Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine–metallo enzyme type.

Biotechnology of Microorganisms Growing– Fundamentals for the Development of a Litter Biodestructor

The purpose of the research work was to select the optimal conditions for the cultivation of microorganisms. As a result of the conducted research work, the modes of growing a nitrogen-fixing culture and a microorganism with high enzymatic activity were selected and worked out. At the same time, the optimal conditions for the cultivation of Azotobacter sp were determined – the temperature optimum for cell accumulation was 30°C, for increased polysaccharide production 20 °C, aeration within 5-10 l/l/min, agitator speed-150 rpm, pH value within 6.0±0.2 units, which allowed to achieve a cell titer of at least 1.0×109 CFU/ml. A cost-effective nutrient medium was selected for growing Pseudomonas sp. molasses-autolysate medium and optimal conditions for growing the culture: cultivation temperature 30-32 °C, aeration 1.0-1.5 l/l/ min, agitator speed 150-200 rpm, pH 6.8-7.2 units, sub-titration 5.0 % KOH, defoaming with adecanol, cultivation time-72 hours, which allowed to achieve a cell titer of at least 1.0×109 CFU/ml.

Overexpression of thermophilic a -amylase in Bacillus licheniformis using a high efficiency chromosomal integration and amplification strategy

Background: The production of industrially important enzymes depends on the development of genetically stable and high-yielding microorganisms. In order to simplify the development of strains able to efficiently overproduce enzymes, a new strategy based on chromosomal integration and amplification in Bacillus sp. was developed.Results: A thermosensitive replicable plasmid pUB-MazF and an integrated expression plasmid pUB'-Ex1 were constructed. pUB-MazF with a thermo-sensitive replicase RepF encoded by gene repF can self-replicate in Bacillus sp. if the cultivation temperature is no more than 35 oC and if the MazF encoded by mazF is not expressed. mazF is controlled by an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducing promoter and MazF is toxic to the cell. When IPTG is present and the MazF encoded by mazF in pUB-MazF is expressed, the host cell should lose the plasmid to survive. At a cultivation temperature over 37 oC, pUB-MazF will not well replicate and is not stable. pUB'-Ex1 has a disrupted repF and cannot self-replicate but can replicate when pUB-MazF is present. By using this approach, pUB'-Ex1 and constructs can replicate and will be forced to integrate into the host chromosome at 37oC or over when a selectable marker is present. The host was cured by MazF expression after induction with IPTG. Bacillus licheniformis BL-UBM integrated with pUB-MazF was transformed with pUB'-amyS derived from pUB'-Ex1 by in-frame cloning of amyS encoding a thermophilic α-amylase from Geobacillus stearothermophilus ATCC 31195. B. licheniformis BL-UBM transformed with pUB'-amyS was cultivated at 42oC with 1 mmol/l IPTG and 500 μg/ml kanamycin and the recombinants expressing high α-amylase activities were selected. All recombinants evaluated were genetically stable. The highest yield of α-amylase of 50 753 U/ml was produced by recombinant BLiS-002 carrying five copies of amyS in a 50-l fermenter. Conclusion: The strategy developed in this study has potential application for convenient and rapid development of strains overexpressing industrially important enzymes.

Feather Biodegradation For Feather Protein Lysate/ Feather Meal Mutual Formations: Optimization Using Crude YNDH Protease/Keratinase; Enzyme Partial Purification and Characterization

Incubation parameters used for the production of a protein lysate from enzymatically degraded waste feather using crude keratinase produced by laceyella sacchari YNDH was optimized using RSM, amino acids quantification was estimated as well. The optimization resulted in total soluble protein 2089.5µg/ml. The optimal conditions were, time 20.2h, feather concentration 3g% and keratinase activity 24.5U/100ml at pH, 10; and cultivation temperature 50oC. The FPL was found enriched with essential and rare amino acids.In parallel, this YNDH enzyme group had been partially purified and some characteristics of it were studied. Crude enzymes were first concentrated with Amicon Ultra 10k centrifugal filter, and then concentrated proteins were applied to "QFF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranged 6 to 10 kDa. The maximum enzyme activity was observed at 70°C and pH 10.4 when measured by both casein and keratin azure as substrates. Interestingly, keratinolytic activity of this group was not affect by EDTA, PMSF and DTT. Generally the overall characters of this group protease/keratinase nearly the same when its activity was measured with both substrates suggesting that all these 3 protein bands working together as a group of keratinases.

Temperature regulates synaptic subcellular specificity mediated by inhibitory glutamate signaling

Environmental factors such as temperature affect neuronal activity and development. However, it remains unknown whether and how they affect synaptic subcellular specificity. Here, using the nematode Caenorhabditis elegans AIY interneurons as a model, we found that high cultivation temperature robustly induces defects in synaptic subcellular specificity through glutamatergic neurotransmission. Furthermore, we determined that the functional glutamate is mainly released by the ASH sensory neurons and sensed by two conserved inhibitory glutamate-gated chloride channels GLC-3 and GLC-4 in AIY. Our work not only presents a novel neurotransmission-dependent mechanism underlying the synaptic subcellular specificity, but also provides a potential mechanistic insight into high-temperature-induced neurological defects.

The influence of cultivation conditions of promising Bacillus subtilis strains on their ability to produce antifungal metabolites

Antifungal compounds, including surfactin and iturin A, are accumulated by B. subtilis BZR336g strain at the cultivation temperature of 20.0-25.0 °C and the nutrient medium acidity pH8.0, for B. subtilis BZR517 strain these parameters are 30.0-35.0 °C and pH8.0-10.0, respectively.

INVESTIGATION OF PRODUCTION-VALUABLE AND TECHNOLOGICAL PROPERTIES OF LEUCONOSTOCS FROM THE REPUBLICAN COLLECTION OF INDUSTRIAL STRAINS OF STARTER CULTURES AND THEIR BACTERIOPHAGES

We investigated the industrial important properties of leuconostocs that make them possible to use it in starter cultures for the dairy industry (fermented and gas-forming activities, resistance for NaCl, pH, sensitivity to bacteriophages, antagonistic activity against coliform bacteria). We have developed the nutritional medium for their cultivation with the justification of the carbohydrate component and identified cultivation temperature of microorganisms.

Production and partial characterization of a crude cold-active cellulase (CMCase) from Bacillus mycoides AR20-61 isolated from an Alpine forest site

Purpose To evaluate the production of a cold-active CMCase (endoglucanase) by Bacillus mycoides AR20-61 isolated from Alpine forest soil and to characterize the crude enzyme. Methods After studying the effect of cultivation parameters (medium composition, temperature, NaCl concentration, pH) on bacterial growth and enzyme production, the crude enzyme was characterized with regard to the effect of pH, temperature, and inhibitors on enzyme activity and stability. Result Optimum growth and enzyme production occurred at 20–25 °C, pH 7, and 1–1.5% (w/v) CMC. Despite high biomass production over the whole growth temperature range (10–35 °C), enzyme production was low at 10 and 35 °C. CMC concentration had a minor effect on growth, independent of the growth temperature, but a significant effect on CMCase production at temperatures ≥ 20 °C. The crude enzyme was active over a broad temperature range (0–60 °C); the apparent optimum temperature for activity was at 40–50 °C. The cultivation temperature influenced the effect of temperature on enzyme activity and stability. A significantly higher thermosensitivity of the enzyme produced at a cultivation temperature of 10 °C compared to that produced at 25 °C was noted at 50 and 65 °C. The enzyme was highly active over a pH range of 4–6 and showed optimum activity at pH 5. No activity was lost after 60 min of incubation at 30 °C and pH 4–9. The CMCase was resistant against a number of monovalent and divalent metal ions, metal-chelating agents, and phenol. Conclusion The CMCase produced by the studied strain is characterized by high activities in the low temperature range (down to 0 °C) and acidic pH range, high stability over a broad pH range, and high resistance against a number of effectors. Our results also demonstrate the different, independent roles of temperature in bacterial growth, enzyme production, nutrient requirements during enzyme production, and enzyme characteristics regarding thermosensitivity, which has not yet been described for cellulases.

PSIX-14 Influence of living yeast of the genus Rhodotorula against probiotic and pathogenic bacteria

The aim of the research was to study the effect of live yeast Rhodotorula spp. (LYR) on the growth and development of microorganisms and microbial profiles in batch culture. A liquid medium was used to prepare the inoculum (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract). The study in vitro was a 3×5 factorial arrangement, including low (5.0) and high media pH (7.5) and temperature (from 20 to 39 ° C). The treatments were LYR with concentrations from 1·103 to 1·1011 CFU/ml. The bacteria were selected: Lactobacillus casei subsp. Rhamnosus ATCC 7469, Bifidobacterium breve ATCC 15701, E. coli ATCC 25922, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 14990. The analyses were carried out separately for each culture and LYR concentration by broth dilution. For incubation were used Saburo agar for LYR and differential-diagnostic media for each bacterial species. After incubation, there was a lack of growth of E. coli, S. aureus and S. epidermidis in LYR concentrations from 1·109 to 1·1011 CFU/ml at the temperature of 28±0.5 °С, while the number of yeast cells did not decrease. At the temperature above 32±0.5 °C there was a decrease in the amount of LYR by more than 1000-fold in the samples of all cultures. The presence of LYR in the medium led to an increase (p≤0.001) in the number of L. casei at pH 5.0 and temperature less than 30±0.5 °С. Unlike L. casei, the viability of the B. breve culture has decreased (P ≤0.05) by 200 times at pH 5.0, but has increased (p≤0.001) by more than 1000 times at pH 7.0. The optimal cultivation temperature was 36±0.5 °С. These results indicate the multidirectional effect of LYR on microorganisms in vitro. This work was supported by the Ministry of Science and Higher Education of Russia (topics GZ AAAA-A18-118021590136-7).