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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/15845

Title: 麥芽寡糖苷海藻糖生成酶的基因重組與重組後酵素之表現與活性分析
Recombination of maltooligosyltrehalose synthase and the expressions and activity assays of resulting recombinant enzymes
Authors: Yu-Jen Jeng
鄭宇軫
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 海藻糖;基因重組
MTSase
Date: 2010
Issue Date: 2011-06-30T08:21:02Z
Abstract: 麥芽寡糖苷海藻糖生成酶 (maltooligosyltrehalose synthase, MTSase) 可催化麥芽寡糖 (maltooligosaccharide) 分子內的轉糖苷反應,而生合成出麥芽寡糖苷海藻糖 (maltooligosyltrehalose)。Sulfolobus solfataricus ATCC 35092 之 treY 基因所表現之 MTSase 蛋白質量較 S. acidocaldarius ATCC 33909 為低,為了改善此缺點,設計以 S. solfataricus ATCC 35092 之 treY 基因序列為基礎,並利用 Splicing Overlap Extension (SOE) 方式將源自嗜高溫古細菌 S. solfataricus ATCC 35092 與 S. acidocaldarius ATCC 33909 之 treY 基因進行五種片段化重組而產生嵌合基因 (chimeric genes)。於五組突變型 MTSase,發現 SA2、SA3 與 SA4 此三組別其重組 MTSase 表現量有明顯上升情形,最適 IPTG 濃度各為 0.2 與 0.1 mM。SA2 組別轉錄後 treY 基因之總 mRNA 也比原生型提昇約三倍之多。但發現表現量提高之組別其重組型 MTSase 之高溫耐受性約下降 15℃。將 SA2 此 MTSase 表現量最好的轉形株再次進行區域轉換突變,將增幅出完整之 PCR 產物命名為 SA21。經定序後,比對 SA2 組別突變前後之胺基酸序列,與 S. solfataricus ATCC 35092 MTSase 的序列比較後,彼此間只有 19 個胺基酸差異。大量培養 SA21 組別,其最適 IPTG 濃度為 0.5 mM,且 MTSase 表現量依然高於原生型,但高溫耐受性同樣下降 15℃。於熱處理階段,原生型與突變型 SA2、SA3、SA4 與 SA21 每克菌重分別產生 4.38、1.77、1.64、1.73 與 2.37 U/g cells。後續以 SA21 組別進行 G107W 與 G131E 兩點定位突變,成功獲得突變之基因後,命名為 SA21-G107W/G131E,其最適 IPTG 濃度為 0.2 mM,突變之 MTSase 表現量也無下降情況。並發現雖經 80℃、30 分鐘之熱處理,SA21-G107W/G131E 組別之突變 MTSase也有被破壞情形,但是受熱破壞的程度較 SA21 有明顯改善的狀況,其酵素活性則能提升至 3.28 U/g cells。
The maltooligosyltrehalose synthase (MTSase) catalyzes an intramolecular transglycosyl reaction of maltooligosaccharide and produced maltooligosyltrehalose. MTSase expression from Sulfolobus solfataricus ATCC 35092 treY gene is much lower than that of S. acidocaldarius ATCC 33909. In order to improve the expression of MTSase from S. solfataricus ATCC 35092 treY gene, Splicing Overlap Extension (SOE) was used to produce five different recombinant treY genes. The expressions of recombinant MTSases of SA2, SA3, and SA4 are increased obviously, and their appropriate IPTG concentrations were 0.2, 0.1 and 0.1 mM, respectively. The amount of mRNA transcribed from SA2 treY gene is higher than that of wild-type. But the thermostabilities of recombinant SA2, SA3, and SA4 MTSases are decreased about 15℃ than that of wild-type MTSase. SOE was carried out again to reduce the sequence differences between SA2 and wild-type MTSases. The amino acid sequence of resulting SA21 MTSase only has 19 amino acids different from that of wild-type. The appropriate IPTG concentration for SA21 induction is 0.5 mM and thermostability of recombinant SA21 MTSase is also decreased about 15℃ than that of wild-type. After heat treatment, the recovered enzyme activities of wild-type, SA2, SA3, SA4, and SA21 are 4.38, 1.77, 1.64, 1.73, and 2.37 U/g cells, respectively. Then, SA21 MTSase was subjected to site-directed mutagenesis to produce SA21-G107W/G131E. The appropriate IPTG concentration is 0.2 mM, and thermostability of recombinant SA21-G107W/G131E MTSase is better than other recombinants. The recovered enzyme activity of SA21-G107W/G131E is 3.28 U/g cells.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M97320038
http://ntour.ntou.edu.tw/ir/handle/987654321/15845
Appears in Collections:[食品科學系] 博碩士論文

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