Experimental studies of two-phase anaerobic digestion of corn steep liquor in semi-continuous automatic and semi-automatic modes of operation of a cascade of two anaerobic bioreactors with monitoring ...and control systems were performed. Corn steep liquor—a waste product from the process of treating corn grain for starch extraction—was used as a substrate in the process of anaerobic digestion with simultaneous hydrogen and methane production. The daily yields of biohydrogen in bioreactor 1 of the cascade (with a working volume of 8 dm3) are variable. In good operation, they are in the range of 0.7 to 1.0 L of biogas from a 1 dm3 working volume of the bioreactor, and the optimal pH is in the range of 5.0–5.5. The concentration of hydrogen in the biogas from the hydrogen bioreactor 1 is in the range of 14–34.7%. The daily yields of biomethane in bioreactor 2 of the cascade (with a working volume of 80 dm3) vary in the range 0.4 to 0.85 L of biogas from a 1 dm3 working volume of the bioreactor, and the concentration of methane in the biogas from bioreactor 2 is high and remains practically constant (in the range 65–69%). At a dilution rate of 0.4 day−1 and an organic loading rate of 20 gL for bioreactor 1, respectively, and a dilution rate of 0.05 day−1 for bioreactor 2, the best results were obtained. The computer control system is presented. Some energetical considerations were discussed.
Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, ...etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed β-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.
Reptiles are known to be asymptomatic carriers of various zoonotic pathogens. A number of Gram-negative opportunistic commensals are causative agents of bacterial infections in immunocompromised or ...stressed hosts and are disseminated by reptiles, whose epidemiological role should not be neglected. Since most studies have focused on exotic species, in captivity or as pet animals, the role of wild populations as a potential source of pathogens still remains understudied. In the present study, we isolated a variety of Gram-negative bacteria from the cloacal microbiota of free-living lizard and tortoise hosts (Reptilia: Sauria and Testudines) from the Bulgarian herpetofauna. We evaluated their pathogenic potential according to their antibiotic susceptibility patterns, biofilm-forming capacity, and extracellular production of some enzymes considered to play roles as virulence factors. To our knowledge, the phenotypic manifestation of virulence factors/enzymatic activity and biofilm formation in wild reptile microbiota has not yet been widely investigated. All isolates were found to be capable of forming biofilms to some extent and 29.6% of them could be categorized as strong producers. Two strains proved to be excellent producers. The majority of the isolated strains showed extracellular production of at least one exoenzyme. The most pronounced pathogenicity could be attributed to the newly isolated
strain due to its multiresistance, excellent biofilm formation, and expression of exoenzymes.
Studies on the gut microbiome of free-living reptiles in Europe are generally fragmentary and still missing in Bulgaria. We aimed to identify and compare the fecal microbiota profiles of five ...syntopic lizard species from three families: the European green lizard (Lacerta viridis), the common wall lizard (Podarcis muralis), the meadow lizard (Darevskia praticola) (Lacertidae), the European snake-eyed skink (Ablepharus kitaibelii) (Scincidae), and the European slow worm (Anguis fragilis) (Anguidae), which coinhabit a low mountainous area in the western part of the country. A high-throughput sequencing of the hypervariable V3-V4 region of the 16S rRNA gene, performed on the Illumina HiSeq2500 platform, was used. The core microbiota of lizard hosts seems to be species-specific. A dynamic phyla proportion between hosts was found. The richest alpha diversity was observed in D. praticola, and the lowest alpha diversity was observed in P. muralis and A. fragilis. Within the three lacertids, the microbiota of D. praticola and L. viridis were more closely related to each other than they were to those of P. muralis. Sharing a largely common trophic resource (all species except A. fragilis are mainly insectivorous) was not an indication of similarity in their gut microbial communities.
The fungal strain, Penicillium griseofulvum P29, isolated from a soil sample taken from Terra Nova Bay, Antarctica, was found to be a good producer of sialidase (P29). The present study was focused ...on the purification and structural characterization of the enzyme. P29 enzyme was purified using a Q-Sepharose column and fast performance liquid chromatography separation on a Mono Q column. The determined molecular mass of the purified enzyme of 40 kDa by SDS-PAGE and 39924.40 Da by matrix desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis correlated well with the calculated mass (39903.75 kDa) from the amino acid sequence of the enzyme. P29 sialidase shows a temperature optimum of 37 °C and low-temperature stability, confirming its cold-active nature. The enzyme is more active towards α(2 → 3) sialyl linkages than those containing α(2 → 6) linkages.
Based on the determined amino acid sequence and 3D structural modeling, a 3D model of P29 sialidase was presented, and the properties of the enzyme were explained. The conformational stability of the enzyme was followed by fluorescence spectroscopy, and the new enzyme was found to be conformationally stable in the neutral pH range of pH 6 to pH 9. In addition, the enzyme was more stable in an alkaline environment than in an acidic environment. The purified cold-active enzyme is the only sialidase produced and characterized from Antarctic fungi to date.
•The purified cold-active enzyme sialidase is isolated from fungal strain, Penicillium griseofulvum P29, isolated from a soil sample from Terra Nova Bay, Antarctic.•The molecular mass of the purified enzyme is 39924.40 Da.•The enzyme is more active towards α(2. → 3) sialyl linkages than those containing α(2 → 6) linkages.•The new enzyme is conformationally stable in the neutral pH range of pH 6 to pH 9.
Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application ...in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain
P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain
P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.
Although investigators have been studying the cold-shock response in a variety of organisms for the last two decades or more, comparatively little is known about the difference between antioxidant ...cell response to cold stress in Antarctic and temperate microorganisms. The change of environmental temperature, which is one of the most common stresses, could be crucial for their use in the biotechnological industry and in ecological research. We compared the effect of short-term temperature downshift on antioxidant cell response in Antarctic and temperate fungi belonging to the genus
Penicillium
. Our study showed that downshift from an optimal temperature to 15° or 6°C led to a cell response typical of oxidative stress: significant reduction of biomass production; increase in the levels of oxidative damaged proteins and accumulation of storage carbohydrates (glycogen and trehalose) in comparison to growth at optimal temperature. Cell response against cold stress includes also increase in the activities of SOD and CAT, which are key enzymes for directly scavenging reactive oxygen species. This response is more species-dependent than dependent on the degree of cold-shock. Antarctic psychrotolerant strain
Penicillium
olsonii
p14 that is adapted to life in extremely cold conditions demonstrated enhanced tolerance to temperature downshift in comparison with both mesophilic strains (Antarctic
Penicillium
waksmanii
m12 and temperate
Penicillium
sp. t35).
Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR ...(leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds α-tubulin and promotes α/β dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds α-tubulin and MTs, and functions in suppression of benomyl sensitivity of
pac2Δ
mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics.
pac2Δ
mutants are sensitive to misfolded protein stress. This is suppressed by ectopic
PAC2
with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.
Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, ...etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed β-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.
Compared to other reptile groups in Europe, lizards have generally been neglected and understudied in terms of microbiota research. In this study, we aimed to isolate, identify and characterize the ...aerobic cloacal microflora of wild-dwelling lizard hosts. We examined a total of 86 individuals from five species belonging to three families: the European green lizard (Lacerta viridis), the common wall lizard (Podarcis muralis), the meadow lizard (Darevskia praticola) (Lacertidae), the European snake-eyed skink (Ablepharus kitaibelii) (Scincidae) and the European slow worm (Anguis fragilis) (Anguidae) which co-occur in a low-mountain region in Western Bulgaria. In general, a similar composition of the resident microbial communities in the cloaca was found, accompanied by variation in the relative abundance of some bacterial taxa between the lizard species. A variety of Gram-negative and Gram-positive bacteria was isolated from the cloacal samples. Some of these bacteria are also known as opportunistic pathogens, both for hosts and humans. The bacterial species Hafnia alvei, Pseudomonas aeruginosa, Klebsiella oxytoca and representatives of Enterobacter spp., Citrobacter spp. and Enterococcus spp. were among the most prevalent.
