Valentin Savov

Role of superoxide dismutase in the oxidation of N-alkanes by yeasts

Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68-72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O2-. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu2+ and Zn2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O2- and high concentrations of Cu2+ and Zn(2+)-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolytica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.

IDENTIFICATION AND CHARACTERIZATION OF α-AMYLASE AND ENDOXYLANASE, PRODUCED BY ASPERGILLUS MUTANT STRAINS

ABSTRACT Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae PP and Aspergillus awamori K-1 because of its ability to produce heterologous proteins in submerged (liquid) cultures. The goal of this investigation was to determine the α-amylase and endoxylanase enzyme production ability and molecular characteristics of fungal strains Aspergillus oryzae PP and Aspergillus awamori K-1 and its mutant strains R5 and A45. The strains were cultivated in liquid culture and maximum enzyme production of parent and mutant strains was determined after 72–96 h of cultivation. Extracellulars α-amylase and endoxylanase were partially purified from the culture filtrates, using molecular sieve chromatography with Gel permeation chromatography. The molecular weight of the partial purified enzymes has been estimated to be 57 kDa for α-amylase and 31 kDa for endoxylanase on SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme α-amylase was 30°C, respectively for endoxylanase was 40°C and the pH optimum was 4.7 and 4.0.

Characterization of Humic System in Fertilizer Raw Materials

The aim of the study was to characterize humus system of natural and artificial products. Humus systems from leonardite, lignite biotransformed with Trichoderma sp. (Plantagra), plant materials after pyrolisis (charcoal) and composts are compared. Humus systems are characterized by Kononova-Belchikova’s method, and heavy metals content was measured by atomic absorption spectroscopy (AAS). Humic acids from the International Humic Substances Society (IHSS) collection are the standards for humus substances quality of compared products. Data obtained for leonardite indicate that the studied substances from factory, Izmir, Turkey contain humic acids over 94%. Compared to the standard, heavy metals content in these materials demonstrate high amounts. Organic carbon content in the composts is very low compared to the leonardite materials and IHSS collection, where the heavy metals content is lower. Biotransformed lignite is characterized with lower content of organic carbon, but humic acids are with high degree of humification. Results obtained show that the fourth studied humus systems may be used in agriculture on base of the high humic acids content. It is recommended to measure heavy metals content before applying the materials in agriculture.

Effect of multiple short highly energetic X-ray pulses on the synthesis of endoglucanase by a mutant strain of Trichoderma reesei-M7.

Bioconversion of cellulose-containing substrate to glucose represents an important area of modern biotechnology. Enzymes for the degradation of the polysaccharide part of biomass have been produced, mostly by fungi belonging to genus Trichoderma. Studies were carried out with the mutant strain Trichoderma reesei-M7, a cellulase producer. Spores of the enzyme producer were irradiated with different doses of characteristic X-ray radiation from metallic tungsten (mainly the W Kα1 and Kα2 lines) with a high dose rate. The latter is a specific property of the dense plasma focus (DPF) device, which has pulsed operation and thus gives short and highly energetic pulses of multiple types of rays and particles. In this case, we focused our study on the influence of hard X-rays. The doses of X-rays absorbed by the spores varied in the range of approximately 5–11,000 mSv measured with thermoluminescent dosimeters (TLD). The influence of the applied doses in combination with exceptionally high dose rates (in the order of tens of millisieverts per microsecond) on the activity of the produced endoglucanase, amount of biomass and extra-cellular protein, was studied in batch cultivation conditions. In the dose range of 200–1200 mSv, some enhancement of endoglucanase activity was obtained: around 18%–32%, despite the drop of the biomass amount, compared with the untreated material.

Characterization of Enzyme Endoxylanase Produced by Mutants Strains of Aspergillus Awamori K-1

ABSTRACT Xylanolytic enzymes of microbial origin have received great attention due to their biotechnological utility and potential applications in a range of industrial processes but the observed effects vary depending on xylanase specificities. The goal of this investigation was to determine the endoxylanase enzyme production ability and molecular characteristics of fungal strains Aspergillus awamori K-1 and its mutant strains A59, A50, A45 and A60. The strains were cultivated in liquid culture with 1% wheat bran and 2% ground corn-cobs as inducer. Maximum enzyme production of parent strain Aspergillus awamori K-1 of 42,35 IU/ml and of mutant strains A59 - 96,91 IU/ml, A50 - 71,67 IU/ml, A45 - 74,04 IU/ml and A60 - 62,59 IU/ml was determined after 72–96 h of cultivation. Extracellular endoxylanase was partially purified from the culture filtrates of five strains, using molecular sieve chromatography with two type of Sephadex—G 50 and G100. The purification conditions were optimized and the main endoxylanase activity was determined in second fraction in case of Sephadex G100. The partial purified enzyme fractions showed the same band for all strains on SDS polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 32 kDa.

Influence of Fe, Cu, Mn, Zn, Co Chelators on the Biotransformation of Humic Substances of Lignite

ABSTRACT Preparations, containing humic substances become more and more popular in modern technologies for organic farming during the past years. The purpose of the study is to determine both the influence of the supporting agent—modified zeolites—during biodestruction of lignite by T.harzianum, and the possibility for using the formed humic substances as a matrix for immobilization of enzyme systems. The influence of chelate form of Fe, Cu, Mn, Zn, Co, Ca, Mg in composition with natural zeolite (clinoptilolite—Eastern Rhodopes, Bulgaria—ammonia form) upon the biotransformation of humic substances from lignite is studied. Lignite from Stanyantsy Mine is inoculated with T.harzianum 105 CFU/g; modified zeolites with concentration 20g/100g lignite are added to the medium. Research data show that N-zeolites, modified with Cu, Zn, Mn and a combination of them, and used as a supporting agent during the biotransformation of lignite by T.harzianum, affect the humification and the formation of enzyme systems depending on the concentration of the saturating micro element. Zinc ions have a lower influence, and manganese ions slow down the humificaion to some extent. Zeolite components, modified with Mn increase the activity of polyphenol oxidase and decrease the activity of peroxidase. Zeolite enrichment with Zn increases the activity of polyphenol oxidase and peroxidase, and the increased concentration of Cuions in zeolites lead to an increased activity of polyphenol oxidase and peroxidase.

Molecular Taxonomic Characterisation of Probiotic Strain Lactobacillus Sp. 50P1

ABSTRACT In recent years interest in the probiotic lactobacilli has been stimulated by the use of these bacteria in products that are claimed to confer health benefits on the consumer. The probiotic effects are usually strain-specific, meaning that a correct identification is important to link the strain to the specific health effect. Taxonomical characterization of probiotic strains only in phenotypic and phisyological characteristics is often with low level of discrimination, probably due to their co-evolution in the same ecological niches. Thus, the nucleotide base techniques provide an accurate basis for phylogenetic analysis and identification. With this aim a probiotic strain Lactobacillus spp. 50P1 was studied. Trough the physiological characterization with API 50 CH the strain 50P1, was determinate as Lactobacillus helveticus, with low probability- 77,5%. Three PCR-based methods, species-specific PCR, of 16S rRNA gene sequencing, and restriction enzyme of 16S rDNA ARDRA analysis were used for reliable taxonomic characterization of probiotic strain Lactobacillus sp. 50P1. The sequence obtained from the strain was compared to those of Lactobacillus species held in GenBank and the belonging of the strain 50P1 to the species Lactobacillus helveticus was confirmed.