g VSS)−1 h−1, respectively, demonstrating that the treatment processes of the pilot plant were effective. Further studies of the effect of chromium III on AUR showed 50% inhibition at a concentration of 85 mg l−1, indicating that this metal was not causing process inhibition during performance operations. Thus, the predenitrification-nitrification process was found to be efficient for simultaneous removal of nitrogen and organic substrates from tannery wastewaters.