Applied Biochemistry and Microbiology最新文献

Plants of the duckweed family (Lemna, Spirodela) are actively used as expression platforms for the production of recombinant proteins due to a number of their advantages over other crops (small size, rapid vegetative reproduction, and high protein content in tissues). Rootless wolffia (Wolffia arrhiza (L.) Horkel ex Wimm) is a promising producer from the Lemnaceae family. Current Wolffia transformation protocols have an efficiency of approximately 0.36%. Based on transient expression, we have identified the most efficient Agrobacterium strain, EHA105, for the W. arrhiza transformation. An effective balance of growth regulators was selected for use at the first passage of cultivation and selection of transformed explants: 2,4-dichlorophenoxyacetic acid, 2.5 mg/L and 6-benzylaminopurine, 1.5 mg/L. The transformation efficiency of W. arrhiza was 0.54%, which is 1.5 times higher than in known protocols.

The ability of the soil-inhabiting ascomycetes Lecanicillium aphanocladii, Talaromyces sayulitensis, Trichoderma harzianum and Fusarium oxysporum to use polyethylene terephthalate (PET) as a sole carbon and energy source has been shown. Utilization of PET by the studied fungi, except for L. aphanocladii, was accompanied by the production of emulsifying compounds. All fungi exhibited the activity of cutinase, the key PET depolymerization enzyme, and a number of oxidoreductases, which apparently catalyze the oxidation of the resulting products: peroxidases in F. oxysporum and T. harzianum, as well as peroxidases and oxidases in L. aphanocladii and Tal. sayulitensis. The data we obtained can be used to develop environmental biotechnologies and contribute to understanding of the processes of degradation/conversion of plastics in natural ecosystems.

To detect and differentiate current influenza B viruses of both evolutionary lineages, the sensitivity of a new variant of microcultural enzyme immunoassay (cell-ELISA) has been assessed using a panel of 16 monoclonal antibodies (mAbs) specific to hemagglutinin of influenza B viruses. Nasopharyngeal samples from patients with acute respiratory viral infections (ARVI) were analyzed. The cell-ELISA was tested for the detection of influenza B viruses in 192 PCR-positive clinical samples. The highest sensitivity in detection and differentiation of influenza B viruses of the Yamagata and Victoria evolutionary lineages was shown for 5B11 and 5B7 mAbs, respectively. The detection of Yamagata influenza B viruses with cell-ELISA in PCR-positive clinical samples obtained in 2018 showed a lower sensitivity (61.7%) compared to traditional methods, that is, isolation of viruses in cell culture with subsequent determination of their belonging to the evolutionary lineage in the hemagglutination inhibition test (HIT) using strain-specific immune sera of animals (95.0%). The sensitivity of cell-ELISA while detecting the more variable Victoria viruses depended on the pathogen circulation time. The level of virus identification in samples collected in 2016–2018 was comparable for cell-ELISA and the traditional method (71–80%). Some of the current viruses circulating in 2020‒2021 did not interact with immune sera obtained to the corresponding reference strains, which made it difficult to identify them by HIT. Thus, the viruses circulating at that time were identified using cell-ELISA with a higher frequency than by isolation and subsequent HIT (59.5 vs. 35.7%, p < 0.05). In addition to the identification of the evolutionary lineage, the use of mAb 9B5 in the cell-ELISA made it possible to detect deletion variants of the Victoria influenza B viruses circulating in Russia in 2018‒2022.

An instrument-free method for detecting the ability of Vibrio cholerae strains to produce toxin has been developed. It is based on dot-ELISA performed on dipstiks. Dipsticks require 100 times less monoclonal antibodies than microplate ELISA.

Based on the Komagataella kurtzmanii yeast, a strain producing Tribolium castaneum recombinant procathepsin (rpTcCathL) has been obtained; this strain provided biosynthesis and secretion of at least 0.5 g/L of the target protein as a result of cultivation in flasks on a medium of complex composition. It was determined that the pH of the yeast culture medium (pH ≥ 6.5) is the key factor that influences the biosynthesis and accumulation of procathepsin. A new chromatographic purification scheme was developed, which allows one to obtain recombinant procathepsin with a yield of about 30% and a purity of over 95%. It was shown that complete autocatalytic activation of the proenzyme is achieved within no more than 30 min at a temperature of 37°C and pH 4.0–4.5. The resulting recombinant cathepsin and activated cathepsin at a concentration of 0.5 mg/mL and below could be stored without detectable changes at a temperature of 4°C for at least 2 weeks. Thus, the research made it possible to develop the fundamentals of the technology for obtaining and storing recombinant preparations of the highly purified major digestive cathepsin L from T. castaneum in the form of a proenzyme and mature cathepsin L.

Two genome editions have resulted in the CHO cell line 4BGD with homozygous knockouts of the BAK1, BAX, DHFR and GLUL genes, and overexpression of the BCL2 and BECN1 genes. This line is capable of long-term growth without changing the culture medium and exhibits a fivefold increase in the integral cell density compared to the parental cell line under the same cultivation regime. Overexpression of the BCL2, BECN1 gene pair led to a significant increase in cell density on days 6‒10 of cultivation compared to the cell line with a knockout of the BAK1, BAX, DHFR and GLUL genes, while the integral viable cell density increases by 34%.

Cloning and expression of the full-length endo-processive-type xyloglucanase from the Trichoderma reesei (TrXeg74A) fungus, as well as its catalytic domain TrXeg74A-CD, in the Penicillium verruculosum B1-537 recipient strain have been carried out. P. verruculosum is a highly effective producer of cellulases. The levels of protein secretion after culturing the obtained recombinant strains in a laboratory fermenter were 35.4 and 31.4 g/L, respectively. TrXeg74A accounted for at least 30% of the total protein, while TrXeg74A-CD was expressed to a much lesser extent. Both forms of the recombinant enzyme were isolated in purified state and their properties were studied. TrXeg74A and TrXeg74A-CD were characterized by a similar degree of processivity when exposed to tamarind xyloglucan and the same Michaelis constant (0.35-0.38 g/L), close to that for the native enzyme (0.30 g/L), while the catalytic constant for TrXeg74A-CD was 1.5 times higher than the corresponding parameter for full-length xyloglucanase. The obtained new recombinant P. verruculosum strains can be useful in the development of composite enzyme preparations for efficient hydrolysis of renewable lignocellulosic raw materials.

The metabiotic properties of six Lactobacillus acidophilus strains, included in symbiotic starter cultures for the production of probiotic lactic acid products, have been studied. The antimicrobial activity spectrum of these strains in relation to contaminants that can develop during fermentation was studied. The time of cultivation of strains required for the maximum accumulation of cells and antimicrobial metabolites was determined. Activity was compared against test microorganisms of living lactobacillus cultures and lysates formed during fermentation or under stressful conditions of the gastrointestinal tract. The bactericidal activity of a number of strains in the lysates was up to 40% higher than in the culture broth. Fungicidal activity was manifested at the later stages of the lactobacilli growth. The most active strains can be recommended as probiotic cultures with metabolic properties suitable for inclusion in complex starter cultures when creating functional food and pharmaceutical products.