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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30297

Title: 探討 Pseudomonas vesicularis MA103 之 a-Amylase 基因轉殖至 Lactococcus lactis NZ3900 表現及轉殖 a-Amylase 生化特性分析
Studies on Cloning and Expression of a-Amylase Gene from Pseudomonas vesicularis MA103 to Lactococcus lactis NZ3900 and Characterization of Recombinant a-Amylase
Authors: 蘇明璁
Contributors: NTOU:Department of Food Science
Keywords: nisin 調控基因表現系統;Lactococcus lactis NZ3900;Pseudomonas vesicularis MA103;乳酸鏈球菌素;a-澱粉酶;Ulva lactuca
NICE system;Lactococcus lactis NZ3900;Pseudomonas vesicularis MA103;nisin;a-amylase;Ulva lactuca
Date: 2011
Issue Date: 2011-11-25T07:38:13Z
Abstract: 本研究目的在探討 Pseudomonas vesicularis MA103 澱粉酶基因 amy-00047 結合乳酸菌鏈球菌素調控表現系統 (nisin-controlled expression system, NICE system) 中之食品級載體 pNZ8149 轉殖到乳酸菌 Lc. lactis NZ3900 中表現,並分析轉殖株添加 nisin 誘導之粗酵素液生化特性和水解不同澱粉之寡醣組成變化。以澱粉酶基因 amy-00047 核甘酸序列設計一組引子對 pNZ8149-Amy-for 與 pNZ8149-Amy-rev 利用 P. vesicularis MA103 genomic DNA 為模板進行 PCR 擴增夾出,被擴增基因序列在 5’ 端與 3’ 端分別帶有 SpeI 與 XbaI 限制酶剪切位。以該兩種限制酶剪切擴增之 amy-00047 基因片段與乳酸菌載體 pNZ8149,透過接合反應 (ligation) 形成重組載體後以電穿孔轉形法轉殖到 Lc. lactis NZ3900 中再以含有乳糖之培養基篩選出九株 lac+ 菌落,其中一株轉殖株命名 Lc. lactis NZ3900-Amy。抽取轉殖株載體以 PCR 擴增與限制酶剪切等實驗結果確認為帶有長度 1,071 bp 之 amy-00047 成功轉殖菌株 Lc. lactis NZ3900-Amy。轉殖菌株 Lc. lactis NZ3900-Amy在含有濃度低於 1000 ng/mL 以下nisin 之 M17 broth中培養不會受到生長抑制。以 10 ng/mL nisin誘導 3 hr 後之胞內 -amylase Pv-AM-R 之酵素活性高於其他濃度 nisin 誘導者,為 2.6 U/mg。而在誘導 8 hr後有最高 -amylase 活性為 4.4 U/mg,胞外酵素活性尚未被測得。胞內amylase 酵素液之最適反應溫度與 pH 值分別為 40oC 與 pH 7.0 ,而Ca2+ 可以使其之相對酵素活性提升 (114%),但Sn2+會使 -amylase Pv-AM-R 失去酵素活性。SDS-PAGE 試驗結果顯示,其分子量約為 40.5 kDa,與自 Amy-00047 基因序列所推估的分子質量相近;在基質特異性的測試中 -amylase Pv-AM-R 對於 potato soluble starch 具有較高的酵素活性 0.36 U/mL。對於藻類多醣熱萃液則未見到顯著的酵素活性產生。在 -amylase Pv-AM-R 水解 2%澱粉水解產物分析上,薄層層析法 (TLC) ,可推測在 40oC 與 pH 7.0 中將 potato soluble starch 水解 24 hr所得寡醣之聚合度 (degree of polymerization, DP) 可能為麥芽糖與名為 DP3 或 DP4 的麥芽寡醣。而在高效能液相層析法 (HPLC) 分析比對標準品後,推測主要的水解寡醣產物為麥芽糖及聚合度大於DP7之麥芽寡糖。本研究主要建立帶有菌株 MA103所產-amylase Pv-AM-R基因重組載體 pNZ8149-amy 於乳酸菌 Lc. lactis NZ3900,該轉殖株經nisin誘導可表現產出胞內-amylase 酵素活性之生產技術。未來可進一步開發生產具生理活性之麥芽寡醣。
The purpose of this thesis is using -amylase gene amy-00047 from Pseudomonas vesicularis MA103 as a reporter gene combine with plasmid pNZ8149 from nisin-controlled expression system (NICE system) and transgenic to Lactococcus lactis NZ3900 host cells, then evaluate the characterization of nisin-induced recombinant crude enzyme, the composition of oligosaccharide from recombinant enzyme-hydrolyzed soluble starch and the bioactivity of hydrolysate. According to amy-00047 amylase gene sequence a pair of primers pNZ8149-Amy-for and pNZ8149-Amy-rev was designed, and the primer pair were used in PCR to acquire 1074 bps recombinant sequence containing -amylase gene amy-00047 with SpeI restriction enzyme site on 5’-ter and XbaI restriction enzyme site on 3’-ter. Using restriction enzyme SpeI and XbaI to insert amy-00047 recombinant sequence into plasmid pNZ8149 and was transformed into Lc. lactis NZ3900 by electroporation. The plasmid accepted colony Lc. lactis NZ3900-Amy were selected by M17L plate and can confirm the length of 1074 bps inserted gene sequence exist by using PCR. The growth of Lc. lactis NZ3900-Amy will be limit by concentration up to 1000 ng/mL of nisin. Using ultrasonic to broken cells successfully obtained single protein band by SDS-PAGE, named as Pv-AM-R and the molecular weight was approximately 39 kDa. Optimal induced recombinant -amylase Pv-AM-R specific activity 2.6 U/mg is obtained by induction at 0.4 to 0.5 OD600nm cell density with 10 ng/mL nisin after 3 hr cultivation at 30oC. The optimal reaction pH and temperature of recombinant amylase Pv-AM-R were pH 7.0 and 40oC. Ca2+ was able to raise the relative activity of Pv-AM-R (114%), on the other side, Sn2+ was with the ability to completely inhibit the enzyme activity of enzymes. On substrate specificity, Pv-AM-R can hydrolyze soluble starch solution, but no obvious hydrolyze showed on Ulva sp. polysaccharide. Analyzing the hydrolysate of Pv-AM-R by HPLC and TLC, compared to the standards, discovered that the main hydrolysate were assumed to be maltose, DP3, DP4 and oligosaccharide greater than DP7. This study provides optimal parameters for the NICE system in Lc. lactis NZ3900 as well as a means to construct genetically modified probiotic bacteria with the ability to produce functional maltooligosaccharides.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98320031
Appears in Collections:[食品科學系] 博碩士論文

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