Cloning And Sequence Analysis Of The Aspergillus Niger Glucose Oxidase Gene

Glucose oxidase GOD (EC 1.1.3.4) which can be specific for β-D-glucose oxidase to generate hydrogen peroxide and gluconic acid, has been widely used in the food industry, feed industry, pharmaceutical industry and bio-sensor. Due to its low purity and activity, how to improve its yield remains a problem. In this experiment one bacterium producing glucose oxidase was screened and identified One glucose oxidase gene fragment was cloned and its DNA sequence was also analyzed. The main results are as follows:1. One strain producing glucose oxidase was screened by Iodine-starch staining method.The genomic DNA was extracted by five different methods. The strain was idetificated by ITS and morphological identification, it belongs to Aspergillus niger.2. A pair of primers was designed by primer premier 5.0 and Oligo7.0 to amplify glucose oxidase gene and a 1400bp DNA fragment was amplified from its genomic DNA. After lignated with pMD20-T vector, transformed into JM109 competent cells, the combinant plasmid was identified by colony PCR, double restriction enzyme digestion and sequencing.3. The results of the NCBI nucleotide blast shows that the amplified fragment was 1444bp, and the similarity with one Aspergillus niger gox gene for glucose oxidase (X16061) was 99%, and the GOD gene fragment was also analyzed by DNAMAN, showed that it belong to the oxidase superfamily, which called Chain A strain of Aspergillus niger, its amino acid homology of GOD was up to 99%.4. Bioinformatics analysis show that its formula of the pupative protein was C2342H3531N641O727S9, its molecular weight was 52.587kDa, theoretical pI was 4.97. It was a hydrophilic proteins without signal peptide and transmembrane domain, had 27.08% helix, 15.42% strand, and 57.50% loop. Tertiary structure prediction showed that proteins contain 12 a-helix and 13 β-folded, and contains two cofactors FAD and NAG, the major functional domains contained in most GOD gene sequence.