Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Applied Microbiology and Biotechnology (v.63, #1)
A new approach for alteration of protease functions: pro-sequence engineering by H. Takagi; M. Takahashi (pp. 1-9).
Several proteases, including the bacterial serine protease subtilisins, require the assistance of the N-terminal pro-sequence of precursors to produce active, mature enzymes. Upon completion of folding, the pro-sequence is autocatalytically degraded because it is not necessary for the activity or stability of folded, mature cognates of the original enzymes. Therefore, the pro-sequence functions as an intramolecular chaperone that guides correct folding of the protease domain. Interestingly, Shinde et al. proposed a new theory of "protein memory" in which an identical polypeptide can fold into an altered conformation with different secondary structure, stability and specificities through a mutated pro-sequence [Shinde et al. (1997) Nature 389:520–522]. We also showed that the autoprocessing efficiency was improved by modifications in the pro-sequence of mutant subtilisins with altered substrate specificity. Further, the pro-sequence from a subtilisin homologue was found to chaperone the intramolecular folding of denatured subtilisin. These results indicate that engineering of the pro-sequence, i.e., site-directed and/or random mutagenesis, chimeras and gene shuffling between members of the family, would be a useful method for improving the functions of autoprocessing proteases. Conventional protein engineering techniques have thus far employed mutagenesis in the protease domain to modify the enzymatic properties. This new approach, which we term "pro-sequence engineering", is not only an important tool for studying the mechanism of protein folding, but also a promising technology for creating unique proteases with various beneficial properties.
Exudate gums: occurrence, production, and applications by D. Verbeken; S. Dierckx; K. Dewettinck (pp. 10-21).
This paper presents a review of the industrially most relevant exudate gums: gum arabic, gum karya, and gum tragacanth. Exudate gums are obtained as the natural exudates of different tree species and exhibit unique properties in a wide variety of applications. This review covers the chemical structure, occurrence and production of the different gums. It also deals with the size and relative importance of the various players on the world market. Furthermore, it gives an overview of the main application fields of the different gums, both food and non-food.
A new labyrinthulid isolate, which solely produces n-6 docosapentaenoic acid by Y. Kumon; R. Yokoyama; T. Yokochi; D. Honda; T. Nakahara (pp. 22-28).
A labyrinthulid strain, L59, was isolated from a leaf floating on seawater collected at the coastal area of Hokkaido Prefecture, Japan. Strain L59 contained only n-6 docosapentaenoic acid (n-6 DPA) among all the long-chain polyunsaturated fatty acids. The proportion of n-6 DPA in the total fatty acids was 48.1% and the total fatty acids content in the cell dry weight was 26.6%. Many oil bodies were observed in the cell, mostly in the vicinity of cell membranes. The strain had spindle-shaped cell bodies and all cells were surrounded by ectoplasmic net elements. It was also clearly classified in the labyrinthulid group by phylogenetic analysis. In the optimum culture condition, using soybean oil and peptone as carbon and nitrogen sources, 0.53 g of n-6 DPA/l was produced at 20 °C in 7 days.
Solid residues from Ruminococcus cellulose fermentations as components of wood adhesive formulations by P. J. Weimer; A. H. Conner; L. F. Lorenz (pp. 29-34).
Residues from the fermentation of cellulose by the anaerobic bacteria Ruminococcus albus (strain 7) or Ruminococcus flavefaciens (strains FD-1 or B34b) containing residual cellulose, bacterial cells and their associated adhesins, were examined for their ability to serve as components of adhesives for plywood fabrication. The residues contained differing amounts of protein (0.4–4.2% of dry weight), but the ratios of monosaccharides recovered following two-stage treatment of the residue with detergent (pH 7) and TFA were similar for all three strains (0.71 glucose:0.18 xylose:0.08 mannose:0.02 galactose), suggesting similarities in exopolysaccharide composition. Three-ply aspen panels prepared with fermentation residues (FR) displayed better shear strength and wood failure under dry conditions than following a vacuum/pressure/soak/dry treatment, but adhesive properties were inferior to those prepared with conventional phenol-formaldehyde (PF) adhesives. However, panels prepared by incorporating the R. albus 7 FR into PF formulation, at 73% by weight of the total adhesive, exhibited shear strength and wood failure similar to that obtained with PF adhesive alone. Use of residues from fermentations by these bacteria as components of adhesives may add value to biomass fermentations aimed primarily at producing ethanol and other chemical products.
Development, validation, and applications of a new laboratory-scale indirect impedancemeter for rapid microbial control by T. Ribeiro; G. Romestant; J. Depoortere; A. Pauss (pp. 35-41).
We introduce a new laboratory-scale impedance-meter which is specially intended for indirect technique. It consists of a software system enabling data acquisition via a connected bus which is wired to the measuring cells. These measuring cells are individual impedance-meters that can be activated independently of one another. In the current configuration, the device is slightly affected by temperature, but it can register as little as 10.9 µmol of CO2. With Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae cultures, the conductance responses were highly replicable and repeatable for inocula concentrations of 1–108 colony-forming units (CFU) ml−1. The main use for such devices could be the detection of contamination in foodstuffs. Several of these foodstuffs, when incubated at 37 °C, spontaneously release quite large amounts of CO2. Our impedancemeter, however, was able to detect an E. coli presence in canned French beans at 2.35×10−2 CFU ml−1 and a S. cerevisiae contamination of apple purée in glass jars at 6.1×10−3 CFU ml−1. The conductance response and the detection time (the time needed for a significant change in conductance) were correlated to the concentration of ampicillin (an antibiotic added to E. coli cultures). The device is thus able to detect the presence of inhibitory compounds in milk or other foodstuffs. Some industrial assays are in process to complement these laboratory tests. Compared with other available techniques for CO2 measurement (manometry, infrared, radioactive labeling), the technique put forward here appears to be the best compromise between sensitivity, technical constraints, and cost. A commercial version of the impedancemeter would enable routine measurements in the quality control of foodstuffs, pharmaceuticals, cosmetics and in R&D laboratories.
Cloning of a gene encoding thermostable cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast by J. Hong; H. Tamaki; K. Yamamoto; H. Kumagai (pp. 42-50).
A gene encoding a cellobiohydrolase (CBH) was isolated from Thermoascus aurantiacus IFO 9748 and designated as cbh1. The deduced amino acid sequence encoded by cbh1 showed high homology with the sequence of glycoside hydrolase family 7. To confirm the sequence of the gene encoding the CBH, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The recombinant enzyme was confirmed as a CBH by analysis of the reaction product and designated as CBHI. Recombinant CBHI retained more than 80% of its initial activity after 1 h of incubation at 65 °C and was stable in the pH range 3.0–9.0. The optimal temperature for enzyme activity was about 65 °C and the optimal pH was about 6.0. The recombinant enzyme was found to be highly glycosylated and this glycosylation was shown to contribute to the thermostability of the enzyme. CBHI expression was shown to be induced at higher temperature in T. aurantiacus.
Biochemical studies on cloned Bacillus sp. BP-7 phenolic acid decarboxylase PadA by N. Prim; F. I. J. Pastor; P. Diaz (pp. 51-56).
Sequence analysis of a Bacillus sp. BP-7 recombinant clone coding for a previously described carboxylesterase revealed the presence of an additional ORF with homology to bacterial hydroxycinnamic acid decarboxylases. Analysis of the amino acid sequence of the encoded enzyme revealed the presence of a single, highly conserved domain of 161 amino acids, with a predicted molecular mass of 19,143 Da and a pI of 5.5. Crude cell extracts from the recombinant clone displayed activity on ferulic, p-coumaric and caffeic acids, with no need for added cofactors. The cloned enzyme, named PadA, displayed maximum activity at 40°C and pH 5.5, being stable over a broad range of pH and up to 45°C. HPLC analysis of the products of catalysis revealed the conversion of phenolic acids to their aromatic 4-vinyl derivatives, with no accumulation of other by-products. PadA was found as a homodimer in the parental Bacillus sp. BP-7 strain and its expression was induced by both hydroxycinnamic acids and their corresponding derivative products. The results obtained suggest that the enzyme could be involved in a stress response for conversion of toxic hydroxycinnamic acids released after plant cell wall degradation.
Enhanced expression and functional characterization of the human ferritin H- and L-chain genes in Saccharomyces cerevisiae by H.-Y. Seo; Y.-J. Chung; S.-J. Kim; C.-U. Park; K.-S. Kim (pp. 57-63).
Enhanced expression of the human ferritin H- and L-chain genes (hfH and hfL) was achieved in Saccharomyces cerevisiae by modifying the N-terminal region of the structural genes. The yeast episomal vector YEp352 with the galactokinase1 (GAL1) promoter was used to construct expression plasmids. The expression of each gene was examined using SDS-PAGE and Western blot analysis. Iron uptake was examined and the cellular iron concentration was increased in S. cerevisiae expressing hfH. When cultured cells were incubated with 14.3 mM Fe2+, the recombinant yeast expressing hfH had a cellular iron concentration 1.5 times greater than that of the control strain. The relationship between the iron taken up by the cells and the expressed proteins was examined. Iron-binding H-chain ferritin (H-ferritin) was seen in the recombinant S. cerevisiae incubated with iron, while small amounts of iron-binding L-chain ferritin (L-ferritin) were observed. Combined, these observations demonstrate that human H-ferritin has a function in iron storage in S. cerevisiae, while L-ferritin does not.
Functional expression of rat adenosine A1 receptor in the dimorphic zygomycete Mucor circinelloides by J. Houghton-Larsen; P. Amstrup Pedersen (pp. 64-67).
We have produced the rat adenosine A1 receptor in Mucor circinelloides using a translational fusion to the endogenous glucoamylase (GlaM) gene. The fusion protein produced from an episomal plasmid was correctly processed as judged by western blotting, since only a 33 kDa band was detected in membrane preparations from M. circinelloides expressing the receptor. This corresponds to the mass of the full-length receptor released from the fused GlaM protein. The presence of a high affinity binding site with a K d value of 0.5 nM for the receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in membrane preparations suggests that the receptor was correctly folded and inserted into the membranes. A receptor expression level of 100–300 fmol/mg total membrane protein was achieved as judged by binding of the antagonist [3H]-DPCPX.
Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by Pseudomonas stutzeri strain JJ by J. A. Dijk; A. J. M. Stams; G. Schraa; H. Ballerstedt; J. A. M. de Bont; J. Gerritse (pp. 68-74).
A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO2 with NO3 − or O2 as electron acceptor, with a preference for O2 if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 °C with a µ max of 0.14 h−1 and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the µ max was 0.31 h−1. NO2 − also served as electron acceptor, but reduction of Fe(OH)3, MnO2, SO4 2−, fumarate or ClO3 − was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.
Degradation of an endocrine disrupting chemical, DEHP [di-(2-ethylhexyl)-phthalate], by Fusarium oxysporum f. sp. pisi cutinase by Y.-H. Kim; J. Lee; S.-H. Moon (pp. 75-80).
The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of di-(2-ethylhexyl)-phthalate (DEHP) was investigated. The DEHP-degradation rate of fungal cutinase was surprisingly high, i.e. almost 70% of the initial DEHP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DEHP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 85% of the DEHP remained even after 3 days of treatment. During the enzymatic degradation of DEHP, several DEHP-derived compounds were detected and time-course changes in composition were also monitored. During degradation with fungal cutinase, most DEHP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. In the degradation by yeast esterase, two organic chemicals were produced from DEHP: IBF and an unidentified compound (X). The final chemical composition after 3 days was significantly dependent on the enzyme used. Fungal cutinase produced IBF as a major degradation compound. However, in the DEHP degradation by yeast esterase, compound X was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis.
Ester synthesis in an aqueous environment by Streptococcus thermophilus and other dairy lactic acid bacteria by S.-Q. Liu; R. Holland; V. L. Crow (pp. 81-88).
The ability of Streptococcus thermophilus ST1 and 19 other dairy lactic acid bacteria (LAB) to synthesize esters was investigated in an aqueous environment. These LAB were able to synthesize esters from alcohols and glycerides via a transferase reaction (alcoholysis) in which fatty acyl groups from glycerides were transferred to alcohols. S. thermophilus ST1 was active on tributyrin and on di- or monoglycerides of up to C10 with ethanol as the acyl acceptor. This strain was also active on a diglyceride of C6 and monoglyceride of C8 with 2-phenyl ethanol as the acyl acceptor. Alcoholysis occurred preferentially over hydrolysis. S. thermophilus ST1 had an apparent K m value of 250 mM for ethanol and an apparent K m value of 1.3 mM for tributyrin, measured against whole cells. Around 80% of both the transferase activity and the esterase activity were detected in the cell-free extract (CFE) of strain ST1. Both activities in the CFEs of five LAB tested were, to a similar degree, enhanced slightly by growth in the presence of ethanol and tributyrin. Using tributyrin and ethanol as substrates, the transferase activities ranged over 0.006–1.37 units/mg cell dry weight among the LAB tested and were both species- and strain-dependent.
The involvement of ATP sulfurylase in Se(VI) and Cr(VI) reduction processes in the fission yeast Schizosaccharomyces pombe by P. Raspor; Š. Fujs; L. Banszky; A. Maraz; M. Batič (pp. 89-95).
The response of Schizosaccharomyces pombe towards the oxyanions selenate [Se(VI)] and dichromate [Cr(VI)] was investigated in order to establish the involvement of the yeast ATP sulfurylase in their reduction. An ATP sulfurylase-defective/selenate-resistant mutant of S. pombe (B-579 Se R -2) and an ATP sulfurylase-active/selenate-sensitive strain of S. pombe (B-579 Se S ) were included in this study. The inhibitory effect of Se(VI) and Cr(VI) oxyanions on growth and bioaccumulation was measured. The sensitive strain showed natural sensitivity to selenate while the resistant mutant tolerated a 100-fold higher concentration of selenate. These results indicate that selenate toxicity to microorganisms is connected with the reduction of selenate to selenite. Both strains showed similar sensitivity to Cr(VI) and in this study there was no evidence that ATP sulfurylase participates in the reduction process of Cr(VI).
Effect of redox potential on stationary-phase xylitol fermentations using Candida tropicalis by J. R. Kastner; M. A. Eiteman; S. A. Lee (pp. 96-100).
Redox potential was used to develop a stationary-phase fermentation of Candida tropicalis that resulted in non-growth conditions with a limited decline in cell viability, a xylitol yield of 0.87 g g−1 (95% of the theoretical value), and a high maximum specific production rate (0.67 g g−1 h−1). A redox potential of 100 mV was found to be optimum for xylitol production over the range 0–150 mV. A shift from ethanol to xylitol production occurred when the redox potential was reduced from 50 mV to 100 mV as cumulative ethanol (Y ethanol) decreased from 0.34 g g−1 to 0.025 g g−1 and Y xylitol increased from 0.15 g g−1 to 0.87 g g−1 (α=0.05). Reducing the redox potential to 150 mV did not improve the fermentation. Instead, the xylitol yield and productivity decreased to 0.63 g g−1 and 0.58 g g−1 h−1 respectively and cell viability declined. The viable, stationary-phase fermentation could be used to develop a continuous fermentation process, significantly increasing volumetric productivity and reducing downstream separation costs, potentially by the use of a membrane cell-recycle reactor.
Methanogen population in a marine biofilm corrosive to mild steel by T. Zhang; H. H. P. Fang; B. C. B. Ko (pp. 101-106).
This study was conducted to analyze the methanogen population in a corrosive marine biofilm based on 16S rDNA analysis, using a PCR-cloning-sequencing approach. There were 80 methanogen clones developed from the PCR-amplified DNA extracted from the biofilm on the mild steel surface. All clones were categorized into one of five operational taxonomy units (OTUs). Two OTUs (comprising 57 clones) were affiliated with the acetotrophic Methanosaeta genus; the remaining three OTUs (23 clones) were affiliated with the hydrogenotrophic genera of Methanogenium, Methanoplanus and Methanocalculus. The hydrogenotrophic methanogens could directly cause metal corrosion through cathodic depolarization, whereas the acetotrophic methanogens grew syntrophically with corrosion-causing sulfate-reducing bacteria, as observed by fluorescent in situ hybridization, and thus contribute indirectly to metal corrosion.