활성이 개선된 알긴산 분해효소 (alyVI) 개발에 관한 연구

Abstract

Alginate is a gelling polysaccharide found in great abundance as part of the cell wall and intracellular material in the brown seaweeds. It is organized in three different types, homopolymeric G blocks, homopolymeric M blocks and heteropolymeric G/M blocks. It can be degraded by the β-elimination of alginate lyases. The gene of alyⅥ is encoding with the alginate lyase of marine vacterium Vibrio sp.QY101. It was cloned by using PCR, then sequenced and expressed in Escherichia coli. Through the assay test of alginate lyase(AlyVI), it was confirmed that 52 amino acids of alyⅥ in N-terminal region behaves as a signal peptide which sends the alyVI enzyme to out side of the plasma membrain in E.coli. The full sequence molecular weight of alyⅥ enzyme was 38kDa. If one of the sequence was eliminated from signal peptide(52 amino acid), the molecular weight was decreased to 34kDa. Several mutants of alyVI (W165A, W165D, W165G, W165R, H171F, H171A, K174E, K174A, K218E, K218H, K218A, K308A, K308E, K308H,Q1 L224V, D226G, D226K, D226A, D226L, F228A, F228G, F228L, F228D, F228K, Y306F, Y312F Q314K, Q314H, L224V/D226G, D226A/F228G, L224V/D226G/F228G) was produced by using site-directed mutagenesis to achieve better activity of alginate lyase. Mutants were transformed and expressed as glutathione S-transferase(GST) fusion enzymes in E.coli and purified by using GSH-agarose affinity chromatography. TBA test was used for an enzyme assay and only 3 samples (L224V/D226G, F228G and D226A) were more enigmatically active than wild type. On the other hand, the Kcat/Km ratios of H200A and N217A were rapidly decreased. We believed that histidine of codon-number 200, asparagin of codon-number 217 and tyrosine of codon-number 306 in alyⅥ are strongly related with the binding site of alginate lyases with alginate. It was found that each amino acid of codon-number 200, codon-number 217 and codon number 306 is contributed to the full catalytic activity of AlyVI enzyme through the 3-dimension-computational study and mutants experiments. Alginate facilitates the attachment of mucoid Pseudomonas aeruginosa strains to the tracheal epithelium and respiratory mucins which protects the bacteria from phagocytes and prevents antibiotic uptake. Once the activity of alginate lyases is grower than wild type of that, it can help the antibiotics to cure the disease like cystic fibro-sis effectively with decomposing alginate.