| Summary: | 碩士 === 大仁科技大學 === 生物科技研究所 === 99 === The bacterial strain PTB3 of Curtobacterium sp. was isolated from wax-apple orchard in Pingtung, Taiwan. It could produce a highly enzyme activity of beta-1,3-glucanase. A 3.8 kb DNA fragment which containing beta-1,3-glucanase gene was isolated from Curtobacterium sp.. The DNA fragment was predicted contain two functional genes, an endo-type gene which could produce beta-1,3-glucanase, and a secretion gene that function was unknown. The endo-type beta-1,3-glucanase gene has been successful to expression, analysis the biochemical characterization and anti-germination activity to phytopathogenic fungal conidia. A homology search using the BLAST program found no similarity between the secreted protein and the known endo-beta-1,3-glucanases, but has significant similarity to proteins of unknown function from Streptomyces spp.. Determination of the nucleotide sequence of the DNA fragment revealed an 1782 bp ORF which may encode a protein of 592 amino acids with a calculated molecular mass of 62.6 kDa. To confirm that the ORF actually function, a DNA fragment containing the ORF was amplified by PCR and transferred into E. coli for protein expression by pET41a, pETDuet, pQE32, or pGEX4T-1 vectors. The fusion protein which was constructed on pGEX4T-1 vector was purified by glutathione S-transferase (GST) column and appeared homogeneous on SDS-PAGE. There were no detectable enzyme activity on different carbohydrate substrates including beta-glucan, starch, cellulose, chitin, and xylan by purified protein. Therefore, the physiological activity of the secreted protein remains to be discovered in further.
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