Vol 55, No 3 (2019)

The interaction of lysozyme with kappa and lambda carrageenans and the thermal stability of the enzyme within a complex are studied via turbidimetry, dynamic light scattering, and differential scanning calorimetry. It is shown that complexes of lysozyme with kappa carrageenan are stabilized by Coulomb and secondary forces, while the complex formation of lysozyme with lambda carrageenan is governed by electrostatic interactions only. The stoichiometric composition of the complexes at a pH of 7.0 and an ionic strength of I = 0.01 is 0.3 : 1.0 (weight of carrageenan/weight of lysozyme) or 1 : 120 (mole of carrageenan/mole of lysozyme) and changes monotonically with an increase in I up to a higher carrageenan content in the mixture. The effect of I on the complex formation has a nonmonotonic character at q = 0.1 and q = 0.3. The complex formation is accompanied by the loss of thermal stability of the enzyme (which is more significant in the case of lambda carrageenan), suggesting that carrageenans have a higher affinity for the unfolded form of lysozyme than for its native form.

To study the characteristics of two malate dehydrogenases (MDHs), their coding genes MIMDH1 and MIMDH2 were cloned and expressed in Escherichiacoli BL21 cells. The molecular weights of both recombinant enzymes partially purified using Ni-NTA affinity chromatography were 32 kDa, and the specific activities of purified MIMDH1 and MIMDH2 were 329.3 and 241.3 U/mg protein, respectively. The optimal temperatures for MIMDH1 and MIMDH2 activities were 55 and 30^oC, respectively, with 70% MIMDH1 activity and ≥70% MIMDH2 activity remaining after 30 min incubation at 45^oC. Addition of 2 mM Zn²⁺ enhanced MIMDH1 activity, whereas the addition of other metal ions resulted in different degrees of inhibition. The inhibitory effect of Co²⁺ was most pronounced on MIMDH1, reaching 82.8% inhibition. Addition of 2 mM Ba²⁺ or Mn²⁺ increased MIMDH2 activity, the addition of other metal ions resulted in different degrees of inhibition. Furthermore, MIMDH1 was stable at pH range of 7.5–8.5, with optimal activity observed at pH of 8.0. MIMDH2 showed similar stability at the same pH range, but was optimal at pH of 7.5. The V_max and K_M values for recombinant MIMDH1 and MIMDH2 catalyzing the reduction of oxaloacetate to malate were 17.66 µmol mg^–1min^–1 and 0.541 mM, 15.59 µmol mg^–1min^–1 and 0.683 mM, respectively.

The paper reports on a study of the effects of different type antioxidant molecules on recombinant, luminescent, bacterial biosensensors. It has been demonstrated that thiamine can activate oxidative stress promoters, mainly in biosensors based on the parental cells of Salmonella typhimurium LT2. We have detected the induction of soxS and katG promoters and activation of the SOS-response system in the analyzed biosensors in the presence of pyridoxine. It has been shown that ascorbic acid has pronounced prooxidant and toxic properties. Other analyzed compounds proved to be less active and did not induce antioxidant and reparatory systems in luminescent biosensors based on the parental cells of Salmonella typhimurium LT2 and Escherichia coli MG1655.

Astaxanthin is a red xanthophyll pigment which is widely commercially used due to its strong antioxidant properties. The heterobasidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the main microorganisms for industrial production of astaxanthin. However, astaxanthin content is low in wild type yeast strain. In our study, we had successfully isolated X. dendrorhous mutant with higher astaxanthin production capacity using chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), through β-ionone screening. The astaxanthin-hyperproducing mutant X. dendrorhous M34 had total carotenoid yield of 602.4 µg/g DW and a 110.8% increment of total carotenoid content compared to wild type. Survival studies indicated that MNNG treatment of yeast led to higher lethality in recipient cells compared to ethyl methanesulfonate (EMS) one. EMS-mutated strains did not show promising carotenoid yield during the screening process by β-ionone and the rate of reverse mutation was very high. In the stability test, no reversion was observed in X. dendrorhous M34 mutant after 10 successive transfers in culture broth. Reversed-phase HPLC showed that the elution profiles of carotenoid extracts of M34 and wild type X. dendrorhous were in agreement with the authentic standard and the major peak in M34 extract was identified as astaxanthin. The stable mutant with high yield of astaxanthin obtained in this work could be a suitable candidate for the industrial-scale production of astaxanthin.

Inactivation of the Kunitz and Bowman-Birk soybean trypsin inhibitors at hydrolysis of soybean proteins by enzymatic complexes was studied. These complexes are derived from king crab hepatopancreas, cod fish pyloric caeca, and a commercial enzymatic complex of protosubtilin. Analysis of the soybean protein hydrolysates showed that these enzymatic complexes completely digested the Kunitz trypsin inhibitor (60–70% of the total trypsin inhibitors) and had almost no effect on the Bowman-Birk trypsin and chymotrypsin inhibitor. All of the enzymatic complexes contain different sets of enzymes with different proteolytic specificity. This allow to make the conclusion that Bowman-Birk inhibitor is highly resistant to proteolysis and is not inactivated at enzymatic hydrolysis.

Five compounds, 4-chloropinselin, chloromonilinic acids B and C, alternariol, and emodin, are the first to be identified in the metabolite composition of Alternaria sonchi, a pathogen of field sowthistle (Sonchus arvensis L.). The metabolite profiles of extracts have been significantly influenced by fermentation and extraction techniques. Solid-state fermentation of A. sonchi S-102 on millet was preferable for the production of alternethanoxin A, chloromonilicin, and 4-chloropinselin with a yield of about 460, 70, and 2760 mg/kg respectively, while the use of pearl barley was optimal for the production of alternethanoxin B (215 mg/kg) and alternethanoxin D (29 mg/kg). Extracts from the culture broth and mycelium of A. sonchi obtained after surface cultivation of the fungus contained 4-chloropinselin, alternariol, emodin, and a number of unidentified biologically active compounds. The extracts display relatively low phytotoxic and antimicrobial activity and only trace levels of alternariol. Thus, there is low toxicological risk for the application of this fungus as a bioherbicide producer.

The correlation between changes in the circular dichroism (CD) spectra of antenna pigments and the photooxidation kinetics of the primary electron donor (P700) in trimeric and monomeric complexes of photosystem 1 (PS1) after incubation at elevated temperatures was studied to assess the thermostability of photosystem I (PSI) complexes from the cyanobacterium Arthrospira platensis. It was shown that heating of the monomers and trimers to 60 and to 70°C, respectively, caused a 10% decrease in the amplitude of the CD signals of antenna chlorophyll. Thermal disturbance of the spatial organization of the antenna chlorophyll correlated with a lower concentration of photoactive P700. A decrease in the initial rate of P700 photooxidation was observed already after 40°C for both monomers and trimers. This decrease occurred simultaneously with a decrease in the intensity of the carotenoid CD band of trimers but not monomers, which is probably due to the different carotenoid compositions of trimers and monomers. It is assumed that the slowdown of P700 photooxidation kinetics in PS1 complexes at temperatures up to 50–60°C is associated not with impairment of energy migration to the reaction center (P700) but rather with an initial delay of P700 photooxidation due to the changes in the state of the acceptor part of PS1, in particular, the secondary acceptor phylloquinone.

The characteristics of biosensors based on screen-printed electrodes containing glucose oxidase, alcohol oxidase, lactate oxidase, and a mixture of glucose oxidase and γ-amylase are studied. The enzymes have been immobilized in a conductive hydrogel of cross-linked bovine serum albumin modified with carbon nanotubes. The developed biosensors are applicable for the quantification of glucose, ethanol, lactic acid, and starch in fermentation media. The analytical and metrological characteristics of these biosensors are determined. The developed biosensor system allows for quantification of glucose, ethanol, lactate, and starch in the concentration range of 0.1–2.7, 1–27, 0.02–73 mM, and 0.6–21 g/L, respectively. Samples of alcoholic and fermented milk products, kvass, and fermentation mass have been analyzed. Statistical analysis of the assay results shows that glucose, ethanol, lactic acid, and starch concentrations determined by biosensors insignificantly differ from those determined by the reference methods. The developed set of biosensors based on specifically selected enzymes can be used for effective monitoring of biotechnological processes as an alternative to the conventional analytical methods.