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Cloning and analysis of Polyhydroxyallkanoate synthase gene (phaC) from the extreme haloarchaea-Haloarcula sp. strain HLR2
the extreme haloarchaea
|摘要:||微生物生長於營養不平衡的狀態下會將體內多餘的碳源以聚羥基烷酯(Polyhydroxyalkanoate, PHA)的方式於體內累積。所累積的PHA會在生物渡過逆境後，再降解為乙醯輔酶A (acetyl-CoA)，並經由代謝途徑提供生物生長所需的碳源及能量來源。利用全基因定序已完成的極端高鹽太古生物Haloarcula marismortui ATCC 43049被命名為PHA合成酶的基因 (poly-β-hydroxyalkanoate synthase, phaC)的ORF序列設計引子，以苗栗縣通霄鎮曬鹽場的鹽山所純化出的Haloarcula sp. strain HLR2的染色體DNA進行聚合酶連鎖反應 ﹙PCR﹚得到其phaCHLR2。phaCHLR2基因全長為1434 bp，G＋C %為61.7 %，預估等電點為3.7，可轉譯出477個胺基酸，預估分子量為53.3 kDa。phaCHLR2與Haloarcula marismortui的PHA synthase胺基酸序列有97.1 %的相似度，且其序列上具有與PHA聚合反應相關的(Cys-162)-(Asp-317)-(His-346)所組成之catalytic triad residues，因此應與真細菌型PHA synthases同歸屬於α/β-hydroxylase superfamily，證實此phaCHLR2確為PHA synthase基因。另與已知的真細菌型PHA合成酶進行胺基酸序列比對，亦發現PhaCHLR2胺基酸序列的Glu-110, Thr-166, Try-263, Val-276, Tyr-283 and Glu-284等6個可能與受質鍵結的位置。此PhaCHLR2是首次被選殖且分析的極端高鹽太古生物的PHA合成酶基因。經由系統演化分析所有已知的PHA合成酶胺基酸序列，發現所有已知太古生物的PHA合成酶獨立成一群，且和真細菌第三型PHA合成酶位於同一群集 (cluster)但不具第三型PHA合成酶特有的Cyanobacterial box。高鹽太古生物的極端酵素具耐鹽、耐高溶劑濃度、抗氧化且高溫穩定的特性，因此，適合工業與胞外 (試管)生產應用，太古生物的PHA合成酶基因的開發研究，不僅可提高phaC基因庫以供構築更多樣化的PhaC及更多新型的PHA，並可開發利用高鹽太古生物的極端酵素。|
Many microorganisms have been demonstrated to transform and accumulate the excess carbon source into the polyhydroxyalkanoate (PHA) granules as energy storage materials while encountering the nutrient-limiting stress. When the supply of the limiting nutrient is restored, the PHA can be depolymerized and subsequently metabolized as carbon and energy source with acetyl-CoA as intermediate. Extreme halophilic archaeon- Haloarcula strain HLR2 was isolated from the salt crystals of the salt mountain at Miao-Li, Taiwan. The unique triangle shape of strain HLR2 grew at 45 ℃ in NHB medium contained 25 % NaCl. Analysis of 16S rDNA sequence revealed that strain HLR2 was closely related to Haloarcula marismortui ATCC 43049. Based on the annoated polyhydroxyalkanoate synthase gene (phaC) of Haloarcula marismortui genome, the specific primers were designed for PCR amplification of phaCHLR2. The phaCHLR2 contains 1434 bp with G+C at 61.7 %. The total 477 amino acids could be translated with predicted molecular weight of 53,300 and the isoelectric point is 3.7. The amino acid similarity of PhaCHLR2 was 97.1 % identity with PhaC of Haloarcula marismortui. The amino acids of (Cys-162)-(Asp-317)-(His-346) was detected at PhaCHLR2 which is correspondent to the (Cys-149)-(Asp-302)-(His-331) occurred at α/β hydrolase superfamily among bacteria that was proposed as the catalytic nucleophile for PHA polymerization. Additionally, six amino acids as possible substrate binding site in bacterial PHA stynthases was also detected at PhaCHLR2 as Glu-110, Thr-166, Try-263, Val-276, Tyr-283 and Glu-284. These results confirmed that phaCHLR2 is a gene for PHA stynthase and this is the first haloarchaeal phaC gene has been cloned, sequenced and expressed in Escherichia coli. Phylogenetic analysis of all known PHA synthases indicated that all archaeal PhaC formed independent group and was clustered with eubacterial type III PHA synthase. The enzymes of extreme halophilic archaea are generally temperature, salt and solvent stable and could be a good resource of industrial enzymes. The investigation of PHA synthase gene (phaC) and protein from the extreme halophilic archaea should increase the PhaC gene library to construct more diverse PHA synthetases and produce more novel PHAs as we desired. Results of these studies will not only increase the diversity of PHAs and its application potentials but also explore the utilization of extremozyme of halophilic archaea.
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