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Applied Microbiology and Biotechnology (v.72, #3)
Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids by T. Zaunmüller; M. Eichert; H. Richter; G. Unden (pp. 421-429).
Heterofermentative lactic acid bacteria (LAB) such as Leuconostoc, Oenococcus, and Lactobacillus strains ferment pentoses by the phosphoketolase pathway. The extra NAD(P)H, which is produced during growth on hexoses, is transferred to acetyl-CoA, yielding ethanol. Ethanol fermentation represents the limiting step in hexose fermentation, therefore, part of the extra NAD(P)H is used to produce erythritol and glycerol. Fructose, pyruvate, citrate, and O2 can be used in addition as external electron acceptors for NAD(P)H reoxidation. Use of the external acceptors increases the growth rate of the bacteria. The bacteria are also able to ferment organic acids like malate, pyruvate, and citrate. Malolactic fermentation generates a proton potential by substrate transport. Pyruvate fermentation sustains growth by pyruvate disproportionation involving pyruvate dehydrogenase. Citrate is fermented in the presence of an additional electron donor to acetate and lactate. Thus, heterofermentative LAB are able to use a variety of unusual fermentation reactions in addition to classical heterofermentation. Most of the reactions are significant for food biotechnology/microbiology.
Keywords: Sugar fermentation; Lactic acid bacteria; Heterofermentative; Organic acids
Antimicrobial activity of resin acid derivatives by Sonia Savluchinske-Feio; Maria João Marcelo Curto; Bárbara Gigante; J. Carlos Roseiro (pp. 430-436).
The wide potential of resin acids as bioactive agents gave rise to a growing effort in the search for new applications of the natural forms and their derivatives. In some of these compounds, the antimicrobial activity is associated to the presence in the molecules of functional groups such as the hydroxyl, aldehyde, and ketone or to their cis or trans configurations. The resin acid family covers a spectrum of antimicrobial activities against several microorganisms, from bacteria to fungi, in which the mode of action was studied by electron microscopy. The morphological alterations are consistent with an unspecific mode of action causing inhibition of the fungal growth or damaging the fungal cells in parallel with a mechanism of resistance based on the retention of the compound by the lipid accumulation. The sterol composition of phytopathogenic fungi Botrytis cinerea and Lophodermium seditiosum treated with methyl cis-7-oxo-deisopropyldehydroabietate revealed the presence of ergosterol (M+ 396) and dihydroergosterol (M+ 398) in both cultures showing that this compound did not interfere with the ergosterol metabolic pathway of both fungi.
Keywords: Resin acids; Antifungal; Antibacterial; Natural products; Phytopathogenic; Dehydroabietic acid
Current state and perspectives of truffle genetics and sustainable biotechnology by Anna Poma; Tania Limongi; Giovanni Pacioni (pp. 437-441).
Mycorrhizal fungi belonging to the genus Tuber produce, after the establishment of a productive interaction with a plant host, hypogeous fruitbodies of great economic value known as ‘‘truffles’’. This review summarizes the state of art on life cycle, genetic, and biotechnological investigations of Tuber spp. The ascocarp formation in truffles is a consequence of the activation of the sexual phase of the biological cycle. The formation of a dikaryotic secondary mycelium and the karyogamy in the ascal cell (followed by meiosis with ascospores formation) have been hypothesized by several authors but some doubts yet arise from the Tuber cycle by considering that a series of abnormalities have been pointed out in respect to other Ascomycetes. It is unclear if binucleated hyphal cells are derived from the fusion of mononucleated cells belonging to mycelia from different mating types or from one only. According to the karyotypes of Tuber melanosporum, Tuber magnatum, and Tuber borchii, the numbers of hyphal chromosomes suggest a chromosome number of eight (2n); these values are in the range of those of several Ascomycetes and observed for Tuber aestivum (2n=10). The importance and growth in interest during the last years in the fungi protoplasts isolation and transformation techniques can be related to current developments in Tuber genetics and biotechnology. T. borchii could be transformed through liposome-mediated delivery of genetic material as mycelial protoplasts isolation and fusion with liposomes has already been established. On the other hand, Agrobacterium-mediated transformation has been successfully established for T. borchii.
Keywords: Truffles; Tuber spp.; Protoplasts; Karyotype; Transformation
Perspectives and advances of biological H2 production in microorganisms by Jens Rupprecht; Ben Hankamer; Jan H. Mussgnug; Gennady Ananyev; Charles Dismukes; Olaf Kruse (pp. 442-449).
The rapid development of clean fuels for the future is a critically important global challenge for two main reasons. First, new fuels are needed to supplement and ultimately replace depleting oil reserves. Second, fuels capable of zero CO2 emissions are needed to slow the impact of global warming. This review summarizes the development of solar powered bio-H2 production processes based on the conversion of photosynthetic products by fermentative bacteria, as well as using photoheterotrophic and photoautrophic organisms. The use of advanced bioreactor systems and their potential and limitations in terms of process design, efficiency, and cost are also briefly reviewed.
Keywords: Hydrogen; Microorganisms; Photosynthesis; Solar energy
Influence of feeding conditions on clavulanic acid production in fed-batch cultivation with medium containing glycerol by Juliana C. Teodoro; Alvaro Baptista-Neto; Isara L. Cruz-Hernández; Carlos O. Hokka; Alberto C. Badino (pp. 450-455).
First, the effect of different levels of nitrogen source on clavulanic acid (CA) production was evaluated in batch cultivations utilizing complex culture medium containing glycerol and three different levels of soy protein isolate (SPI). Cellular growth, evaluated in terms of the rheological parameter K, was highest with a SPI concentration of 30 g.L−1 (4.42 g.L−1 N total). However, the highest production of CA (380 mg.L−1) was obtained when an intermediate concentration of 20 g.L−1 of SPI (2.95 g.L−1 total N) was used. To address this, the influences of volumetric flow rate (F) and glycerol concentration in the complex feed medium (CsF) in fed-batch cultivations were investigated. The best experimental condition for CA production was F=0.01 L.h−1 and CsF=120 g.L−1, and under these conditions maximum CA production was practically twice that obtained in the batch cultivation. A single empirical equation was proposed to relate maximum CA production with F and CsF in fed-batch experiments.
A novel approach for improving the productivity of ubiquinone-10 producing strain by low-energy ion beam irradiation by Shao-Bin Gu; Jian-Ming Yao; Qi-Peng Yuan; Pei-Jian Xue; Zhi-Ming Zheng; Li Wang; Zeng-Liang Yu (pp. 456-461).
Agrobacterium tumefaciens ATCC4452 cells were irradiated by nitrogen ion beam, a new mutagen, with energy of 10 keV and fluence ranging from 2.6×1014 ions/cm2 to 6.5×1015 ions/cm2. A similar “saddle shape” survival curve due to ion beam irradiation appeared again in this study. Some mutants with high yield of ubiquinone-10 were induced by ion implantation. High mutation rate and wide mutation spectrum were also observed in the experiment. These results suggested that the mutagenic effect of such low-energy ion influx into bacterium cells could result from multiple processes involving direct collision of particles with cytoplasm, nucleolus, and cascade atomic and molecular reactions due to plentiful primary and secondary particles.
Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice by Su-Jung Kim; Do Youn Jun; Chae Ha Yang; Young Ho Kim (pp. 462-470).
To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST–Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracelluar secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.
High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression by Zhinan Xu; Zhixia Zhong; Lei Huang; Li Peng; Fang Wang; Peilin Cen (pp. 471-479).
Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37°C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34°C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl β-d-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.
Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium by E. Efremenko; O. Spiricheva; S. Varfolomeyev; V. Lozinsky (pp. 480-485).
Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.
Biotransformation of tolbutamide to 4′-hydroxytolbutamide by the fungus Cunninghamella blakesleeana by Haihua Huang; Xiuwei Yang; Qiang Li; Lu Sun; Dafang Zhong (pp. 486-491).
The hypoglycemic drug tolbutamide is commonly used as a probe drug to evaluate CYP2C9 enzyme activity in terms of production of 4′-hydroxytolbutamide. In the present study, an initial screening of seven filamentous fungi was carried out to identify which was most competent to transform tolbutamide into 4′-hydroxytolbutamide. From this screening, the fungus Cunninghamella blakesleeana AS 3.910 was selected as a suitable bioconverter. At a concentration of 1.2 mg ml−1, the growing fungus transformed 95.0% of tolbutamide into 4′-hydroxytolbutamide in 96 h. With resting culture, the yield could reach 91.7% and exceeded 91.0% even when the tolbutamide concentration was increased to 4.0 mg ml−1. On scale-up to 3 l buffer containing 12.0 g tolbutamide, 90% of tolbutamide was transformed into 4′-hydroxytolbutamide in 96 h. Work-up of the broth by column chromatography and recrystallization yielded 6.5 g (53.9% recovered) of 4′-hydroxytolbutamide with a purity of more than 99%. These results suggest C. blakesleeana AS 3.910 is a useful biosynthetic tool in the preparation of 4′-hydroxytolbutamide.
Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris by Narendar K. Khatri; Frank Hoffmann (pp. 492-498).
The yeast Pichia pastoris is a suitable production system for recombinant proteins due to its strong methanol-inducible AOX1 promoter. A key parameter of the production process is the specific methanol uptake rate. To control the methanol uptake and simultaneously maintain a constant methanol concentration during the production phase, two strategies were developed to generate purposeful oxygen limitation and to feed-forward control the specific methanol uptake rate into the optimum range. First, the cell density at induction was adjusted by prolonged preinduction glycerol feeding. Alternatively, the airflow rate was restricted and increased in parallel with the biomass. While the product accumulation started 20 h earlier with the first approach, the specific production rate of a single-chain antibody fragment was three times higher in the latter case. After 70 h of production, both schemes yielded product concentrations in the gram-per-liter range. Moreover, they release the requirement for dosage of pure oxygen and thereby can facilitate the scale-up of the production process. The different production profiles indicate that the impact of specific methanol uptake rate on protein production by recombinant P. pastoris depends on the control mode.
Purification and characterization of a novel l-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli by Isamu Tashima; Takashi Yoshida; Yoshihiro Asada; Tetsuo Ohmachi (pp. 499-507).
l-2-Amino-Δ2-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu2+ and Mn2+ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K m (0.35 mM) and V max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine.
A laccase from the medicinal mushroom Ganoderma lucidum by H. X. Wang; T. B. Ng (pp. 508-513).
A protein demonstrating laccase activity and potent inhibitory activity towards human immunodeficiency virus (HIV)-1 reverse transcriptase (IC50 1.2 μM) was isolated from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. The laccase had a novel N-terminal sequence and a molecular mass of 75 kDa, which is higher than the range (55–56 kDa) reported for most other mushroom laccases. It was isolated by sequential chromatography on DEAE-cellulose and Affi-gel blue gel and adsorption on Con A-Sepharose. Unlike some of the previously isolated laccases, it was adsorbed only on Con A-Sepharose. The enzyme required a pH of 3–5 and a temperature of 70°C to exhibit maximal activity. Minimal activity was detected at pH 6 and 7. Activity was undetectable at pH 8 and 9 and after exposure to 100°C for 10 min.
A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration by Tsuyoshi Mouri; Junji Michizoe; Hirofumi Ichinose; Noriho Kamiya; Masahiro Goto (pp. 514-520).
A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.
Development of an expression vector for Metarhizium anisopliae based on the tef-1α homologous promoter by Luciano Nakazato; Valéria Dutra; Leonardo Broetto; Charley Christian Staats; Marilene Henning Vainstein; Augusto Schrank (pp. 521-528).
The high-conserved translation elongation factor 1 α (tef-1α) gene from the enthomopathogenic fungus Metarhizium anisopliae was characterized to select the promoter region. A 640-bp DNA fragment upstream to the start codon was employed to drive the expression of the reporter protein sGFP or a dominant selectable marker, the gene bar (resistance to ammonium glufosinate). Transformants carrying this homologous promoter system showed no difference in virulence bioassays against the cattle tick Boophilus microplus comparing to the M. anisopliae wild-type strain. Moreover, GFP fluorescence was detected during tick infection bioassay.
Characterization of the T7 promoter system for expressing penicillin acylase in Escherichia coli by Yali Xu; Stefan Rosenkranz; Chiao-Ling Weng; Jeno M. Scharer; Murray Moo-Young; C. Perry Chou (pp. 529-536).
The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the T7 promoter in Escherichia coli. PAC, with its complex formation mechanism, serves as a unique target protein for demonstration of several key strategies for enhancing recombinant protein production. The current T7 system for pac overexpression was fraught with various technical hurdles. Upon the induction with a conventional inducer of isopropyl-β-d-thiogalactopyranoside (IPTG), the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies and various negative cellular responses such as growth inhibition and cell lysis. The expression performance could be improved by the coexpression of degP encoding a periplasmic protein with protease and chaperone activities. In addition to IPTG, arabinose was shown to be another effective inducer. Interestingly, arabinose not only induced the current T7 promoter system for pac expression but also facilitated the posttranslational processing of proPAC for maturation, resulting in significant enhancement for the production of PAC. Glycerol appeared to have an effect similar to, but not as significant as, arabinose for enhancing the production of PAC. The study highlights the importance of developing suitable genetically engineered strains with culture conditions for enhancing recombinant protein production in E. coli.
Analysis of genetic diversity in Ganoderma population with a novel molecular marker SRAP by Shu-Jing Sun; Wei Gao; Shu-Qian Lin; Jian Zhu; Bao-Gui Xie; Zhi-Bin Lin (pp. 537-543).
Genetic marker technology designed to detect naturally occurring polymorphisms at the DNA level had become an invaluable and revolutionizing tool for both applied and basic studies of fungi. To eliminate the confusion on the taxonomy of Ganoderma strains, in this study, a collection of 31 accessions representative of morphotypes and some unclassified types was used for analyzing molecular diversity using a novel molecular marker sequence-related amplified polymorphism (SRAP). This collection included commercial cultivars and wild varieties that represented the great diversification of types from different countries and regions. The experimental results showed that 50 out of 95 combinations of primers turned out to be polymorphic, and 85 polymorphism bands were obtained using six combinations. Based on the appearances of markers, the genetic similarity coefficients were calculated, and genetic variations were observed (0∼1) among the 31 different Ganoderma strains. The group of Ganoderma lucidum showed significant differences from the group of Ganoderma sinense. Moreover, G. lucidum in China was also different from G. lucidum in Yugoslavia. At the same time, cluster analysis successfully categorized these 31 Ganoderma strains into five groups. These results revealed the genetic diversity of Ganoderma strains and their correlation with geographic environments. It also suggested SRAP marker could be used in the taxonomic analysis of fungi. To our knowledge, this is the first application of SRAP marker on the systematics of Ganoderma strains within basidiomycetes.
Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy by M. M. Harmsen; C. B. van Solt; A. M. van Zijderveld-van Bemmel; T. A. Niewold; F. G. van Zijderveld (pp. 544-551).
We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use.
Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 by Xin Liu; Akio Tani; Kazuhide Kimbara; Fusako Kawai (pp. 552-559).
Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 degrading short ethoxy (EO) chain-nonylphenol (NP) [NPEOav2.0 containing NP mono- ∼ tetraethoxylates (NP1EO ∼ NP4EO); average 2.0 EO units] were isolated by enrichment cultures. Both strains grew on NP but not on octyl- and nonylphenol polyethoxylates (NPEOs) (average 10 EO units). Growth and degradation of NPEOav2.0 was increased with increased concentrations of yeast extract (0.02–0.5%) in a culture medium. Culture supernatants of both strains grown on NPEOav2.0 were analyzed by high-performance liquid chromatography, showing degradation of NP4EO–NP1EO. The metabolites from nonylphenol diethoxylate (NP2EO) by resting cells of both strains were identified by gas chromatography–mass spectrometry as nonylphenoxyethoxyacetic acid, NP1EO, nonylphenoxyacetic acid (NP1EC), and NP, while those from NP1EO were identified as NP1EC and NP. Cell-free extracts from strain AS08 grown on NPEOav2.0 dehydrogenated NPEOs, NPEOav2.0, NP2EO, NP1EO, and PEG 400, but the extracts were inactive toward di- ∼ tetraethylene glycol. Aldehydes were formed in the reaction mixture of each substrate with cell-free extracts. From these results, the aerobic metabolic pathway for short EO chain-NP is proposed: A terminal alcohol group of the EO chain is oxidized to a carboxylic acid via an aldehyde, and then one EO unit is removed. This process is repeated until NP is produced.
Asr1, an alcohol-responsive factor of Saccharomyces cerevisiae, is dispensable for alcoholic fermentation by Shingo Izawa; Kayo Ikeda; Takeomi Kita; Yoshiharu Inoue (pp. 560-565).
Yeast Asr1 is the first reported protein whose intracellular distribution changes specifically in response to alcohol (Betz et al. (2004) J Biol Chem 279:28174–28181). It was reported that Asr1 is required for tolerance to alcohol and plays an important role in the alcohol stress response. Therefore, Asr1 is of interest to brewers and winegrowers attempting to improve the techniques of alcoholic fermentation. We verified the importance of Asr1 in the alcohol stress response during alcoholic fermentation. Although we reconfirmed the alcohol-responsive changes in the intracellular localization of Asr1, we could not detect the effects of Asr1-deficiency on Japanese sake brewing or winemaking. In addition, we could not reconfirm the hypersensitivity of Asr1-deficient mutants to alcohol and sodium dodecyl sulfate. Instead, we conclude that Asr1 is not required and nor important for tolerance to alcohol stress.
Metabolism of 4,4′-dichlorobiphenyl by white-rot fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142 by Ichiro Kamei; Riichiro Kogura; Ryuichiro Kondo (pp. 566-575).
Degradation experiment of model polychlorinated biphenyl (PCB) compound 4,4′-dichlorobiphenyl (4,4′-DCB) and its metabolites by the white-rot fungus Phanerochaete chrysosporium and newly isolated 4,4′-DCB-degrading white-rot fungus strain MZ142 was carried out. Although P. chrysosporium showed higher degradation of 4,4′-DCB in low-nitrogen (LN) medium than that in potato dextrose broth (PDB) medium, Phanerochaete sp. MZ142 showed higher degradation of 4,4′-DCB under PDB medium condition than that in LN medium. The metabolic pathway of 4,4′-DCB was elucidated by the identification of metabolites upon addition of 4,4′-DCB and its metabolic intermediates. 4,4′-DCB was initially metabolized to 2-hydroxy-4,4′-DCB and 3-hydroxy-4,4′-DCB by Phanerochaete sp. MZ142. On the other hand, P. chrysosporium transformed 4,4′-DCB to 3-hydroxy-4,4′-DCB and 4-hydroxy-3,4′-DCB produced via a National Institutes of Health shift of 4-chlorine. 3-Hydroxy-4,4′-DCB was transformed to 3-methoxy-4,4′-DCB; 4-chlorobenzoic acid; 4-chlorobenzaldehyde; and 4-chlorobenzyl alcohol in the culture with Phanerochaete sp. MZ142 or P. chrysosporium. LN medium condition was needed to form 4-chlorobenzoic acid, 4-chlorobenzaldehyde, and 4-chlorobenzyl alcohol from 3-hydroxy-4,4′-DCB, indicating the involvement of secondary metabolism. 2-Hydroxy-4,4′-DCB was not methylated. In this paper, we proved for the first time by characterization of intermediate that hydroxylation of PCB was a key step in the PCB degradation process by white-rot fungi.
Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405 by Rumana Islam; Nazim Cicek; Richard Sparling; David Levin (pp. 576-583).
We have investigated hydrogen (H2) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H2-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H2, carbon dioxide (CO2), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H2/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H2, CO2, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H2-production continued even in stationary phase and H2/CO2 ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H2 production rate of 14.6 mmol g dry cell−1 h−1 was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.
Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant by Raku Shinkyo; Masaki Kamakura; Shin-ichi Ikushiro; Kuniyo Inouye; Toshiyuki Sakaki (pp. 584-590).
Among polychlorinated dibenzo-p-dioxins (PCDDs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is the most toxic one. Recently, we reported that rat CYP1A1 mutant, F240A, expressed in yeast showed metabolic activity toward 2,3,7,8-TetraCDD. In this study, we successfully expressed N-terminal truncated P450s (Δ1A1 and ΔF240A) in Escherichia coli cells. Kinetic analysis using membrane fractions prepared from the recombinant E. coli cells revealed that ΔF240A has enzymatic properties similar to F240A expressed in yeast. The metabolism of PCDDs by recombinant E. coli cells expressing both ΔF240A and human NADPH-P450 reductase was also examined. When 2,3,7-TriCDD was added to the E. coli cell culture at a final concentration of 10 μM, approximately 90% of the 2,3,7-TriCDD was converted into multiple metabolites within 8 h. These results indicate the possible application of prokaryotic cells expressing ΔF240A to the bioremediation of PCDD-contaminated soil.
Influence of temperature on process efficiency and microbial community response during the biological removal of chlorophenols in a packed-bed bioreactor by H. Zilouei; A. Soares; M. Murto; B. Guieysse; B. Mattiasson (pp. 591-599).
Two reactors, initially operated at 14 and 23±1°C (RA and RB, respectively), were inoculated with a bacterial consortium enriched and acclimatized to the respective temperatures over 4 months. The biofilms, formed in the reactors, were studied using scanning electron microscopy, cultivation of the biofilm microflora, and physiological analysis of the isolates. Two bacteria able to mineralize chlorophenols under a large range of temperature (10–30°C) were isolated from the biofilm communities of reactors RA and RB and characterized as Alcaligenaceae bacterium R14C4 and Cupriavidus basilensis R25C6, respectively. When temperature was decreased by 10°C, the chlorophenols removal capacity was reduced from 51.6 to 22.8 mg l−1 h−1 in bioreactor RA (from 14 to 4°C) and from 59.3 to 34.7 mg l−1 h−1 in bioreactor RB (from 23±1 to 14°C). Fluorescence in situ hybridization (FISH) of the biofilm communities showed that, in all temperatures tested, the β-proteobacteria were the major bacterial community (35–47%) followed by the γ-proteobacteria (12.0–6.5%). When the temperature was decreased by 10°C, the proportions of γ-proteobacteria and Pseudomonas species increased significantly in both microbial communities.
Convenient treatment of acetonitrile-containing wastes using the tandem combination of nitrile hydratase and amidase-producing microorganisms by Erina Kohyama; Akihiro Yoshimura; Daisuke Aoshima; Toyokazu Yoshida; Hiroyoshi Kawamoto; Toru Nagasawa (pp. 600-606).
This study aimed to construct an acetonitrile-containing waste treatment process by using nitrile-degrading microorganisms. To degrade high concentrations of acetonitrile, the microorganisms were newly acquired from soil and water samples. Although no nitrilase-producing microorganisms were found to be capable of degrading high concentrations of acetonitrile, the resting cells of Rhodococcus pyridinivorans S85-2 containing nitrile hydratase could degrade acetonitrile at concentrations as high as 6 M. In addition, an amidase-producing bacterium, Brevundimonas diminuta AM10-C-1, of which the resting cells degraded 6 M acetamide, was isolated. The combination of R. pyridinivorans S85-2 and B. diminuta AM10-C-1 was tested for the conversion of acetonitrile into acetic acid. The resting cells of B. diminuta AM10-C-1 were added after the first conversion involving R. pyridinivorans S85-2. Through this tandem process, 6 M acetonitrile was converted to acetic acid at a conversion rate of >90% in 10 h. This concise procedure will be suitable for practical use in the treatment of acetonitrile-containing wastes on-site.
Biodegradation of a phenolic mixture in a solid–liquid two-phase partitioning bioreactor by George P. Prpich; Andrew J. Daugulis (pp. 607-615).
A solid–liquid two-phase partitioning bioreactor (TPPB) in which the non-aqueous phase consisted of polymer (HYTREL) beads was used to degrade a model mixture of phenols [phenol, o-cresol, and 4-chlorophenol (4CP)] by a microbial consortium. In one set of experiments, high concentrations (850 mg l−1 of each of the three substrates) were reduced to sub-inhibitory levels within 45 min by the addition of the polymer beads, followed by inoculation and rapid (8 h) consumption of the total phenolics loading. In a second set of experiments, the beneficial effect of using polymer beads to launch a fermentation inhibited by high substrate concentrations was demonstrated by adding 1,300 and 2,000 mg l−1 total substrates (equal concentrations of each phenolic) to a pre-inoculated bioreactor. At these levels, no cell growth and no degradation were observed; however, after adding polymer beads to the systems, the ensuing reduced substrate concentrations permitted complete destruction of the target molecules, demonstrating the essential role played by the polymer sequestering phase when applied to systems facing inhibitory substrate concentrations. In addition to establishing alternative modes of TPPB operation, the present work has demonstrated the differential partitioning of phenols in a mixture between the aqueous and polymeric phases. The polymeric phase was also observed to absorb a degradation intermediate (arising from the incomplete biodegradation of 4CP), which opens the possibility of using solid–liquid TPPBs during biosynthetic transformation to sequester metabolic byproducts.
Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth by Chengli Yang; Jianmin Xing; Yueping Guan; Huizhou Liu (pp. 616-622).
An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.