Applied Biochemistry and Microbiology最新文献

The biotechnological production of citric acid (CA) is one of the largest and fastest-growing sectors in the global biotechnology market. It is produced in crystalline form and as citrate salts and is widely employed in the food industry, household chemicals, cosmetics, metallurgy, and the petroleum extraction sector. Global CA production exceeds two million tons per year and is increasing by approximately 4% annually. Traditional biotechnological CA production relies on the utilization of mycelial fungi, specifically Aspergillus niger as the producer. Various alternative CA producers, including yeast and bacteria-based systems, have been described. However, from an economic standpoint, they lag behind industrial Aspergillus niger producers. Among the most promising, environmentally friendly, and high-tech CA producers are strains of the yeast Yarrowia lipolytica. The development of competitive biotechnologies for CA production using Y. lipolytica yeast strains with glucose as a carbon source has become feasible due to advancements in understanding the genetic control of CA transport and metabolism within the cell.

The second part of this review is concerned with analysis of the possible effect of flavonoids on the SARS-CoV-2 enzymes directly involved in the infectious cycle of the virus, taking into account the peculiarities of host cell functioning. The results of virtual screening of flavonoids in conditions of molecular docking and molecular modeling are considered in this review. In the experiments in silico, binding of flavonoids to three SARS-CoV-2 enzymes was analyzed: 3-chymotrypsin-like protease (3CL^pro), papain-like protease (PL^pro), and RNA-dependent RNA polymerase (RdRp). A high affinity of binding of flavonoids to these viral proteins (most pronounced for flavonols, flavones, and flavan-3-ols and often superior to the control antiviral drugs) was demonstrated. The process of penetration of SARS-CoV-2 into the cell and host cell components involved in the infectious cycle of the virus, as well as the possible effect of flavonoids on them, are considered separately.

Abstract—Calcium–magnesium silicate ceramic diopside is an effective carrier of BMP-2 and, in combination with a bovine demineralized bone matrix as a scaffold, can induce effective reparative osteogenesis on the model of craniotomy in mice. It was of interest to investigate the efficacy of combined usage of BMP-2-loaded diopside with recombinant spidroins for the induction of osteogenesis. For this purpose, porous implants were prepared based on recombinant spidroin rS1/9, into which BMP-2 was introduced either as BMP-2 loaded diopside particles suspended in hyaluronic acid or by adsorption. Neo-osteogenesis with bone marrow formation occurred in both groups with BMP-2, most pronounced in the group with BMP-2 and diopside on the model of critical size defects in the cranium in mice. Thus, the use of 3D scaffolds based on recombinant spidroin with BMP2-loaded diopside particles in hyaluronic acid can lead to effective induction of osteogenesis. The spidroin scaffolds themselves represent a sufficiently effective carrier for BMP-2.

A method for molecular serotyping of Bacillus turingiensis strains has been developed. It is based on comparison of nucleotide sequences of conserved regions of the flaA gene in five B. turingiensis serovars: sotto, kurstaki, israilensis, berliner, and morrisoni. For amplification of the target DNA fragment, a set of primers universal for all studied serovars of B. turingiensis was designed. To assign a strain to a particular serovar, a new algorithm based on the degree of homology of the conserved flaA region was developed. Validation of the method was carried out on 11 strains of B. turingiensis from the collection of the National Bioresource Center All-Russia National Collection of Industrial Microorganisms (VKPM). The results obtained by molecular serotyping fully coincided with the data of serological methods.

The first part of this review is devoted to an analysis of the beneficial effect of flavonoids on the prevention, course, and outcomes of COVID-19. In addition to the well-known antioxidant, anti-inflammatory, immunomodulatory, antithrombotic, and antitumor effects, flavonoids, secondary metabolites of many plants, exhibit antiviral activity and low cytotoxicity. The basic stages of SARS-CoV-2 replication and COVID-19 pathogenesis are considered to identify possible targets for flavonoids. The effect of these polyphenolic compounds on SARS-CoV-2 both in in vitro systems and in animal models that adequately reproduce COVID-19 symptoms have been analyzed. The results of the first clinical trials on the prevention of this disease and the feasibility of using flavonoids, mainly quercetin, as adjuvant therapy are discussed.

Surfactin is a powerful surfactant of biological origin with a wide range of potential applications in industry and medicine. The main factor limiting its active utilization is the high cost of production. Due to the complexity of the structure, the chemical synthesis of surfactin is unprofitable, so the main approach to its production is microbiological synthesis. This work demonstrated for the first time the ability of B. subtilis NCIB 3610 to synthesize surfactin. This strain possesses properties lost by most B. subtilis model laboratory strains. The nucleotide sequences of genomes of B. subtilis NCIB 3610 and B. subtilis PY79 strains have been obtained by nanopore sequencing. А comparative analysis of the obtained genomes with genome of B. subtilis 168, genetically modified derivatives of which are patented surfactin producers, has been performed. No mutations affecting surfactin biosynthesis were found in the NCIB 3610 strain, while in the PY79 strain mutations were identified in genes involved in cellular processes competing with biosynthesis of the surfactin. Thus, the results obtained in this work make it possible to consider B. subtilis NCIB 3610 and PY79 as alternative base strains for the genetic engineering design of surfactin superproducers.

In order to increase the lytic activity of the enzyme, structures of the recombinant modified CHAP domain of the phage K endolysin to Staphylococcus aureus have been developed, including (starting from the N-terminus) a sequence of various cationic peptides HB(X) fused with the sequence of the CHAP domain through the GSG₄S linker region. Genetic engineering constructs encoding the new HB(X)-CHAP were obtained, which were cloned and expressed in the recipient strain E. coli BL21(DE3). All variants of HB(X)-CHAP, as well as the control variant of the CHAP domain, were isolated and purified using a single technique involving a combination of cation and anion exchange chromatography. The lytic activity of the obtained enzymes was studied by the turbidimetric method using an autoclavable culture of S. aureus. For the two most promising HB(X)-CHAP variants from the point of view of further use, the main physicochemical characteristics are determined. It was shown that the presence of the GSG₄S linker site in the structure of the molecule led to at least a twofold increase in the activity in the lysis of S. aureus cells, while the cationic peptides did not have a positive effect on the lytic activity of endolysin against Staphylococcus aureus. The obtained data may allow a rational approach to the issue of choosing an approach to optimizing the structure in the future and will expand the possibilities for designing endolysins in order to create an effective drug.

The adaptation of the N. crassa lipoxygenase (LOX) in response to heat (45°C) and cold (4°C) shock was studied. The difference was revealed in the dynamics of the LOX activity depending on the growing conditions of the mycelium. After incubation of the surface culture at 45°C, a gradual increase in the specific activity of the enzyme was observed with maximum at 2–3 h, followed by a subsequent decrease to the initial level. Under the same conditions, in a submerged culture, a decrease in the LOX activity was observed after 5 min; however, after 1 h of incubation, the enzyme activity also reached the initial level. The sensitivity of the N. crassa LOX to elevated temperatures is very high, since it is noted only in a narrow temperature range: the effect was detected at 45^oC; however, the enzymatic activity did not change in the culture incubated at 42°C, but, on the other hand, LOX was completely inactivated in the mycelium at 48°C. When the fungal cells were exposed to cold, the specific LOX activity increased after 1 h, then decreased to the initial level (2–3 h) and increased again, reaching a maximum after 18 h. When two stress factors, cold and starvation, acted simultaneously on the N. crassa cells, the treatment with cold had a decisive effect on the LOX activity, which was especially noticeable after 8 h of incubation.

This paper presents the results of a study of the dynamics of growth and metabolism of Lacticaseibacillus casei production strains in model milk media and semi-hard cheeses. In dairy media under optimal conditions, the maximum number of viable cells of the studied Lacticaseibacillus casei strains was achieved on day 3; an increase in titratable acidity continued for up to 7‒10 days. In model dairy media simulating cheese ripening conditions, the growth and metabolism (glycolysis, proteolysis, and production of volatile aroma compounds) of Lacticaseibacillus casei slowed down. In semi-hard cheeses, the Lacticaseibacillus casei culture (as an independent acid-generating starter component), did not provide the required intensity of acid formation during the production process. However, as an adjunct culture, it intensified proteolysis and the processes of gas and aroma formation during ripening, thus, improving the organoleptic characteristics of cheeses.

With high-throughput sequencing of the variable region V3–V4 of the 16S rRNA gene, the study of the full phylogenetic composition of microbial communities developed on the surface of titanium plates exposed in the water column of the coastal zone of the Black and White Seas was carried out. The presence of potentially corrosive microorganisms from various physiological groups, such as sulfate-reducing bacteria, acidophilic iron-oxidizing bacteria and archaea, sulfur-oxidizing and nitrifying bacteria, was shown in the fouling. In the fouling of titanium plates exposed in the Black Sea, the most common microorganisms were uncultivated sulfate-reducing bacteria of the order Desulfotomaculales, which accounted for 8.13% of all 16S rRNA gene sequence reads, as well as acidophilic iron-oxidizing bacteria of the genera Acidiferrobacter (5.47%), Acidithiobacillus (4.52%), and Acidiphilium (2.55%). Acidophilic archaea accounted for up to 7.97% of all reads. In the fouling of titanium plates exposed in the White Sea, the most common were also acidophilic bacteria from the orders Acidiferrobacterales and Acidithiobacillales (7.68%), as well as acidophilic archaea from the order Thermoplasmatales (7.43%). Uncultivated sulfate-reducing bacteria of the order Desulfotomaculales were also represented in relatively high numbers (6.61% of all reads).