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26.  Enzymatical and microbial degradation of cyclic dipeptides (diketopiperazines) 
AMB Express  2013;3:51.
Diketopiperazines (DKPs) are cyclic dipeptides, representing an abundant class of biologically active natural compounds. Despite their widespread occurrence in nature, little is known about their degradation. In this study, the enzymatical and microbial cleavage of DKPs was investigated. Peptidase catalyzed hydrolysis of certain DKPs was formerly reported, but could not be confirmed in this study. While testing additional peptidases and DKPs no degradation was detected, indicating peptidase stability of the peptide bond in cyclic dipeptides. Besides confirmation of the reported degradation of cyclo(l-Asp-l-Phe) by Paenibacillus chibensis (DSM 329) and Streptomyces flavovirens (DSM 40062), cleavage of cyclo(l-Asp-l-Asp) by DSM 329 was detected. Other DKPs were not hydrolyzed by both strains, demonstrating high substrate specificity. The degradation of cyclo(l-Asp-l-Phe) by DSM 40062 was shown to be inducible. Three strains, which are able to hydrolyze hydantoins and dihydropyrimidines, were identified for the degradation of DKPs: Leifsonia sp. K3 (DSM 27212) and Bacillus sp. A16 (DSM 25052) cleaved cyclo(dl-Ala-dl-Ala) and cyclo(l-Gly-l-Phe), and Rhizobium sp. NA04-01 (DSM 24917) degraded cyclo(l-Asp-l-Phe), cyclo(l-Gly-l-Phe) and cyclo(l-Asp-l-Asp). The first enantioselective cleavage of cyclo(dl-Ala-dl-Ala) was detected with the newly isolated strains Paenibacillus sp. 32A (DSM 27214) and Microbacterium sp. 40A (DSM 27211). Cyclo(l-Ala-d-Ala) and cyclo(l-Ala-l-Ala) were completely degraded, whereas the enantiomer cyclo(d-Ala-d-Ala) was not attacked. Altogether, five bacterial strains were newly identified for the cleavage of DKPs. These bacteria may be of value for industrial purposes, such as degradation of undesirable DKPs in food and drugs and production of (enantiopure) dipeptides and amino acids.
PMCID: PMC3852281  PMID: 24001323
Diketopiperazines; Cyclic dipeptides; Peptide bond; Degradation; Hydrolysis; Biotransformation; Cyclic amidases; Peptidases
27.  Fed-batch production of MCL-PHA with elevated 3-hydroxynonanoate content 
AMB Express  2013;3:50.
With no inhibition of β-oxidation, Pseudomonas putida KT2440 produces medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) with approximately 65 mol% 3-hydroxynonanoate (HN) from nonanoic acid. Production of PHA with higher HN content and an adjustable monomeric composition was obtained using acrylic acid, a fatty acid β-oxidation inhibitor, together with nonanoic acid and glucose as co-substrates in fed-batch fermentations. Different monomeric compositions were obtained by varying the feeding conditions to impose different specific growth rates and inhibitor feed concentrations. At a nonanoic acid: glucose: acrylic acid feed mass ratio of 1.25: 1: 0.05 and a specific growth rate of 0.15 h-1, 71.4 g L-1 biomass was produced containing 75.5% PHA with 89 mol% HN at a cumulative PHA productivity of 1.8 g L-1 h-1.
PMCID: PMC3846598  PMID: 23987136
PHA; Fed-batch; Acrylic acid; Pseudomonas putida; β-oxidation inhibition
28.  Properties of the newly isolated extracellular thermo-alkali-stable laccase from thermophilic actinomycetes, Thermobifida fusca and its application in dye intermediates oxidation 
AMB Express  2013;3:49.
Laccases are diphenol oxidases that have numerous applications to biotechnological processes. In this study, the laccase was produced from the thermophilic actinomycetes, Thermobifida fusca BCRC 19214. After 36 h of fermentation in a 5-liter fermentor, the culture broth accumulated 4.96 U/ml laccase activity. The laccase was purified 4.64-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Q-Sepharose FF and Sephacryl™ S-200 column chromatography. The overall yield of the purified enzyme was 7.49%. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sephacryl™ S-200 was found to be 73.3 kDa and 24.7 kDa, respectively, indicating that the laccase from T. fusca BCRC 19214 is a trimer. The internal amino acid sequences of the purified laccase, as determined by LC-MS/MS, had high homology with a superoxide dismutase from T. fusca YX. Approximately 95% of the original activity remained after treatment at 50°C for 3 h. and approximately 75% of the original activity remained after treatment at pH 10.0 for 24 h. This laccase could oxidize dye intermediates, especially 2,6-dimethylphenylalanine and p-aminophenol, to produce coloring. This is the first report on laccase properties from thermophilic actinomycetes. These properties suggest that this newly isolated laccase has potential for specific industrial applications.
PMCID: PMC3846457  PMID: 23985268
Laccase; Dye intermediate; Thermophilic; Thermobifida fusca; LC-MS/MS
29.  Production of 2,3-butanediol from cellulose and Jatropha hulls after ionic liquid pretreatment and dilute-acid hydrolysis 
AMB Express  2013;3:48.
Abundant Jatropha waste is a promising renewable feedstock for the production of sugars and 2,3-butanediol fermentation. To obtain high yield of water-soluble products and high concentration of reducing-sugars, ionic liquid (IL) pretreatment and dilute acid hydrolysis at 150°C were combined in this work. The destruction of crystalline structure and increase surface area of biomasses after IL-pretreatment, made their hydrolysis more efficient. Compared with original cellulose, after IL-pretreatment, both the yield and concentration of reducing-sugars increased by 139%, and the water-soluble products yield increased by 128% after hydrolysis. Compared with water-washed Jatropha hulls, after IL-pretreatment, the yield and concentration of reducing-sugars increased by 80% and 76%, respectively, and the water-soluble products yield increased by 70% after hydrolysis. IL-pretreatment benefited the fermentation of Jatropha hull hydrolysate with 66.58% diol yield and its productivity increased from 0.35 to 0.40 g/(L · h).
PMCID: PMC3751892  PMID: 23958155
Ionic liquid; Hydrolysis; 2,3-Butanediol; Fermentation; Klebsiella oxytoca; Jatropha hulls
30.  Cellulolytic potential of thermophilic species from four fungal orders 
AMB Express  2013;3:47.
Elucidation of fungal biomass degradation is important for understanding the turnover of biological materials in nature and has important implications for industrial biomass conversion. In recent years there has been an increasing interest in elucidating the biological role of thermophilic fungi and in characterization of their industrially useful enzymes. In the present study we investigated the cellulolytic potential of 16 thermophilic fungi from the three ascomycete orders Sordariales, Eurotiales and Onygenales and from the zygomycete order Mucorales thus covering all fungal orders that include thermophiles. Thermophilic fungi are the only described eukaryotes that can grow at temperatures above 45°C. All 16 fungi were able to grow on crystalline cellulose but their secreted enzymes showed widely different cellulolytic activities, pH optima and thermostabilities. Interestingly, in contrast to previous reports, we found that some fungi such as Melanocarpus albomyces readily grew on crystalline cellulose and produced cellulases. These results indicate that there are large differences in the cellulolytic potential of different isolates of the same species. Furthermore, all the selected species were able to degrade cellulose but the differences in cellulolytic potential and thermostability of the secretome did not correlate to the taxonomic position. PCR amplification and sequencing of 22 cellulase genes from the fungi showed that the level of thermostability of the cellulose-degrading activity could not be inferred from the phylogenetic relationship of the cellulases.
PMCID: PMC3766086  PMID: 23958135
Thermophilic fungi; Endoglucanase; Cellobiohydrolase; Cellulolytic potential
31.  Changes and recovery of soil bacterial communities influenced by biological soil disinfestation as compared with chloropicrin-treatment 
AMB Express  2013;3:46.
Soil bacterial composition, as influenced by biological soil disinfestation (BSD) associated with biomass incorporation was investigated to observe the effects of the treatment on the changes and recovery of the microbial community in a commercial greenhouse setting. Chloropicrin (CP) was also used for soil disinfestation to compare with the effects of BSD. The fusarium wilt disease incidence of spinach cultivated in the BSD- and CP-treated plots was reduced as compared with that in the untreated control plots, showing effectiveness of both methods to suppress the disease. The clone library analyses based on 16S rRNA gene sequences showed that members of the Firmicutes became dominant in the soil bacterial community after the BSD-treatment. Clone groups related to the species in the class Clostridia, such as Clostridium saccharobutylicum, Clostridium tetanomorphum, Clostridium cylindrosporum, Oxobacter pfennigii, etc., as well as Bacillus niacini in the class Bacilli were recognized as the most dominant members in the community. For the CP-treated soil, clones affiliated with the Bacilli related to acid-tolerant or thermophilic bacteria such as Tuberibacillus calidus, Sporolactobacillus laevolacticus, Pullulanibacillus naganoensis, Alicyclobacillus pomorum, etc. were detected as the major groups. The clone library analysis for the soil samples collected after spinach cultivation revealed that most of bacterial groups present in the original soil belonging to the phyla Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes, Planctomycetes, TM7, etc. were recovered in the BSD-treated soil. For the CP-treated soil, the recovery of the bacterial groups belonging to the above phyla was also noted, but some major clone groups recognized in the original soil did not recover fully.
PMCID: PMC3751922  PMID: 23958081
Anaerobic bacteria; Biological soil disinfestation (BSD); Clone library; Chloropicrin; Clostridial group; Wilt disease of spinach
32.  Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system 
AMB Express  2013;3:45.
Antibacterial factor 2 (ABF-2) is a 67-residue antimicrobial peptide derived from the nematode Caenorhabditis elegans. Although it has been reported that ABF-2 exerts in vitro microbicidal activity against a range of bacteria and fungi, the structure of ABF-2 has not yet been solved. To enable structural studies of ABF-2 by NMR spectroscopy, a large amount of isotopically labeled ABF-2 is essential. However, the direct expression of ABF-2 in Escherichia coli is difficult to achieve due to its instability. Therefore, we applied a coexpression method to the production of ABF-2 in order to enhance the inclusion body formation of ABF-2. The inclusion body formation of ABF-2 was vastly enhanced by coexpression of aggregation-prone proteins (partner proteins). By using this method, we succeeded in obtaining milligram quantities of active, correctly folded ABF-2. In addition, 15 N-labeled ABF-2 and a well-dispersed heteronuclear single quantum coherence (HSQC) spectrum were also obtained successfully. Moreover, the effect of the charge of the partner protein on the inclusion body formation of ABF-2 in this method was investigated by using four structurally homologous proteins. We concluded that a partner protein of opposite charge enhanced the formation of an inclusion body of the target peptide efficiently.
PMCID: PMC3751704  PMID: 23945047
Antimicrobial peptide; Coexpression; Inclusion bodies; Refolding; NMR; HSQC
33.  Antimicrobial action and anti-corrosion effect against sulfate reducing bacteria by lemongrass (Cymbopogon citratus) essential oil and its major component, the citral 
AMB Express  2013;3:44.
The anti-corrosion effect and the antimicrobial activity of lemongrass essential oil (LEO) against the planktonic and sessile growth of a sulfate reducing bacterium (SRB) were evaluated. Minimum inhibitory concentration (MIC) of LEO and its major component, the citral, was 0.17 mg ml-1. In addition, both LEO and citral showed an immediate killing effect against SRB in liquid medium, suggesting that citral is responsible for the antimicrobial activity of LEO against SRB. Transmission electron microscopy revealed that the MIC of LEO caused discernible cell membrane alterations and formed electron-dense inclusions. Neither biofilm formation nor corrosion was observed on carbon steel coupons after LEO treatment. LEO was effective for the control of the planktonic and sessile SRB growth and for the protection of carbon steel coupons against biocorrosion. The application of LEO as a potential biocide for SRB growth control in petroleum reservoirs and, consequently, for souring prevention, and/or as a coating protection against biocorrosion is of great interest for the petroleum industries.
PMCID: PMC3751693  PMID: 23938023
Lemon grass essential oil; Citral; Antimicrobial activity; Anti-corrosion; Souring prevention; Sulfate reducing bacteria
34.  Characterization of erythrose reductases from filamentous fungi 
AMB Express  2013;3:43.
Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected.
PMCID: PMC3751045  PMID: 23924507
Trichoderma reesei; Aspergillus niger; Fusarium graminearum; Erythrose reductase; Erythritol
35.  Conversion of C6 and C5 sugars in undetoxified wet exploded bagasse hydrolysates using Scheffersomyces (Pichia) stipitis CBS6054 
AMB Express  2013;3:42.
Sugarcane bagasse is a potential feedstock for cellulosic ethanol production, rich in both glucan and xylan. This stresses the importance of utilizing both C6 and C5 sugars for conversion into ethanol in order to improve the process economics. During processing of the hydrolysate degradation products such as acetate, 5-hydroxymethylfurfural (HMF) and furfural are formed, which are known to inhibit microbial growth at higher concentrations. In the current study, conversion of both glucose and xylose sugars into ethanol in wet exploded bagasse hydrolysates was investigated without detoxification using Scheffersomyces (Pichia) stipitis CBS6054, a native xylose utilizing yeast strain. The sugar utilization ratio and ethanol yield (Yp/s) ranged from 88-100% and 0.33-0.41 ± 0.02 g/g, respectively, in all the hydrolysates tested. Hydrolysate after wet explosion at 185°C and 6 bar O2, composed of mixed sugars (glucose and xylose) and inhibitors such as acetate, HMF and furfural at concentrations of 3.2 ± 0.1, 0.4 and 0.5 g/l, respectively, exhibited highest cell growth rate of 0.079 g/l/h and an ethanol yield of 0.39 ± 0.02 g/g sugar converted. Scheffersomyces stipitis exhibited prolonged fermentation time on bagasse hydrolysate after wet explosion at 200°C and 6 bar O2 where the inhibitors concentration was further increased. Nonetheless, ethanol was produced up to 18.7 ± 1.1 g/l resulting in a yield of 0.38 ± 0.02 g/g after 82 h of fermentation.
PMCID: PMC3750711  PMID: 23895663
Scheffersomyces (Pichia) stipitis; Cellulosic ethanol; Sugarcane bagasse; Wet explosion pretreatment; Inhibitors; Xylose fermentation
36.  Mercurial-resistance determinants in Pseudomonas strain K-62 plasmid pMR68 
AMB Express  2013;3:41.
We report the complete nucleotide sequence of plasmid pMR68, isolated from Pseudomonas strain K-62, two plasmids contribute to broad-spectrum mercury resistance and that the mer operon from one of them (pMR26) has been previously characterized. The plasmid was 71,020 bp in length and contained 75 coding regions. Three mer gene clusters were identified. The first comprised merR-orf4-orf5-merT1-merP1-merF-merA-merB1, which confers bacterial resistance to mercuric ions and organomercury. The second and third clusters comprised merT2-merP2, which encodes a mercury transport system, and merB2, which encodes an organomercurial lyase, respectively. The deduced amino acid sequences for the proteins encoded by each of the mer genes identified in pMR68 bore greater similarity to sequences from Methylobacterium extorquens AM1 than to those from pMR26, a second mercury-resistance plasmid from Pseudomonas strain K-62. Escherichia coli cells carrying pMKY12 (containing merR-orf4-orf5-merT1-merP1-merF-merA-merB1 cloned from pMR68) and cells carrying pMRA114 (containing merR-merT-merP-merA-merG-merB1 cloned from plasmid pMR26) were more resistant to, and volatilized more, mercury from mercuric ions and phenylmercury than the control cells. The present results, together with our earlier findings, indicate that the high phenylmercury resistance noted for Pseudomonas strain K-62 seems to be achieved by multiple genes, particularly by the multiple merB encoding organomercurial lyase and one merG encoding cellular permeability to phenylmercury. The novel mer gene identified in pMR68 may help us to design new strategies aimed at the bioremediation of mercurials.
PMCID: PMC3737084  PMID: 23890172
Mercury resistance; mer operon; Plasmid pMR68; Pseudomonas strain K-62
37.  Engineered Pichia pastoris for enhanced production of S-adenosylmethionine 
AMB Express  2013;3:40.
A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.
PMCID: PMC3750815  PMID: 23890127
S-adenosylmethionine synthetase; Pichia pastoris; Heterologous host; Bioreactor
38.  Increasing protein production by directed vector backbone evolution 
AMB Express  2013;3:39.
Recombinant protein production in prokaryotic and eukaryotic organisms was a key enabling technology for the rapid development of industrial and molecular biotechnology. However, despite all progress the improvement of protein production is an ongoing challenge and of high importance for cost-effective enzyme production. With the epMEGAWHOP mutagenesis protocol for vector backbone optimization we report a novel directed evolution based approach to increase protein production levels by randomly introducing mutations in the vector backbone. In the current study we validate the epMEGAWHOP mutagenesis protocol for three different expression systems. The latter demonstrated the general applicability of the epMEGAWHOP method. Cellulase and lipase production was doubled in one round of directed evolution by random mutagenesis of pET28a(+) and pET22b(+) vector backbones. Protease production using the vector pHY300PLK was increased ~4-times with an average of ~1.25 mutations per kb vector backbone. The epMEGAWHOP does not require any rational understanding of the expression machinery and can generally be applied to enzymes, expression vectors and related hosts. epMEGAWHOP is therefore from our point of view a robust, rapid and straight forward alternative for increasing protein production in general and for biotechnological applications.
PMCID: PMC3750827  PMID: 23890095
Directed evolution; epMEGAWHOP; Recombinant protein production; Lipase expression; Cellulase expression; Protease expression
39.  Optimization of macroelement concentrations, pH and osmolarity for triacylglycerol accumulation in Rhodococcus opacus strain PD630 
AMB Express  2013;3:38.
The refinement of biodiesel or renewable diesel from bacterial lipids has a great potential to make a contribution for energy production in the future. This study provides new data concerning suitable nutrient concentrations for cultivation of the Gram-positive Rhodococcus opacus PD630, which is able to accumulate large amounts of lipids during nitrogen limitation. Enhanced concentrations of magnesium have been shown to increase the final optical density and the lipid content of the cells. Elevated phosphate concentrations slowed down the onset of the accumulation phase, without a clear effect on the final optical density and the cell’s lipid content. A robust growth of R. opacus was possible in the presence of ammonium concentrations of up to 1.4 g l-1 and sucrose concentrations of up to 240 g l-1, with an optimum regarding growth and lipid storage observed in the range of 0.2 to 0.4 g l-1 ammonium and 20 to 40 g l-1 sucrose, respectively. Moreover, R. opacus showed tolerance to high salt concentrations.
PMCID: PMC3723911  PMID: 23855965
Biodiesel; Biofuels; Lipids; Rhodococcus opacus; Triacylglycerols
40.  Chitosan against cutaneous pathogens 
AMB Express  2013;3:37.
Propionibacterium acnes and Staphylococcus aureus are cutaneous pathogens that have become increasingly resistant to antibiotics. We sought to determine if chitosan, a polymer of deacetylated chitin, could be used as a potential treatment against these bacteria. We found that higher molecular weight chitosan had superior antimicrobial properties compared to lower molecular weights, and that this activity occurred in a pH dependent manner. Electron and fluorescence microscopy revealed that chitosan forms aggregates and binds to the surface of bacteria, causing shrinkage of the bacterial membrane from the cell wall. Of special relevance, clinical isolates of P. acnes were vulnerable to chitosan, which could be combined with benzoyl peroxide for additive antibacterial effect. Chitosan also demonstrated significantly less cytotoxicity to monocytes than benzoyl peroxide. Overall, chitosan demonstrates many promising qualities for treatment of cutaneous pathogens.
PMCID: PMC3720194  PMID: 23829873
Chitosan; Benzoyl peroxide; Acne vulgaris; Propionibacterium acnes; Staphylococcus aureus; Antibacterial
41.  Bacillus subtilis natto: a non-toxic source of poly-γ-glutamic acid that could be used as a cryoprotectant for probiotic bacteria 
AMB Express  2013;3:36.
It is common practice to freeze dry probiotic bacteria to improve their shelf life. However, the freeze drying process itself can be detrimental to their viability. The viability of probiotics could be maintained if they are administered within a microbially produced biodegradable polymer - poly-γ-glutamic acid (γ-PGA) - matrix. Although the antifreeze activity of γ-PGA is well known, it has not been used for maintaining the viability of probiotic bacteria during freeze drying. The aim of this study was to test the effect of γ-PGA (produced by B. subtilis natto ATCC 15245) on the viability of probiotic bacteria during freeze drying and to test the toxigenic potential of B. subtilis natto. 10% γ-PGA was found to protect Lactobacillus paracasei significantly better than 10% sucrose, whereas it showed comparable cryoprotectant activity to sucrose when it was used to protect Bifidobacterium breve and Bifidobacterium longum. Although γ-PGA is known to be non-toxic, it is crucial to ascertain the toxigenic potential of its source, B. subtilis natto. Presence of six genes that are known to encode for toxins were investigated: three component hemolysin (hbl D/A), three component non-haemolytic enterotoxin (nheB), B. cereus enterotoxin T (bceT), enterotoxin FM (entFM), sphingomyelinase (sph) and phosphatidylcholine-specific phospholipase (piplc). From our investigations, none of these six genes were present in B. subtilis natto. Moreover, haemolytic and lecithinase activities were found to be absent. Our work contributes a biodegradable polymer from a non-toxic source for the cryoprotection of probiotic bacteria, thus improving their survival during the manufacturing process.
PMCID: PMC3720193  PMID: 23829836
Probiotics; γ-PGA; Cryoprotectant; Toxicity; Bifidobacteria; Lactobacillus
42.  Efficient direct ethanol production from cellulose by cellulase- and cellodextrin transporter-co-expressing Saccharomyces cerevisiae 
AMB Express  2013;3:34.
Efficient degradation of cellulosic biomass requires the synergistic action of the cellulolytic enzymes endoglucanase, cellobiohydrolase, and β-glucosidase. Although there are many reports describing consolidation of hydrolysis and fermentation steps using recombinant Saccharomyces cerevisiae that express cellulolytic enzymes, the efficiency of cellulose degradation has not been sufficiently improved. Although the yeast S. cerevisiae cannot take up cellooligosaccharide, some fungi can take up and assimilate cellooligosaccharide through a cellodextrin transporter. In this study, a S. cerevisiae strain co-expressing genes for several cell surface display cellulases and the cellodextrin transporter was constructed for the purpose of improving the efficiency of direct ethanol fermentation from phosphoric acid swollen cellulose (PASC). The cellulase/cellodextrin transporter-coexpressing strain produced 1.7-fold more ethanol (4.3 g/L) from PASC during a 72-h fermentation than did a strain expressing cellulase only (2.5 g/L). Direct ethanol production from PASC by the recombinant S. cerevisiae strain was improved by co-expression of cellulase display and cellodextrin transporter genes. These results suggest that cellulase- and cellodextrin transporter-co-expressing S. cerevisiae could be a promising technology for efficient direct ethanol production from cellulose.
PMCID: PMC3699431  PMID: 23800294
Bioethanol; Cellulase; Yeast; Cellodextrin transporter; Cell surface display; Cellulose
43.  Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and anti-staphylococcal biofilm agents 
AMB Express  2013;3:35.
The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.
The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.
PMCID: PMC3702452  PMID: 23800329
Biofilm; Staphylococci; Antimicrobial peptides (AMP); Innate immunity
44.  Cloning, overexpression and biocatalytic exploration of a novel Baeyer-Villiger monooxygenase from Aspergillus fumigatus Af293 
AMB Express  2013;3:33.
The presence of several putative Baeyer-Villiger Monooxygenases (BVMOs) encoding genes in Aspergillus fumigatus Af293 was demonstrated for the first time. One of the identified BVMO-encoding genes was cloned and successfully overexpressed fused to the cofactor regenerating enzyme phosphite dehydrogenase (PTDH). The enzyme named BVMOAf1 was extensively characterized in terms of its substrate scope and essential kinetic features. It showed high chemo-, regio- and stereoselectivity not only in the oxidation of asymmetric sulfides, (S)-sulfoxides were obtained with 99% ee, but also in the kinetic resolution of bicyclo[3.2.0]hept-2-en-6-one. This kinetic resolution process led to the production of (1S,5R) normal lactone and (1R,5S) abnormal lactone with a regioisomeric ratio of 1:1 and 99% ee each. Besides, different reaction conditions, such as pH, temperature and the presence of organic solvents, have been tested, revealing that BVMOAf1 is a relatively robust biocatalyst.
PMCID: PMC3762062  PMID: 23767684
Eukaryotic BVMO; Aspergillus; Baeyer-Villiger oxidation; Kinetic resolution; Sulfide oxidation
45.  Psychrotrophic yeast Yarrowia lipolytica NCYC 789 mediates the synthesis of antimicrobial silver nanoparticles via cell-associated melanin 
AMB Express  2013;3:32.
A psychrotrophic marine strain of the ascomycetous yeast Yarrowia lipolytica (NCYC 789) synthesized silver nanoparticles (AgNPs) in a cell-associated manner. These nanostructures were characterized by UV-Visible spectroscopy and scanning electron microscope-energy dispersive spectrometer (SEM-EDS) analysis. The brown pigment (melanin) involved in metal-interactions was obtained from the cells. This extracted pigment also mediated the synthesis of silver nanoparticles that were characterized by a variety of analytical techniques. The melanin-derived nanoparticles displayed antibiofilm activity. This paper thus reports the synthesis of AgNPs by the biotechnologically important yeast Y. lipolytica; proposes a possible mechanism involved in the synthetic process and describes the use of the bio-inspired nanoparticles as antibiofilm agents.
PMCID: PMC3702394  PMID: 23758863
Yarrowia lipolytica; Silver nanoparticles; Melanin; Antibiofilm activity
46.  Effect of immobilized cells in calcium alginate beads in alcoholic fermentation 
AMB Express  2013;3:31.
Saccharomyces cerevisiae cells were immobilized in calcium alginate and chitosan-covered calcium alginate beads and studied in the fermentation of glucose and sucrose for ethanol production. The batch fermentations were carried out in an orbital shaker and assessed by monitoring the concentration of substrate and product with HPLC. Cell immobilization in calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads in eight sequential fermentation cycles of 10 h each. The final concentration of ethanol using free cells was 40 g L-1 and the yields using glucose and sucrose as carbon sources were 78% and 74.3%, respectively. For immobilized cells in calcium alginate beads, the final ethanol concentration from glucose was 32.9 ± 1.7 g L-1 with a 64.5 ± 3.4% yield, while the final ethanol concentration from sucrose was 33.5 ± 4.6 g L-1 with a 64.5 ± 8.6% yield. For immobilized cells in chitosan-covered calcium alginate beads, the ethanol concentration from glucose was 30.7 ± 1.4 g L-1 with a 61.1 ± 2.8% yield, while the final ethanol concentration from sucrose was 31.8 ± 6.9 g L-1 with a 62.1 ± 12.8% yield. The immobilized cells allowed eight 10 h sequential reuse cycles to be carried out with stable final ethanol concentrations. In addition, there was no need to use antibiotics and no contamination was observed. After the eighth cycle, there was a significant rupture of the beads making them inappropriate for reuse.
PMCID: PMC3695878  PMID: 23721664
Immobilized cells; Fermentation; Ethanol; Glucose; Sucrose; Alginate
47.  Evaluation of the inhibitory effects of chloroform on ortho-chlorophenol- and chloroethene-dechlorinating Desulfitobacterium strains 
AMB Express  2013;3:30.
Organohalide-respiring Desulfitobacterium strains are believed to play an important role in the bioremediation and natural attenuation of chlorinated aliphatic and aromatic hydrocarbons. However, several studies have reported that chloroform significantly inhibits microbial reductive dechlorination of chloroethene. In this study, we examined the effect of chloroform on several Desulfitobacterium strains, including ortho-chlorophenol-dechlorinating Desulfitobacterium dehalogenans JW/IU-1 and Desulfitobacterium hafniense DCB-2, and also the chloroethene-dechlorinating strain D. hafniense TCE1. In medium containing 3-chloro-4-hydroxyphenylacetate as an electron acceptor, chloroform inhibited the growth of strains JW/IU-1 and DCB-2. Although chloroform did not directly inhibit dechlorination of 3-chloro-4-hydroxyphenylacetate by resting cells, cells cultivated with chloroform showed decreased dechlorination activity. Moreover, transcription of the gene encoding the reductive dehalogenase CprA decreased significantly in cells cultivated with chloroform. These results indicate that chloroform inhibits the growth and dechlorination activity of strains JW/IU-1 and DCB-2 via inhibition of cprA transcription. In contrast, cultivation of strain TCE1 in the presence of chloroform gave rise to a PceA reductive dehalogenase gene-deletion variant of strain TCE1; a similar phenomenon was observed in our previous study of chloroethene-dechlorinating D. hafniense strain Y51. Our results suggest that chloroform extensively inhibits the dechlorination activity of Desulfitobacterium strains, and that the inhibitory mechanism appears to differ between ortho-chlorophenol dechlorinators and chloroethene dechlorinators.
PMCID: PMC3679974  PMID: 23705686
Desulfitobacterium; Reductive dechlorination; Organohalide respiration; Growth inhibition; Chloroform; Chlorophenol; Chloroethene
48.  Co-cultivation of Lactobacillus zeae and Veillonella cricetifor the production of propionic acid 
AMB Express  2013;3:29.
In this work a defined co-culture of the lactic acid bacterium Lactobacillus zeae and the propionate producer Veillonella criceti has been studied in continuous stirred tank reactor (CSTR) and in a dialysis membrane reactor. It is the first time that this reactor type is used for a defined co-culture fermentation. This reactor allows high mixing rates and working with high cell densities, making it ideal for co-culture investigations. In CSTR experiments the co-culture showed over a broad concentration range an almost linear correlation in consumption and production rates to the supply with complex nutrients. In CSTR and dialysis cultures a strong growth stimulation of L. zeae by V. criceti was shown. In dialysis cultures very high propionate production rates (0.61 g L-1h-1) with final titers up to 28 g L-1 have been realized. This reactor allows an individual, intracellular investigation of the co-culture partners by omic-technologies to provide a better understanding of microbial communities.
PMCID: PMC3699425  PMID: 23705662
Propionic Acid; Lactic Acid; Lactobacillus zeae; Veillonella criceti; Dialysis chamber reactor; Co-culture; Fermentation
49.  Biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC 533 
AMB Express  2013;3:28.
The potential of Lactobacillus johnsonii NCC 533 to metabolize chlorogenic acids from green coffee extract was investigated. Two enzymes, an esterase and a hydroxycinnamate decarboxylase (HCD), were involved in this biotransformation. The complete hydrolysis of 5-caffeoylquinic acid (5-CQA) into caffeic acid (CA) by L. johnsonii esterase occurred during the first 16 h of reaction time. No dihydrocaffeic acid was identified in the reaction mixture. The decarboxylation of CA into 4-vinylcatechol (4-VC) started only when the maximum concentration of CA was reached (10 μmol/ml). CA was completely transformed into 4-VC after 48 h of incubation. No 4-vinylphenol or other derivatives could be identified in the reaction media. In this study we demonstrate the capability of L. johnsonii to transform chlorogenic acids from green coffee extract into 4-VC in two steps one pot reaction. Thus, the enzymatic potential of certain lactobacilli might be explored to generate flavor compounds from plant polyphenols.
PMCID: PMC3679781  PMID: 23692950
Chlorogenic acid; 4-vinylcatechol; Esterase; Decarboxylase; L. johnsonii
50.  Tailoring fungal morphology of Aspergillus niger MYA 135 by altering the hyphal morphology and the conidia adhesion capacity: biotechnological applications 
AMB Express  2013;3:27.
Current problems of filamentous fungi fermentations and their further successful developments as microbial cell factories are dependent on control fungal morphology. In this connection, this work explored new experimental procedures in order to quantitatively check the potential of some culture conditions to induce a determined fungal morphology by altering both hyphal morphology and conidia adhesion capacity. The capacity of environmental conditions to modify hyphal morphology was evaluated by examining the influence of some culture conditions on the cell wall lytic potential of Aspergillus niger MYA 135. The relative value of the cell wall lytic potential was determined by measuring a cell wall lytic enzyme activity such as the mycelium-bound β-N-acetyl-D-glucosaminidase (Mb-NAGase). On the other hand, the quantitative value of conidia adhesion was considered as an index of its aggregation capacity. Concerning microscopic morphology, a highly negative correlation between the hyphal growth unit length (lHGU) and the specific Mb-NAGase activity was found (r = -0.915, P < 0.001). In fact, the environment was able to induce highly branched mycelia only under those culture conditions compatible with specific Mb-NAGase values equal to or higher than 190 U gdry.wt-1. Concerning macroscopic morphology, a low conidia adhesion capacity was followed by a dispersed mycelial growth. In fact, this study showed that conidia adhesion units per ml equal to or higher than 0.50 were necessary to afford pellets formation. In addition, it was also observed that once the pellet was formed the lHGU had an important influence on its final diameter. Finally, the biotechnological significance of such results was discussed as well.
PMCID: PMC3679960  PMID: 23688037
Aspergillus niger; Culture conditions; β-N-Acetyl-D-Glucosaminidase; Hyphal morphology; Conidia adhesion; Pellets formation; Metabolite production

Results 26-50 (192)