Applied Biochemistry and Microbiology

Belenkaya, S. V.; Balabova, D. V.; Belov, A. N.; Koval, A. D.; Shcherbakov, D. N.; Elchaninov, V. V.

doi: 10.1134/S0003683820040031pmid: N/A

The review discusses the basic biochemical properties of the known recombinant chymosins that are currently used in cheesemaking or may potentially be used as industrial milk coagulants. The parameters of the Michaelis–Menten kinetics, milk-clotting activity, proteolytic activity, specificity, and the dependence of coagulation ability on pH and calcium-ion concentration are considered.

Khmelevtsova, L. E.; Sazykin, I. S.; Azhogina, T. N.; Sazykina, M. A.

doi: 10.1134/S0003683820030059pmid: N/A

This review considers papers on the diversity of the structure and functions of prokaryotic peroxidases. The most significant groups of peroxidases found in bacteria, such as catalases (Kats), catalase peroxidases (CPs), di-heme cytochrome c peroxidases (DiHCcPs), dye-decolorizing peroxidases (DyPs), are described. The data on their use in biotechnology are presented. Their participation in the biodegradation of recalcitrant substrates by microorganisms and the possibility of their use in bioremediation are discussed.

Ostrik, A. A.; Salina, E. G.; Skvortsova, Y. V.; Grigorov, A. S.; Bychenko, O. S.; Kaprelyants, A. S.; Azhikina, T. L.

doi: 10.1134/S0003683820040122pmid: N/A

The regulatory mechanisms of pathogenic bacteria contribute to their survival under stress conditions in the host environment, which allows them to avoid the immune system of the macroorganism. Small, noncoding RNAs were previously found to regulate some metabolic processes of Mycobacterium tuberculosis. We have revealed that the viability of M. tuberculosis in host-like conditions depends on the expression level of small RNAs. Strains overexpressing small RNA MTS1338 and MTS0997 in M. tuberculosis were produced, and their survival under stressful conditions in vitro and in infected human macrophages were studied. We found that overexpression of the small, noncoding RNA MTS1338 increased bacterial resistance to the stressful effects of hydrogen peroxide, nitric oxide, an acidic environment, and a long-term lack of nutrients at different growth phases and contributed to the viability of bacteria in infected macrophages. An overexpression of the small, noncoding RNA MTS0997 did not significantly affect cell viability under stress conditions. Thus, the two studied small RNAs play different roles in the mycobacterial adaptation to intracellular stresses during infection.

Nesterova, L. Yu.; Tsyganov, I. V.; Tkachenko, A. G.

doi: 10.1134/S0003683820040110pmid: N/A

Biogenic polyamines affect the properties of surface of Mycobacterium smegmatis cells. The presence of spermidine and spermine in the culture medium had a significant effect on the cell-surface charge and sliding motility of mycobacteria but did not affect the cell-surface hydrophobicity. Cell aggregation in M. smegmatis and the capacity for biofilm formation increased under polyamine treatment. Polyamines also decreased the antibiotic susceptibility of both planktonic forms and biofilms. For the first time, spermine has been shown to enhance the antimycobacterial activity of rifampicin, which is of interest for the treatment of mycobacterial infections.

Kim, P.; Kaszuba, A.; Jang, H.-I.; Kim, Y.-I.

doi: 10.1134/S0003683820040092pmid: N/A

The cyanobacterial circadian clock is the most well-understood and simplest biological time-keeping system. Its oscillator consists of three proteins: KaiA, KaiB, and KaiC. When combined together in a test tube, the Kai proteins produce a free-running 24-h cycle of rhythmic auto-phosphorylation and auto-dephosphorylation. To generate a robust circadian rhythm of the in vitro reaction mixture, KaiC, the core oscillator protein, must be purified with an untraditional approach, since even the smallest amount of impurity can hinder its post-translational activities. Until recently, series of fast protein liquid chromatography (FPLC) columns (glutathione S-transferase (GST), anion exchange (Q), and desalting columns) have been used to purify the oscillator proteins, often requiring laborious elution processes. Although the common methodology has already been established, whether the purified KaiC can produce robust oscillations remains to be verified. Here we emphasize the significance of eliminating the Q step and lengthening the step of removing the non-specifically bound impurities on the GST column for generating a rhythmic KaiC phosphorylation in vitro. These findings demonstrate the potential for shortening the amount of time and effort it takes to purify proteins without compromising its quality.

Lin, B.; Zheng, X.; Zheng, S.; Luo, M.; Lin, Z.

doi: 10.1134/S0003683820040109pmid: N/A

The ammonia-secreting performance is one of the key factors for determining nitrogen-fixing capacity of the cell. Klebsiella variicola GN02 is an endophytic nitrogen-fixing bacterium isolated from the roots of mature Pennisetum giganteum z.x.lin. In order to reveal the key factor which influences the ammonia secretion in fermentation process of K. variicola, GC-TOF-MS metabolomics was used. The changes of OD600, ammonia nitrogen content and intracellular metabolites of bacteria were measured simultaneously during fermentation, and a total of 172 intracellular metabolites were detected. Then some methods were used to analysis these high-throughput data. Principal component analysis (PCA) showed that the substances with more contribution to major component exhibited an order as mannitol, glycerol, glucose-1-phosphate, 2‑hydroxypyridine, glutamic acid, fructose-6-phosphate, aniline, lyxose and norleucine. Orthogonal projection to latent structures (OPLS) results revealed that the most important substances for the secretion of ammonia from K. variicola GN02 were mannitol, Gly-Pro, fructose-6-phosphate, and other 12 substances including glycerol, Glu and diacylglycerol having a significant impact with variable importance plot (VIP) values higher than 1, which can be used as the key metabolites for the secretion of ammonia from bacteria. Heatmap clustering analysis showed that mannitol, Gly-Pro and fructose-6-phosphate were strongly expressed at different fermentation stages as the major metabolites. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that fructose-mannose metabolism and amino acid metabolism are both metabolic pathways with the greatest impact on the secretion of ammonia in bacteria. Mannitol, fructose-6-phosphate and Glu are at the core hub of the metabolic process in bacteria, and Gly-Pro is the major nitrogen-related metabolite synthesized.

Baş, L.; Otur, Ç.; Kurt-Kızıldoğan, A.

doi: 10.1134/S000368382004002Xpmid: N/A

Tunicamycin is a nucleoside type antibiotic with a potent antibacterial activity. Tunicamycin gene cluster in Streptomyces clavuligerus lacks a cluster-situated regulator (CSR). Therefore, there is no information about its regulation in the cell. To have an insight about the regulation of tunicamycin biosynthesis, the possible effects of BldG pleiotropic regulator involved in the control of secondary metabolite production in S. clavuligerus were investigated. To overexpress bldG in the cell, strains containing multiple copies of the gene expressed from PglpF promoter of S. clavuligerus pLB1, and an additional bldG integrated in the chromosome of S. clavuligerus pLB2, were constructed. S. clavuligerus pLB1 and S. clavuligerus pLB2 fermentations resulted in 16.4- and 13.8-fold higher specific tunicamycin titers, respectively, in comparison to wild type by confirming quantitative reverse-transcription PCR (qRT-PCR) data. However, qRT-PCR expression analysis of tunicamycin genes in S. clavuligerus ΔbldG constructed by Bignell with coworkers [1] showed that gene expressions at T36 (except for SCLAV_4274 and SCLAV_4275) were from 3.6- to 57.9-fold reduced compared to wild type. The tunicamycin titers were lower in S. clavuligerus ΔbldG than in wild type, as well. Consequently, the data presented here is the first report indicating a positive role of BldG on tunicamycin.

Sharma, V.; Upadhyay, L. S. B.; Vasanth, D.

doi: 10.1134/S0003683820040146pmid: N/A

This study presents the exploration of laccase-like enzyme produced by thermophilic bacterial strains present in the Tattapani hotspring located in Chhattisgarh, India. Two bacterial isolates namely TPNR1 and TPNR6 were found to be positive for laccase-like enzyme production on screening with guaiacol as a substrate. The biochemical and 16S rRNA gene sequence analysis indicated that the isolates have similarity with Bacillus licheniformis strains and, thus, named as B. licheniformis TPNR1 and B. licheniformis TPNR6. The activity of crude enzymes was estimated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as a substrate and measured as 3.1 U/mL and 7.1 U/mL for TPNR1 and TPNR6, respectively. The SDS-PAGE of purified enzymes showed that the enzymes are monomers with molecular weight of 44 (TPNR1) and 38 (TPNR6) kDa. The native-PAGE technique followed by activity staining using ABTS confirms the presence of purified active enzyme with laccase-like activity in both cases. Kinetic studies displayed that catalytic efficiency of the enzymes was higher for the substrate ABTS than 2,6-dimethoxyphenol for both enzymes. The maximum enzyme activity was observed at 50°C for both enzymes while the optimal pH for TPNR1 and TPNR6 was 5.0 and 6.0, respectively. The TPNR1 exhibited half-life of 4 h at 70°C and was stable at 60°C for 180 min with a residual activity of 79%. Similarly, the TPNR6 possessed half-life of 3.1 h at 70°C and retained 88% of its activity at 60°C for 180 min. In addition, the catalytic efficiency of the enzymes was tested by decolourization of toxic dyes which showed that the both enzymes are highly potential to degrade them.

Kamal, N.; Tarafdar, A.; Sinha, A.; Kumar, V.

doi: 10.1134/S0003683820040067pmid: N/A

A gram-positive gabapentin-degrading bacteria strain N.ISM.1 was isolated from the pharmaceutical plant soil. According to the analysis of 16S rRNA gene sequence, the strain was categorized as a Micrococcus luteus. Though the strain N.ISM.1 is capable of degrading gabapentin (GABA), this compound cannot provide enough carbon source for the isolated strain. M. luteus N.ISM.1 degrades GABA in the presence of glucose. The greater the concentration of secondary simpler carbon source (glucose) for cometabolism, the greater the biodegradation of GABA is. Around 97% of GABA was degraded within a course of 40 days after using 150 mg/L glucose as cometabolic simpler carbon source. M. luteus N.ISM.1 is a promising microorganism for the biodegradation of an anticonvulsant drug GABA contaminated environment and with the aid of cometabolic activity of glucose (150 mg/L) the degradation achieved perfection. GABA biodegradation metabolites were identified by a liquid chromatography-electrospray ionization tandem mass spectrometry. This is the very first report of GABA biodegradation as per our concern.

Khlupova, M. E.; Shumakovich, G. P.; Vasil’eva, I. S.; Zaitseva, E. A.; Morozova, O. V.; Yaropolov, A. I.

doi: 10.1134/S0003683820040080pmid: N/A

The copolymers of aniline (ANI) and 2-aminophenethyl alcohol (APA) were synthesized on a poly (2-acrylamido-2-methyl-1-propanesulfonic) acid (PAMPS) template with the fungal laccase Trametes hirsuta, which has a high redox potential, as the catalyst. Atmospheric oxygen was the oxidizing agent. The copolymer/PAMPS complexes were pseudo-soluble and were characterized by various physicochemical methods. The electrical conductivity of the copolymers depended on the initial molar ratio of the monomers. The copolymer synthesized at an APA : ANI ratio of 2 : 8 possessed the best characteristics. It is shown that the laccase/mediator system can be to oxidize the primary alcohol groups of the copolymer to reactive aldehyde groups.