Molecular methods have become non-replaceable for the improvement of food production and its control. The Department for Biochemistry and Genetic Engineering (DBGE) as a part of the Faculty of Agriculture Sciences and Food is a crucial entity using molecular techniques in different areas of agriculture and livestock production. DBGE is mainly dealing with marker assisted selection (MAS), evolutionary studies; determine the origin of food products; enzyme analysis in food processing; transcriptional and translational analyses; molecular detection of plant viruses; and GMO analysis. The target molecules were proteins, RNA and DNA. MAS is systematically applied in animal breeding in Macedonia using RYR-1, and S1-casein genes. Evolutionary studies were performed using DNA microsatellites to estimate genetic distance among autochthonic strains of sheep and tomato varieties. The identification of food products origin and gender identification in fish were done using protein profiling. Gene expression was studied analysing different growth factors and inhibitors at RNA level. More than 3.000 plant and animal samples were analysed in the Laboratory for biochemistry and molecular biology. GMO laboratory as a part of the DBGE is the first authorized facility for GMO testing of food and feed samples. In more than 100 samples RT PCR methods were used.
Тhermophilic bacteria Bacillus caldolyticus isolated from the hot spring in Bansko, Republic of Macedonia, were used for the isolation of DNA polymerase. Bacterial cells were disrupted by sonication and the first step in the purification of DNA polymerase was 40% ammonium sulfate precipitation. This was followed by chromatographic procedures on Sephadex G-50, DE-52 and CM-52 cellulose. DNA polymerase activity was analyzed at each step of purification using the incorporation of 3 H dATP in activated calf thymus DNA. The purity of the DNA polymerase was analyzed on SDS PAGE. Optimal conditions of activity were determined for temperature, pH, dNTP and Mg ++ concentration. In addition, the effect of ethanol and EDTA as possible inhibitors of polymerase activity was also analyzed. The optimal temperature of DNA polymerase was 66 ºC; the optimal pH was 7.2, optimal MgCl 2 concentration was 2.5 mM, and the optimal substrate concentration was 2.5 × 10 -6 M dNTP. The inhibitory effect of EDTA and ethanol on DNA polymerase was above 10 mM and 10%, respectively. Keywords: Bacillus caldolyticus; DNA polymerase; purification; optimization; inhibitors ПРОЧИСТУВАЊЕ И ОПТИМИЗИРАЊЕ НА УСЛОВИТЕ ЗА ДНК-ПОЛИМЕРАЗА ИЗОЛИРАНА ОД ТЕРМОФИЛНАТА БАКТЕРИЈА BACILLUS CALDOLYTICUSТермофилната бактерија Bacillus caldolyticus изолирана од термалниот извор во Банско, Република Македонија, беше користена за изолација на ДНК-полимераза. Бактериските клетки се лизирани со ултразвук и првиот чекор во прочистувањето на ДНК-полимеразата беше исталожување со 40% амониум сулфат. Потоа следуваа хроматографски процедури на Sephadex G-50, DE-52 и CM-52 целулоза. Активноста на ДНК-полимеразата беше анализирана во секој степен на прочистувањето со инкорпорација на 3 H dATP во активирана ДНК од телешки тимус. Чистотата на ДНК-полимеразата беше анализирана со користење на SDS PAGE. Оптималните услови за активноста беа одредени за температура, pH, dNTP и Mg ++ . Дополнително беа анализирни ефектите на етанолот и EDTA како можни инхибитори на ДНК-полимеразната активност. Оптималната температура за оваа ДНК-полимераза е 66 ºC, оптималната pH е 7.2, оптималната концентрација на MgCl 2 е 2.5 mM, додека оптималната концентрација на dNTP е 2.5 × 10 -6 M. EDTA покажува инхибиторен ефект врз ДНК-полимеразата во концентрација над 10 mM, додека етанолот во концентрација над 10%.
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