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Applied Microbiology and Biotechnology (v.61, #4)
Biotransformation of limonene by bacteria, fungi, yeasts, and plants by W. A. Duetz; H. Bouwmeester; J. B. van Beilen; B. Witholt (pp. 269-277).
The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (α-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.
Aminoacyl-tRNA synthetases and their inhibitors as a novel family of antibiotics by S. Kim; S. W. Lee; E.-C. Choi; S. Y. Choi (pp. 278-288).
The emergence of multidrug-resistant strains of pathogenic microorganisms and the slow progress in new antibiotic development has led in recent years to a resurgence of infectious diseases that threaten the well-being of humans. The result of many microorganisms becoming immune to major antibiotics means that fighting off infection by these pathogens is more difficult. The best strategy to get around drug resistance is to discover new drug targets, taking advantage of the abundant information that was recently obtained from genomic and proteomic research, and explore them for drug development. In this regard, aminoacyl-tRNA synthetases (ARSs) provide a promising platform to develop novel antibiotics that show no cross-resistance to other classical antibiotics. During the last few years there has been a comprehensive attempt to find the compounds that can specifically target ARSs and inhibit bacterial growth. In this review, the current status in the development of ARS inhibitors will be briefly summarized, based on their chemical structures and working mechanisms.
Triacylglycerol biosynthesis in yeast by D. Sorger; G. Daum (pp. 289-299).
Triacylglycerol (TAG) is the major storage component for fatty acids, and thus for energy, in eukaryotic cells. In this mini-review, we describe recent progress that has been made with the yeast Saccharomyces cerevisiae in understanding formation of TAG and its cell biological role. Formation of TAG involves the synthesis of phosphatidic acid (PA) and diacylglycerol (DAG), two key intermediates of lipid metabolism. De novo formation of PA in yeast as in other types of cells can occur either through the glycerol-3-phosphate- or dihydroxyacetone phosphate-pathways—each named after its respective precursor. PA, formed in two steps of acylation, is converted to DAG by phosphatidate phosphatase. Acylation of DAG to yield TAG is catalyzed mainly by the two yeast proteins Dga1p and Lro1p, which utilize acyl-CoA or phosphatidylcholine, respectively, as acyl donors. In addition, minor alternative routes of DAG acylation appear to exist. Endoplasmic reticulum and lipid particles (LP), the TAG storage compartment in yeast, are the major sites of TAG synthesis. The interplay of these organelles, formation of LP, and enzymatic properties of enzymes catalyzing the synthesis of PA, DAG, and TAG in yeast are discussed in this communication.
Biodegradation of microbial and synthetic polyesters by fungi by D. Y. Kim; Y. H. Rhee (pp. 300-308).
A variety of biodegradable polyesters have been developed in order to obtain useful biomaterials and to reduce the impact of environmental pollution caused by the large-scale accumulation of non-degradable waste plastics. Polyhydroxyalkanoates, poly(ε-caprolactone), poly(l-lactide), and both aliphatic and aromatic polyalkylene dicarboxylic acids are examples of biodegradable polyesters. In general, most aliphatic polyesters are readily mineralized by a number of aerobic and anaerobic microorganisms that are widely distributed in nature. However, aromatic polyesters are more resistant to microbial attack than aliphatic polyesters. The fungal biomass in soils generally exceeds the bacterial biomass and thus it is likely that fungi may play a considerable role in degrading polyesters, just as they predominantly perform the decomposition of organic matter in the soil ecosystem. However, in contrast to bacterial polyester degradation, which has been extensively investigated, the microbiological and environmental aspects of fungal degradation of polyesters are unclear. This review reports recent advances in our knowledge of the fungal degradation of microbial and synthetic polyesters and discusses the ecological importance and contribution of fungi in the biological recycling of waste polymeric materials in the biosphere.
Immobilization of xylan-degrading enzymes from Melanocarpus albomyces IIS 68 on the smart polymer Eudragit L-100 by I. Roy; A. Gupta; S. K. Khare; V. S. Bisaria; M. N. Gupta (pp. 309-313).
Xylanase of Melanocarpus albomyces IIS 68 was immobilized on Eudragit L-100. The latter is a copolymer of methacrylic acid and methyl methacrylate and is a pH-sensitive smart polymer. The immobilization was carried out by gentle adsorption and an immobilization efficiency of 0.82 was obtained. The enzyme did not leach off the polymer even in the presence of 1 M NaCl and 50% ethylene glycol. The K m of the enzyme changed from 5.9 mg ml–1 to 9.1 mg ml–1 upon immobilization. The V max of the immobilized enzyme showed an increase from 90.9 µmol ml–1 min–1 (for the free enzyme) to 111.1 µmol ml–1 min–1. The immobilized enzyme could be reused up to ten times without impairment of the xylanolytic activity. The immobilized enzyme was also evaluated for its application in pre-bleaching of eucalyptus kraft pulp.
Identifiability and retrievability of unique parameters describing intrinsic Andrews kinetics by E. A. Seagren; H. Kim; B. F. Smets (pp. 314-322).
A key factor contributing to the variability in the microbial kinetic parameters reported from batch assays is parameter identifiability, i.e., the ability of the mathematical routine used for parameter estimation to provide unique estimates of the individual parameter values. This work encompassed a three-part evaluation of the parameter identifiability of intrinsic kinetic parameters describing the Andrews growth model that are obtained from batch assays. First, a parameter identifiability analysis was conducted by visually inspecting the sensitivity equations for the Andrews growth model. Second, the practical retrievability of the parameters in the presence of experimental error was evaluated for the parameter estimation routine used. Third, the results of these analyses were tested using an example data set from the literature for a self-inhibitory substrate. The general trends from these analyses were consistent and indicated that it is very difficult, if not impossible, to simultaneously obtain a unique set of estimates of intrinsic kinetic parameters for the Andrews growth model using data from a single batch experiment.
Studies on a thermostable α-amylase from the thermophilic fungus Scytalidium thermophilum by A. C. M. M. Aquino; J. A. Jorge; H. F. Terenzi; M. L. T. M. Polizeli (pp. 323-328).
An α-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60°C, respectively. In the absence of substrate the purified α-amylase was stable for 1 h at 50°C and had a half-life of 12 min at 60°C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca2+ (up to 10 mM), but HgCl2 and CuCl2 inhibited its activity. The α-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an α-amylase (1,4-α-glucan glucanohydrolase).
Significantly enhanced stability of glucose dehydrogenase by directed evolution by S.-H. Baik; T. Ide; H. Yoshida; O. Kagami; S. Harayama (pp. 329-335).
An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution. The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes. Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the pTrc99A expression vector and expressed in the host, Escherichia coli. In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B. megaterium IWG3, was also subjected to directed evolution by the family shuffling method. A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries. This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes. The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl. In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66°C), while the half-life of isolated mutant DN-46 was 540 min at 66°C, i.e., 415-fold more thermostable than mutant F20. The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.
Cloning, expression and characterisation of two tyrosinase cDNAs from Agaricus bisporus by H. J. Wichers; K. Recourt; M. Hendriks; C. E. M. Ebbelaar; G. Biancone; F. A. Hoeberichts; H. Mooibroek; C. Soler-Rivas (pp. 336-341).
Using primers designed on the basis of sequence homologies in the copper-binding domains for a number of plant and fungal tyrosinases, two tyrosinase encoding cDNAs were cloned from an Agaricus bisporus U1 cDNA-library. The sequences AbPPO1 and AbPPO2 were, respectively, 1.9 and 1.8 kb in size and encoded proteins of approximately 64 kDa. The cDNAs represent different loci. Both AbPPO1 and AbPPO2 occur as single copies on the genomes of the U1 parental strains H39 and H97. The genomic size of AbPPO1 and AbPPO2 is minimally 2.3 and 2.2 kb, respectively. Alignment and phylogenetic analysis of 35 tyrosinase and polyphenol oxidase sequences of animal, plant, fungal, and bacterial origin indicated conserved copper-binding domains, and stronger conservation within genera than between them. The translation products of AbPPO1 and AbPPO2 possess putative N-glycosylation and phosphorylation sites and are recognised by antibodies directed against a 43-kDa tyrosinase. The observations are consistent with previously proposed maturation and activation models for plant and fungal tyrosinases.
Biodegradation of phenanthrene by Pseudomonas sp. strain PP2: novel metabolic pathway, role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation by Y. Prabhu; P. S. Phale (pp. 342-351).
Pseudomonas sp. strain PP2 isolated in our laboratory efficiently metabolizes phenanthrene at 0.3% concentration as the sole source of carbon and energy. The metabolic pathways for the degradation of phenanthrene, benzoate and p-hydroxybenzoate were elucidated by identifying metabolites, biotransformation studies, oxygen uptake by whole cells on probable metabolic intermediates, and monitoring enzyme activities in cell-free extracts. The results obtained suggest that phenanthrene degradation is initiated by double hydroxylation resulting in the formation of 3,4-dihydroxyphenanthrene. The diol was finally oxidized to 2-hydroxymuconic semialdehyde. Detection of 1-hydroxy-2-naphthoic acid, α-naphthol, 1,2-dihydroxy naphthalene, and salicylate in the spent medium by thin layer chromatography; the presence of 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-2,3-dioxygenase activity in the extract; O2 uptake by cells on α-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate and catechol; and no O2 uptake on o-phthalate and 3,4-dihydroxybenzoate supports the novel route of metabolism of phenanthrene via 1-hydroxy-2-naphthoic acid → [α-naphthol] → 1,2-dihydroxy naphthalene → salicylate → catechol. The strain degrades benzoate via catechol and cis,cis-muconic acid, and p-hydroxybenzoate via 3,4-dihydroxybenzoate and 3-carboxy-cis,cis-muconic acid. Interestingly, the culture failed to grow on naphthalene. When grown on either hydrocarbon or dextrose, the culture showed good extracellular biosurfactant production. Growth-dependent changes in the cell surface hydrophobicity, and emulsification activity experiments suggest that: (1) production of biosurfactant was constitutive and growth-associated, (2) production was higher when cells were grown on phenanthrene as compared to dextrose and benzoate, (3) hydrocarbon-grown cells were more hydrophobic and showed higher affinity towards both aromatic and aliphatic hydrocarbons compared to dextrose-grown cells, and (4) mid-log-phase cells were significantly (2-fold) more hydrophobic than stationary phase cells. Based on these results, we hypothesize that growth-associated extracellular biosurfactant production and modulation of cell surface hydrophobicity plays an important role in hydrocarbon assimilation/uptake in Pseudomonas sp. strain PP2.
Corn fiber hydrolysis by Thermobifida fusca extracellular enzymes by D. Irwin; T. D. Leathers; R. V. Greene; D. B. Wilson (pp. 352-358).
Thermobifida fusca was grown on cellulose (Solka-Floc), xylan or corn fiber and the supernatant extracellular enzymes were concentrated. SDS gels showed markedly different protein patterns for the three different carbon sources. Activity assays on a variety of synthetic and natural substrates showed major differences in the concentrated extracellular enzyme activities. These crude enzyme preparations were used to hydrolyze corn fiber, a low-value biomass byproduct of the wet milling of corn. Approximately 180 mg of reducing sugar were produced per gram of untreated corn fiber. When corn fiber was pretreated with alkaline hydrogen peroxide, up to 429 mg of reducing sugars were released per gram of corn fiber. Saccharification was enhanced by the addition of β-glucosidase or by the addition of a crude xylanase preparation from Aureobasidium sp.
Dual influence of the carbon source and l-methionine on the synthesis of sulphur compounds in the cheese-ripening yeast Geotrichum candidum by K. Arfi; R. Tâche; H. E. Spinnler; P. Bonnarme (pp. 359-365).
The effect of the carbon source and l-methionine on the ability of Geotrichum candidum to produce volatile sulphur compounds (VSC) was studied. This yeast was cultivated in a synthetic medium supplemented with various carbon sources and l-methionine at different concentrations. Both glycerol and glucose significantly increased VSC production by G. candidum. Unlike the effect on the l-methionine- and 4-methylthio-2-oxobutyric acid-demethiolating activities, the supply of a carbon source had a dramatic effect on the activity of aminotransferase, a key enzyme in l-methionine catabolism. An increase in the initial concentration of l-methionine resulted in a rise in the production of sulphur compounds (VSC, 4-methylthio-2-oxobutyric acid) but had limited effect on l-methionine-catabolising enzyme activities. Evidence for the existence of a dual effect of the carbon source and l-methionine on VSC biosynthesis was obtained in this study.
Pseudomonas putida as the dominant toluene-degrading bacterial species during air decontamination by biofiltration by S. Roy; J. Gendron; M.-C. Delhoménie; L. Bibeau; M. Heitz; R. Brzezinski (pp. 366-373).
The microbial communities established in three laboratory-scale compost matrix biofilters fed with toluene were characterized. The biofilters were operated for 7 weeks at inlet concentrations of toluene ranging over 250–500 ppm with daily irrigation, using a nutrient solution containing variable concentrations of nitrogen, supplied as urea, and other inorganic salts. The indigenous microflora of the compost included toluene-degrading species, making inoculation unnecessary. The numerically predominant toluene-degrading strains were isolated from the most diluted positive wells of most-probable-number counts on mineral medium with toluene as sole carbon source and identified by rRNA 16S gene sequencing. On the basis of sequence similarity, all the isolated strains were assigned to the species Pseudomonas putida, although some variations were observed in their respective sequences. It is concluded that the mode of biofilter operation including a daily supply of non-carbon nutrients created an environment favoring the constant numerical predominance of this fast-growing toluene-degrading species.
Lignin degradation in a compost environment by the deuteromycete Paecilomyces inflatus by B. Kluczek-Turpeinen; M. Tuomela; A. Hatakka; M. Hofrichter (pp. 374-379).
Two strains of the deuteromycete Paecilomyces inflatus were isolated from compost samples consisting of municipal wastes, paper and wood chips. Lignin degradation by P. inflatus was studied following the mineralization of a synthetic 14Cβ-labeled lignin (side-chain labeled dehydrogenation polymer, DHP). Approximately 6.5% of the synthetic lignin was mineralized during solid-state cultivation of the fungus in autoclaved compost; and 15.5% was converted into water-soluble fragments. Laccase was the only ligninolytic enzyme detectable when the isolates were grown in autoclaved compost. Production of the enzyme was growth-associated and dependent on the culture conditions. The optimal pH for laccase production was between 4.5 and 5.5 and the optimal temperature was around 30 °C. Activity levels of laccase increased in the presence of low-molecular-mass aromatic compounds, such as veratryl alcohol, veratric acid, vanillin and vanillic acid.
Biodegradation of chloronaphthalenes and polycyclic aromatic hydrocarbons by the white-rot fungus Phlebia lindtneri by T. Mori; S. Kitano; R. Kondo (pp. 380-383).
The biodegradation of chloronaphthalene (CN) and polycyclic aromatic hydrocarbons by the white-rot fungus Phlebia lindtneri, which can degrade dichlorinated dioxins and non-chlorinated dioxin-like compounds, was investigated. Naphthalene, phenanthrene, 1-chloronaphthalene (1-CN) and 2-chloronaphthalene (2-CN) were metabolized by the fungus to form several oxidized products. Naphthalene and phenanthrene were metabolized to the corresponding hydroxylated and dihydrodihydroxylated metabolites. 2-CN was metabolized to 3-chloro-2-naphtol, 6-chloro-1-naphtol and two other chloronaphtols, CN-dihydrodiols and CN-diols. Significant inhibition of the degradation of these substrates was observed when they were incubated with the cytochrome P-450 monooxygenase inhibitors 1-aminobenzotriazole and piperonyl butoxide. These results suggest that P. lindtneri initially oxidizes these substrates by a cytochrome P-450 monooxygenase.