Abstract:
:Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating α-factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: preproα-AAO > preαproK-AAO > preKproα-AAO > preproK-AAO. The chimeric preαproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[α1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preαproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Viña-Gonzalez J,Gonzalez-Perez D,Ferreira P,Martinez AT,Alcalde Mdoi
10.1128/AEM.01966-15subject
Has Abstractpub_date
2015-09-01 00:00:00pages
6451-62issue
18eissn
0099-2240issn
1098-5336pii
AEM.01966-15journal_volume
81pub_type
杂志文章abstract::The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against several kinds of...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.00662-17
更新日期:2017-06-30 00:00:00
abstract::Quantitative analysis of genes that code for Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemic...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.01482-09
更新日期:2010-02-01 00:00:00
abstract::A gram-negative bacterium which hydrolyzed aryl N-methylcarbamate insecticides was isolated from an agricultural soil which quickly degraded these pesticides. This organism, designated strain ER2, grew on carbofuran as a sole source of carbon and nitrogen with a doubling time of 3 h in a mineral salts medium. The arom...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.59.10.3339-3349.1993
更新日期:1993-10-01 00:00:00
abstract::A yellow-pigmented marine bacterium, designated strain SD-21, was isolated from surface sediments of San Diego Bay, San Diego, Calif., based on its ability to oxidize soluble Mn(II) to insoluble Mn(III, IV) oxides. 16S rRNA analysis revealed that this organism was most closely related to members of the genus Erythroba...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/aem.67.9.4024-4029.2001
更新日期:2001-09-01 00:00:00
abstract::The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20 degrees C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.70.10.5750-5755.2004
更新日期:2004-10-01 00:00:00
abstract::Quantitative assessment of growth of filamentous microorganisms, such as streptomycetes, is generally restricted to determination of dry weight. Here, we describe a straightforward methylene blue-based sorption assay to monitor microbial growth quantitatively, simply, and rapidly. The assay is equally applicable to un...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.00778-13
更新日期:2013-07-01 00:00:00
abstract::A dot blot hybridization procedure with 32P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.54.5.1158-1162.1988
更新日期:1988-05-01 00:00:00
abstract::Twenty Gateway-compatible destination vectors were constructed. The vectors comprise fluorescent and epitope fusion tags, various drug markers, and replication origins that should make them useful for exploring existing microbial ORFeomes. In an attempt to validate several of these vectors, we observed polar and oscil...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.01873-06
更新日期:2007-02-01 00:00:00
abstract::The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66 degrees S, 143 degrees E; depth range, 709 to 964 m) were studied. Correlations were found between microbial biomass and aminopeptidase and chitinase rates,...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/aem.69.5.2448-2462.2003
更新日期:2003-05-01 00:00:00
abstract::Soils harbor enormously diverse bacterial populations, and soil bacterial communities can vary greatly in composition across space. However, our understanding of the specific changes in soil bacterial community structure that occur across larger spatial scales is limited because most previous work has focused on eithe...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.00335-09
更新日期:2009-08-01 00:00:00
abstract::The influence of trichloroethene (TCE; 0 to 65 mg/liter) and 1,1,1-trichloroethane (1,1,1-TCA; 0 to 103 mg/liter) on methane consumption of a mixed culture of methane-oxidizing bacteria was studied in laboratory batch experiments. Increasing concentrations of TCE or 1,1,1-TCA resulted in decreasing methane consumption...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.56.8.2488-2493.1990
更新日期:1990-08-01 00:00:00
abstract::The wastewater of livestock slaughterhouses is considered a source of antimicrobial-resistant bacteria with clinical relevance and may thus be important for their dissemination into the environment. To get an overview of their occurrence and characteristics, we investigated process water (n = 50) from delivery and unc...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.02748-19
更新日期:2020-04-01 00:00:00
abstract::The interactions of bacteria isolated from corroded copper coupons on thin films of copper evaporated onto germanium internal reflection elements were evaluated nondestructively in real time by attentuated total reflectance Fourier transform infrared spectroscopy. The films were stable in the presence of flowing or st...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.57.7.1956-1962.1991
更新日期:1991-07-01 00:00:00
abstract::Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotell...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.58.4.1313-1320.1992
更新日期:1992-04-01 00:00:00
abstract::An NAD(P)H-nicotine blue (quinone) oxidoreductase was discovered as a member of the nicotine catabolic pathway of Arthrobacter nicotinovorans. Transcriptional analysis and electromobility shift assays showed that the enzyme gene was expressed in a nicotine-dependent manner under the control of the transcriptional acti...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.02668-06
更新日期:2007-04-01 00:00:00
abstract::The genus of Marinobacter is one of the most ubiquitous in the global oceans and assumed to significantly impact various biogeochemical cycles. The genome structure and content of Marinobacter aquaeolei VT8 was analyzed and compared with those from other organisms with diverse adaptive strategies. Here, we report the ...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.01866-10
更新日期:2011-04-01 00:00:00
abstract::An assay has been developed to detect bacterial glycosidases in colonies grown on the surface of agar plates. Advantages of this technique over previously described methods include elimination of the need for replica plating, better visualization of chromagenic reaction products, and a simple permeabilization step to ...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.47.1.208-209.1984
更新日期:1984-01-01 00:00:00
abstract::Antibodies against a microcystin (MCYST) leucine-arginine variant (MCYST-LR) were demonstrated 4 weeks after immunization of rabbits with either MCYST-LR-polylysine- or MCYST-LR-ethylenediamine-modified bovine serum albumin. A radioimmunoassay (RIA), a direct competitive enzyme-linked immunosorbent assay (ELISA), and ...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.55.8.1928-1933.1989
更新日期:1989-08-01 00:00:00
abstract::The size frequency distribution of planktonic cells of purple sulfur phototrophic bacteria was measured at several depths in a bacterial layer of Lake Cisó (Spain). The bacterioplankton was dominated by Chromatium minus (87 to 94% of the total biomass). The largest cells of C. minus were found in the top part of the b...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.53.4.864-871.1987
更新日期:1987-04-01 00:00:00
abstract::Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8. Samples containing L. pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B. After incubation at 35 degrees C for 3 days,...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.55.6.1640-1641.1989
更新日期:1989-06-01 00:00:00
abstract::A rich history of investigation documents various Drosophila-yeast mutualisms, suggesting that Drosophila suzukii similarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts of D. suzukii. Terminal ...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.00841-12
更新日期:2012-07-01 00:00:00
abstract::Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.05097-11
更新日期:2011-09-01 00:00:00
abstract::A homokaryotic, clavine alkaloid-producing strain of ergot, Claviceps sp. strain SD 58, was used in an attempt to demonstrate parasexuality. Genetically marked auxotrophic strains were produced by mutation with N-methyl-N'-nitro-N-nitrosoguanidine. Protoplast fusion of pairs of unlike doubly auxotrophic strains and is...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.53.1.70-73.1987
更新日期:1987-01-01 00:00:00
abstract::The fate of selenium in the environment is controlled, in part, by microbial selenium oxyanion reduction and Se(0) precipitation. In this study, we identified a genetic regulator that controls selenate reductase activity in the Se-reducing bacterium Enterobacter cloacae SLD1a-1. Heterologous expression of the global a...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.02542-06
更新日期:2007-03-01 00:00:00
abstract::Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca, the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 ut...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.02656-18
更新日期:2019-02-20 00:00:00
abstract::Cadmium is an important heavy metal pollutant. For this study, we investigated the effects of cadmium exposure on the oxidative stress responses of Xanthomonas campestris, a soil and plant pathogenic bacterium. The exposure of X. campestris to low concentrations of cadmium induces cross-protection against subsequent k...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.71.4.1843-1849.2005
更新日期:2005-04-01 00:00:00
abstract::The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraos...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.49.4.765-771.1985
更新日期:1985-04-01 00:00:00
abstract::Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyan...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.01062-15
更新日期:2015-08-01 00:00:00
abstract::Both the periplasmic and the extracellular cellodextrinases from Bacteroides succinogenes S85 grown on Avicel microcrystalline cellulose were purified to homogeneity by column chromatography and characterized. Over 70% of the total cellobiosidase activity displayed by cells was accounted for by these enzymes. The peri...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.54.6.1488-1493.1988
更新日期:1988-06-01 00:00:00
abstract::Characteristics of an obligately methylotrophic coccoid methanogen (strain GS-16) previously isolated from estuarine sediment are described. Growth was demonstrated on dimethyl sulfide (DMS) or trimethylamine (TMA), but not on methane thiol, methane thiol plus hydrogen, dimethyl disulfide, or methionine. DMS-grown cel...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.55.4.994-1002.1989
更新日期:1989-04-01 00:00:00