Increase in glucoamylase productivity of <Emphasis Type="Italic">Aspergillus awamori</Emphasis> strain by combination of radiation mutagenesis and plasmid transformation methods

Increase in the expression level of amylolytic genes activator protein encoded by amyR gene was shown to result in enhancement of glucoamylase productivity of A. awamori strain by 30%. However, the same effect equal to 30% increase can be achieved by introduction of extra copies of gla gene encoding glucoamylase. These two effects were not additive, which gave the possibility to suggest an additional limitation in the regulation mechanism of glucoamylase gene expression in A. awamori strain while introducing an additional copies of amyR and gla genes.     

Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.     

Creation of a heterologous gene expression system on the basis of <Emphasis Type="Italic">Aspergillus awamori</Emphasis> recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of glucoamylase. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and Aspergillus niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6–14% range of total protein.     

Comparative study of fungicidal and herbicidal activities of salts of polyenic macrolide antibiotics]

The fungicidal and herbicidal activities of water soluble salts of levorin, nystatin, mycoheptin and amphotericin B, polyenic macrolide antibiotics, were studied. The biological tests showed that the antibiotics had fungicidal and low herbicidal activities. They also had a growth regulating action.     

Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.     

Evaluation of axillary lymph nodes from the results of radioisotopic study

The purpose of the investigation was to study the pattern of 99mTc (MIBI) technitrile uptake in the metastatically involved lymph nodes in breast cancer, by applying the plain scintigraphic technique. The scintigraphic study of the breast and regional metastatic areas by means of the radionuclide 99mTc (MIBI) technitrile was made on a Millennium GE tomographic gamma chamber. The radiotracer 550 (MBq) dissolved in 10-20 ml of saline solution was intravenously injected into the arm cubital vein contralaterally to the lesion. Following 20 minutes of injection of the agent, plain scintigraphy was carried out in three standard projections: frontal and two oblique ones. The scintigraphy was performed using a high-resolution collimator recording a 128 x 128 matrix image. The detector was maximally approximated to the organ being examined. A plain scintigraphic scan was recorded on each side for 10 minutes. By the degree of axillary lymph nodal involvement, the patients were divided into 3 groups in accordance with the international TNM classification: N0 (n = 55), N1 (n = 13), N2 (n = 4). Among 72 patients, axillary lymph nodes could be detected in 6, in 2 of them changes were not diagnosed by X-ray and ultrasound studies. The final pathomorphological study of intraoperative materials revealed axillary lymph nodal metastatic involvement in 17 patients. A micrometastasis in one lymph node was found in 1 patient. None of the radionuclide studies showed tumor spread in 5 cases. The sensitivity, specificity, and precision of the technique were 35.3, 100, and 84.7%, respectively. Thus, a combination of the high cost of the procedure, its radiation load on a patient, and its low sensitivity make the use of plain scintigraphy of axillary regions inexpedient in the complex of studies of the extent of breast cancer in the present development of technology.     

Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.