Metagenomics datasets of water and sediments from eutrophication-impacted artificial lakes in South Africa.

We present metagenomes of 16 samples of water and sediment from two lakes, collected from eutrophic and non-eutrophic areas, including pooled samples enriched with phosphate and nitrate. Additionally, we assembled 167 bacterial metagenome-assembled genomes (MAGs). These MAGs were de-replicated into 83 unique genomes representing different species found in the lakes. All the MAGs exhibited >70% completeness and <10% contamination, with 79 MAGs being classified as 'nearly complete' (completeness >90%), while 54 falling within 80-90% range and 34 between 75-80% complete. The most abundant MAGs identified across all samples were Proteobacteria (n = 80), Firmicutes_A (n = 35), Firmicutes (n = 13), and Bacteriodota (n = 22). Other groups included Desulfobacteria_I (n = 2), Verrucomicrobiota (n = 4), Campylobacterota (n = 4) and Actinobacteriota (n = 6). Importantly, phylogenomic analysis identified that approximately 50.3% of the MAGs could not be classified to known species, suggesting the presence of potentially new and unknown bacteria in these lakes, warranting further in-depth investigation. This study provides valuable new dataset on the diverse and often unique microbial communities living in polluted lakes, useful in developing effective strategies to manage pollution.

Unlocking water potential in drylands: Quicklime and fly ash enhance soil microbiome structure, ecological networks and function in acid mine drainage water-irrigated agriculture.

In water-stressed regions, treated acid mine drainage (AMD) water for irrigated agriculture is a potential solution to address freshwater scarcity. However, a significant knowledge gap exists on the short and long-term effects of treated AMD water on soil health. This study used high-throughput Illumina sequencing and predictive metagenomic profiling to investigate the impact of untreated AMD (AMD), quicklime- (A1Q and A2Q) and quicklime and fly ash-treated AMD water (AFQ) irrigation on soil bacterial diversity, co-occurrence networks and function. Results showed that untreated AMD water significantly increased soil acidity, electrical conductivity (EC), sulfate (SO42-), and heavy metals (HM), including reduced microbial diversity, disrupted interaction networks, and functional capacity. pH, EC, Cu, and Pb were identified as key environmental factors shaping soil microbial diversity and structure. Predominantly, Pseudomonas, Ralstonia picketti, Methylotenera KB913035, Brevundimonas vesicularis, and Methylobacteriumoryzae, known for their adaptability to acidic conditions and metal resistance, were abundant in AMD soils. However, soils irrigated with treated AMD water exhibited significantly reduced acidity (pH > 6.5), HM and SO42- levels, with an enrichment of a balanced bacterial taxa associated with diverse functions related to soil health and agricultural productivity. These taxa included Sphingomonas, Pseudoxanthomonas, Achromobacter, Microbacterium, Rhodobacter, Clostridium, Massillia, Rhizobium, Paenibacillus, and Hyphomicrobium. Moreover, treated AMD water contributed to higher connectivity and balance within soil bacterial co-occurrence networks compared to untreated AMD water. These results show that quicklime/fly ash treatments can help lessen impacts of AMD water on soil microbiome and health, suggesting its potential for irrigated agriculture in water-scarce regions.

High-throughput amplicon sequencing datasets of microbial community in soils irrigated by quicklime and fly ash-treated acid mine drainage water.

In water-stressed regions, the use of treated acid mine drainage (AMD) water for irrigated agriculture has been suggested as an alternative to address the shortage of fresh water sources. However, the short and long-term impact of using such (un)treated AMD water on soil health, particularly the microbiome structure and functional capacity, is not known. We present high-throughput amplicon sequence (HTS) datasets of purified microbial metacommunity DNA of soils under Irish potato production irrigated by quicklime and fly ash treated AMD water. The irrigation treatments included quicklime treated AMD water (A1Q and A2Q; n = 16), and quicklime and fly ash-treated AMD water (AFQ; n = 5), untreated AMD water (uAMD; n = 7) and control group using tap water (n = 5). The V1-V3 hypervariable region of the 16S rRNA gene from each sample were sequenced on an Illumina MiSeq to generate these HTS datasets. The raw sequences underwent quality-checking, demultiplexing into FASTQ files, and processing using MOTHUR pipeline (v1.40.0). Th quality reads classified into taxonomic ids (phylum, class, order, family, and genus) using the Naïve Bayesian classifier algorithm against the SILVA database (v132) and were assigned to operational taxonomic units (OTUs) based on the pairwise distance matrix (Euclidean distance matrix). The applicability of the HTS datasets was confirmed by microbial taxa at the phylum level. All HTS datasets are available through the BioSample Submission Portal under the BioProject ID PRJNA974836 (https://www.ncbi.nlm.nih.gov/bioproject/974836).

Sediment microbiome diversity and functional profiles of unprotected arid-tropical natural wetlands in South Africa revealed by shotgun metagenomics data.

The Limpopo province, located in the arid-tropical region in northeastern South Africa, is renowned for its diverse natural wetlands, some of which are currently unprotected. These wetlands play a crucial role in preserving biodiversity, purifying water, controlling floods, and supporting agricultural production for rural communities. Unfortunately, human activities such as agricultural effluents, run-offs, domestic wastewater, and plastics pollution, along with the impacts of climate change, are mounting pressures on these ecosystems. However, there is limited information on the microbial ecology of natural wetlands in this region, considering the changing anthropogenic activities. The data presented represents the first report on the microbial and functional diversity of sediment microbiomes associated with unprotected arid-tropical natural wetlands in South Africa. Metagenomic shotgun sequencing was performed on sediment samples from ten different wetlands using the Illumina NextSeq 2000 platform. Taxonomic profiling of 328,625,930 high-quality sequencing reads using the MetaPhlAn v3.0 pipeline revealed that Bacteria were the most abundant kingdom (54.5 %), followed by Viruses (0.40 %), Archaea (0.01 %), and Eukaryota (0.36 %). Among bacteria, the most prevalent taxa belonged to the phylum Proteobacteria, particularly the classes Gammaproteobacteria and Betaproteobacteria, which accounted for 83 % of bacterial sequences. The Terrabacteria group, consisting of the phyla Firmicutes and Actinobacteria, made up 3 % of the bacterial population. The abundance of these top bacterial taxa varied across different wetland samples, both at the genus and species levels. In addition, hierarchical clustering based on Bray-Curtis dissimilarity distances of fungal, protist, archaea, and virus species showed distinct clustering of sediment samples from different wetlands. Functional annotation of the metagenomes identified 1224-1702 enzyme classes, 84,833-198,397 gene families, and 280-400 pathways across the various wetland sediments. The data provide crucial baseline information on the microbial and functional diversity of sediment communities in arid tropical wetlands. This knowledge will contribute to a better understanding of these unique environments and can aid in their management and conservation efforts in rural South Africa.

Bioaccumulation, Bioindication and Health Risk Assessment of Heavy Metals in Cape Horse Mackerel (Trachurus trachurus) and Slinger Seabream (Chrysoblephus puniceus) in the Durban Basin and Cape Vidal, South Africa.

The bioaccumulation of heavy metals (HMs) in marine fish is a growing global concern due to potential human health risks. The study analyzed HM in the muscle tissue, gills, and gut of adult male and female cape horse mackerel and slinger seabream caught in the polluted Durban Basin and pristine Cape Vidal from April 2018 to February 2019. Results revealed interspecific, spatial, and organ-specific variability in HM levels. In the Durban Basin, slinger seabream had bioaccumulation (in mg/kg) of As (2.3 ± 0.2), Cr (2.6 ± 0.2), Ni (2.0 ± 0.1), and Pb (4.1 ± 0.3) while cape horse mackerel had Ni (1.6 ± 0.2), Pb (4.7 ± 0.6), and Zn (52 ± 3.01) exceeding World Health Organization (WHO) regulatory limits. Metal pollution index (MPI) values were also higher in Durban Basin (> 5.13) than Cape Vidal (< 3.32) for both species' muscles. Liver and gills of slinger seabream and gut of cape horse mackerel exhibited higher HM accumulation patterns proportionate to the environmental concentrations, indicating the bioindicative potential of HM pollution by the two species. Risk assessment indicated that both fish species had target hazard quotient > 1 for Cr, and target cancer risk < 10-4 for Pb, implying significant potential non-carcinogenic and carcinogenic health risks associated with fish consumption from the Durban Basin. The study recommends daily consumption limits of 16 g/day for slinger seabream and 14 g/day for cape horse mackerel to ensure health safety. The findings contribute to the understanding of HM pollution in the Durban Basin and provide important information for decision-makers and policymakers in developing effective strategies to mitigate and manage HM contamination in fish populations.

Multivariate analysis of enriched landfill soil consortia provide insight on the community structural perturbation and functioning during low-density polyethylene degradation.

Plastic-enriched sites like landfills have immense potential for discovery of microbial consortia that can efficiently degrade plastics. In this study, we used a combination of culture enrichment, high-throughput PacBio sequencing of 16 S rRNA and the ITS gene, Fourier transform infrared (FTIR), and scanning electron microscopy (SEM) to examine the compositional and diversity perturbations of bacterial and fungal consortia from landfill soils and their impact on low-density polyethylene (LDPE) film biodegradation over a 90-day period. Results showed that enrichment cultures effectively utilized LDPE as a carbon source for cellular growth, resulting in significant weight reduction (22.4% and 55.6%) in the films. SEM analysis revealed marked changes in the micrometric surface characteristics (cracks, fissures, and erosion) and biofilm formation in LDPE films. FTIR analyses suggested structural and functional group modification related to C-H (2831-2943 cm⁻¹), and CH2 (1400 cm⁻¹) stretching, CO and CC (680-950 cm⁻¹) scission, and CO incorporation (3320-3500 cm⁻¹) into the carbon backbone, indicative of LDPE polymer biodegradation. Enrichment cultures had lower diversity and richness of microbial taxa compared to soil samples, with LDPE as a carbon source having a direct influence on the structure and functioning of the microbial consortia. A total of 26 bacterial and 12 fungal OTU exhibiting high relative abundance and significant associations (IndVal > 0.7, q < 0.05) were identified in the enrichment culture. Bacterial taxa such as unclassified Parvibaculum FJ375498, Achromobacter xylosoxidans, unclassified Chitinophagaceae PAC002331, unclassified Paludisphaera and unclassified Comamonas JX898122, and six fungal species (Galactomyces candidus, Trichosporon chiropterorum, Aspergillus fumigatus, Penicillium chalabudae, Talaromyces thailandensis, and Penicillium citreosulfuratum) were identified as the putative LDPE degraders in the enrichment microbial consortium cultures. PICRUSt2 metagenomic functional profiling of taxonomic bacterial taxa abundances in both landfill soil and enrichment microbial consortia also revealed differential enrichment of energy production, stress tolerance, surface attachment and motility pathways, and xenobiotic degrading enzymes important for biofilm formation and hydrolytic/oxidative LDPE biodegradation. The findings shed light on the composition and structural changes in landfill soil microbial consortia during enrichment with LDPE as a carbon source and suggest novel LDPE-degrading bacterial and fungal taxa that could be explored for management of polyethylene pollution.

Contribution of pollution gradient to the sediment microbiome and potential pathogens in urban streams draining into Lake Victoria (Kenya).

In sub-Saharan Africa (SSA), urban rivers/streams have long been subjected to anthropogenic pollution caused by urbanization, resulting in significantly altered chemical and biological properties of surface water and sediments. However, little is known about the diversity and structure of river microbial community composition and pathogens, as well as how they respond to anthropogenic inputs. High-throughput 16S rRNA amplicon sequencing and PICRUSt predictive function profiling were used in this study to conduct a comprehensive analysis of the spatial bacterial distribution and metabolic functions in sediment of two urban streams (Kisat and Auji) flowing through Kisumu City, Kenya. Results revealed that sediment samples from the highly urbanized mid and lower stream catchment zones of both streams had significantly higher levels of total organic carbon (TOC), total nitrogen (TN), total phosphorous (TP) than the less urbanized upper catchment zone, and were severely polluted with toxic heavy metals lead (Pb), cadmium (Cd), and copper (Cu). Differential distribution of Actinobacteria, Proteobacteria, Chloroflexi, and Verrucomicrobia in sediment bacterial composition was detected along stream catchment zones. The polluted mid and lower catchment zones were rich in Actinobacteria and Proteobacteria, as well as a variety of potential pathogenic taxa such as Corynebacterium, Staphylococcus, Cutibacterium, Turicella, Acinetobacter, and Micrococcus, as well as enteric bacteria such as Faecalibacterium, Shewanella, Escherichia, Klebsiella, Enterococcus, Prevotella, Legionella, Vibrio and Salmonella. Furthermore, PICRUSt metabolic inference analysis revealed an increasing enrichment in the sediments of genes associated with carbon and nitrogen metabolism, disease pathogenesis, and virulence. Environmental factors (TOC, Pb, Cd, TN, pH) and geographical distance as significant drivers of sediment bacterial community assembly, with the environmental selection to play a dominant role. In polluted river catchment zone sediment samples, Pb content was the most influential sediment property, followed by TOC and Cd content. Given the predicted increase in urbanization in SSA, further alteration of surface water and sediment microbiome due to urban river pollution is unavoidable, with potential long-term effects on ecosystem function and potential health hazards. As a result, this study provides valuable information for ecological risk assessment and management of urban rivers impacted by diffuse and point source anthropogenic inputs, which is critical for future proactive and sustainable urban waste management, monitoring, and water pollution control in low-income countries.

Pathogenic and Endosymbiotic Bacteria and Their Associated Antibiotic Resistance Biomarkers in Amblyomma and Hyalomma Ticks Infesting Nguni Cattle (Bos spp.).

Deciphering the interactions between ticks and their microbiome is key to revealing new insights on tick biology and pathogen transmission. However, knowledge on tick-borne microbiome diversity and their contribution to drug resistance is scarce in sub-Saharan Africa (SSA), despite endemism of ticks. In this study, high-throughput 16S rRNA amplicon sequencing and PICRUSt predictive function profiling were used to characterize the bacterial community structure and associated antibiotic resistance markers in Amblyomma variegatum, A. hebraeum, and Hyalomma truncatum ticks infesting Nguni cattle (Bos spp.). Twenty-one (seven families and fourteen genera) potentially pathogenic and endosymbiotic bacterial taxa were differentially enriched in two tick genera. In H. truncatum ticks, a higher abundance of Corynebacterium (35.6%), Porphyromonas (14.4%), Anaerococcus (11.1%), Trueperella (3.7%), and Helcococcus (4.7%) was detected. However, Rickettsia (38.6%), Escherichia (7%), and Coxiellaceae (2%) were the major differentially abundant taxa in A. variegatum and A. hebraeum. Further, an abundance of 50 distinct antibiotic resistance biomarkers relating to multidrug resistance (MDR) efflux pumps, drug detoxification enzymes, ribosomal protection proteins, and secretion systems, were inferred in the microbiome. This study provides theoretical insights on the microbiome and associated antibiotic resistance markers, important for the design of effective therapeutic and control decisions for tick-borne diseases in the SSA region.

Local Geomorphological Gradients and Land Use Patterns Play Key Role on the Soil Bacterial Community Diversity and Dynamics in the Highly Endemic Indigenous Afrotemperate Coastal Scarp Forest Biome.

Southern Afrotemperate forests are small multi-layered and highly fragmented biodiversity rich biomes that support unique flora and fauna endemism. However, little is known about the microbial community and their contribution to these ecosystems. In this study, high throughput sequencing analysis was used to investigate the soil bacterial community structure and function, and understand the effect of local topography/geomorphological formations and land use patterns on a coastal scarp forest. Soil samples were collected from three forest topography sites: upper (steeper gradients, 30-55°; open canopy cover, <30%), mid (less steep, 15-30°; continuous forest canopy, >80%), and lower (flatter gradient, <15°; open canopy cover, 20-65%), and from the adjacent sugarcane farms. Results indicated that forest soils were dominated by members of phyla Proteobacteria (mainly members of α-proteobacteria), Actinobacteria, Acidobacteria, Firmicutes, and Planctomycetes, while Actinobacteria and to a lesser extent ß-proteobacteria and γ-proteobacteria dominated SC soils. The core bacterial community clustered by habitat (forest vs. sugarcane farm) and differed significantly between the forest topography sites. The Rhizobiales (genera Variibacter, Bradyrhizobium, and unclassified Rhizobiales) and Rhodospirallales (unclassified Rhodospirillum DA111) were more abundant in forest mid and lower topographies. Steeper forest topography (forest_upper) characterized by the highly leached sandy/stony acidic soils, low in organic nutrients (C and N) and plant densities correlated to significant reduction of bacterial diversity and richness, associating significantly with members of order Burkholderiales (Burkholderia-Paraburkholderia, Delftia, and Massilia) as the key indicator taxa. In contrast, changes in the total nitrogen (TN), soil organic matter (SOM), and high acidity (low pH) significantly influenced bacterial community structure in sugarcane farm soils, with genus Acidothermus (Frankiales) and uncultured Solirubrobacterales YNFP111 were the most abundant indicator taxa. Availability of soil nutrients (TN and SOM) was the strongest driver of metabolic functions related to C fixation and metabolism, N and S cycling; these processes being significantly abundant in forest than sugarcane farm soils. Overall, these results revealed that the local topographical/geomorphological gradients and sugarcane farming affect both soil characteristics and forest vegetation (canopy coverage), that indirectly drives the structure and composition of bacterial communities in scarp forest soils.

Shifts in Bacterial Diversity During the Spontaneous Fermentation of Maize Meal as Revealed by Targeted Amplicon Sequencing.

Maize meal was allowed to undergo uncontrolled fermentation in the laboratory, in simulation of the traditional method of fermentation as practised in most African households. During the fermentation process, samples were collected daily for 11 days. Physico-chemical analysis of the fermenting slurry and metagenomics analysis of the microbial community using 16S rRNA demonstrated an interrelationship between the changes in the properties of the fermentation environment and the successional interplay of the microbial community. The first 24 h of fermentation at pH of 6.5 was characterised by the proliferation of probiotic Lactobacillus and Bifidobacterium, with their relative abundance being 40.7% and 29.9%, respectively. However, prolonged fermentation and a drop in pH from 5.3 to 3.7 caused a decline and finally an absence of these probiotic bacteria which were replaced by Clostridium spp. with a relative abundance of between 97% and 99% from day 5 to day 11. This study demonstrated that prolonged fermentation of maize meal is not ideally suited for the proliferation of probiotic nutritionally beneficial bacteria.

Azole antifungal resistance in fungal isolates from wastewater treatment plant effluents.

Wastewater treatment plants (WWTPs) can be significant sources of antifungal resistant fungi, which can disseminate further in the environment by getting into rivers together with effluents discharged from WWTPs and pose a risk for human health. In this study, the presence of azole resistance was determined in fungal isolates from treated effluents of two WWTPs using the standard microdilution method from Clinical and Laboratory Standards Institute (CLSI). A total of 41 fungal isolates representing 23 fungal species and 16 fungal genera were obtained. Fungal genera related to the known human and/or plant pathogens such as Aspergillus, Fusarium, and Candida were detected. Among the observed species, the susceptibility of Aspergillus fumigatus and Fusarium oxysporum was tested against fluconazole (FCZ), ketoconazole (KTZ), itraconazole (ITZ), and voriconazole (VCZ). The isolate A. fumigatus was susceptible to KTZ, ITZ, and VCZ, while it showed resistance against FCZ. On the contrast, the isolate F. oxysporum showed resistance to KTZ, ITZ, and VCZ. Comparatively, VCZ showed highest activity against both A. fumigatus and F. oxysporum. Analysis of the gene Cyp51A for the A. fumigatus isolate showed no evidence of drug resistance that could be related to point mutations and/or tandem repeats in the gene. To the best of our knowledge, this is the first susceptibility test study on A. fumigatus and F. oxysporum isolates from the WWTPs of South Africa. In conclusion, this study indicated an urgent need for thorough investigation with larger group of fungal isolates from different regions of South Africa to broadly understand the role of WWTPs in the dissemination of azole antifungal drug resistance.

Physicochemical characterization of the pelotherapeutic and balneotherapeutic clayey soils and natural spring water at Isinuka traditional healing spa in the Eastern Cape Province of South Africa.

Isinuka Springs at Port St Johns in the Eastern Cape Province of South Africa is a traditional spa sacred to the AmaMpondo tribe of the Xhosa speaking people. The bathing pond is considered to have healing powers both spiritually and therapeutically. Hundreds of people flock into the spiritual pond every weekend for both recreational and its spiritual healing power. In this study, we present the metal concentrations of the bathing pond (sediments and water samples), the hole drinking water as well as sediments from a cave situated at the bottom of the hill harbouring the bathing pond. Our results show that the geophagic clays from the cave and bathing pond has elevated concentrations of earth metals (up to 134,506 mg kg-1 for calcium), trace metals (up to 36,272 mg kg-1 for iron) and toxic metals (up to 25 mg kg-1 for lead). The levels of both essential and toxic metals in the drinking water were above the recommended daily limits except for zinc and copper. Aluminium, a metal with antibacterial activity was as high as 71,792 mg kg-1 in pond sediments. Even though the results show elevated concentrations especially for toxic metals, the study observes that the spa remains limited in potential for metal toxipathy because the frequency of contact with the pond is minimal estimated at once a week by traditional healers and once a month for locals while visitors from other parts of the province rarely come back.

Anabaena sp. DyP-type peroxidase is a tetramer consisting of two asymmetric dimers.

DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.

Antioxidant/Prooxidant and antibacterial/probacterial effects of a grape seed extract in complex with lipoxygenase.

In an attempt to determine the antioxidant/prooxidant, antibacterial/probacterial action of flavan-3-ols and procyanidins from grape seeds, pure catechin (CS), and an aqueous grape seed extract (PE), were applied in the absence and presence of pure lipoxygenase (LS) or in extract (LE) to leucocyte culture, Escherichia coli B 41 and Brevibacterium linens, and observed whether there was any effect on lipid peroxidation, cytotoxicity, or growth rate. Short time periods of coincubation of cells with the polyphenols, followed by the exposure to LS and LE, revealed a high level of lipid peroxidation and a prooxidative effect. Longer coincubation and addition of LS and LE resulted in the reversal of the prooxidant action either to antioxidant activity for CS + LS and PE + LS or to the control level for CS + LE and PE + LE. Lipid peroxidation was significantly reduced when cells were exposed to polyphenols over a longer period. Longer exposure of E. coli to CS or PE followed by addition of LS for 3 h resulted in bactericidal activity. Significant stimulatory effect on microbial growth was observed for PE + LS and PE + LE treatments in B. linens, illustrating the potential probacterial activity in B. linens cultures. Lipoxygenase-polyphenols complex formation was found to be responsible for the observed effects.

L-aspartate dehydrogenase: features and applications.

L-amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of L-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD(+)) or nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors. These enzymes have been studied widely because of their potential applications in the synthesis of amino acids for use in production of pharmaceutical peptides, herbicides and insecticides, in biosensors or diagnostic kits, and development of coenzyme regeneration systems for industrial processes. This article presents a review of the currently available data about the recently discovered amino acid dehydrogenase superfamily member L-aspartate dehydrogenase (L-AspDH), their relevant catalytic properties and speculated physiological roles, and potential for biotechnological applications. The proposed classification of L-AspDH on the basis of bioinformatic information and potential role in vivo into NadB (NAD biosynthesis-related) and non-NadB type is unique. In particular, the mesophilic non-NadB type L-AspDH is a novel group of amino acid dehydrogenases with great promise as potential industrial biocatalysts owing to their relatively high catalytic properties at room temperature. Considering that only a few L-AspDH homologs have been characterized so far, identification and prodigious enzymological research of the new members will be necessary to shed light on the gray areas pertaining to these enzymes.

A novel L-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for L-aspartate production.

L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for L-aspartate (L-Asp) and oxaloacetate (OAA) of 127 and 147 U mg(-1), respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T (m) value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K (m) values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The L-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of L-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of L-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of L-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.

Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues.

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H(2)O(2). The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H(2)O(2) stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H(2)O(2), respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H(2)O(2) and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.

Molecular characterization of a novel peroxidase from the cyanobacterium Anabaena sp. strain PCC 7120.

The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35 degrees C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 muM.min(-1), respectively. The apparent K(m) and k(cat)/K(m) values for Reactive Blue 5 were 3.6 muM and 1.2 x 10(7) M(-1) s(-1), respectively, while the apparent K(m) and k(cat)/K(m) values for H(2)O(2) were 5.8 muM and 6.6 x 10(6) M(-1) s(-1), respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to approximately 50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.