基因重組腈水解酶轉化亞氨基二乙腈產生亞氨基二乙酸和以奈米金粒子檢測亞氨基二乙腈;Conversion of Iminodiacetonitrile to Iminodiacetic acid using recombinant nitrilase and detection of Iminodiacetonitrile using gold nanoparticles

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Title:	基因重組腈水解酶轉化亞氨基二乙腈產生亞氨基二乙酸和以奈米金粒子檢測亞氨基二乙腈;Conversion of Iminodiacetonitrile to Iminodiacetic acid using recombinant nitrilase and detection of Iminodiacetonitrile using gold nanoparticles
Authors:	陳俊夆;Chen, Jun-Feng
Contributors:	化學工程與材料工程學系
Keywords:	腈水解酶;亞氨基二乙酸;亞氨基二乙腈;奈米金粒子;Nitrilase;iminodiacetic acid;iminodiacetonitrile;gold nanoparticles
Date:	2025-07-22
Issue Date:	2025-10-17 11:25:10 (UTC+8)
Publisher:	國立中央大學
Abstract:	現時生產亞氨基二乙酸Iminodiacetic(IDA) ，需要消耗能源加熱和加入酸和鹼的條件下生產，並有多餘的副產物，正需要改善，而本實驗將以基因工程改造之腈水解酶進行生產，我們以重組蛋白法將含有6個His-Tag的基因片段NitA轉殖入E.coli BL21後進行表達，表達成功後，培養細菌成長，再以超音波震盪打破細菌細胞壁後，以金屬親和性純化層析法對酵素粗液進行純化，再以SDS膠體電泳法測得為43kDa的腈水解酶，並以NADH分光光度法量測該酵素對亞氨基二乙腈具有活性，以腈水解酶轉化50mM 亞氨基二乙腈(IDAN)測得吸收度0.601 獲得35.6mM IDA，轉化率71.2%，實驗發現TB給予更多碳源和氮源能於12小時的培養時間快速拉高OD600，這適合用於親和性金屬純化法生產腈水解酶，再以濕式合成法以檸檬酸鈉還原四氯金酸，得到粒徑平均19.36nm奈米金粒子，再將奈米金粒子和亞氨基二乙腈溶液混合後，從紫紅色快速變化為透明無色，但和IDA溶液接觸不會變色，故能利用此性質去檢測判定產物是否含有未反應完的IDAN，並發現原因是奈米金粒子混合IDAN後，團聚造成表面電荷環境改變，造成顏色從紫紅色改變至藍灰色直至變透明，並析出黑色團聚物，並以分光光度法和Tauc plot發現其混合後最大吸收波長從520nm移動至640nm和能隙由2.537eV改變至3.085eV，所以顏色由紫紅色改變至藍灰色直至變透明。;The current production of iminodiacetic acid (IDA) requires energy-intensive heating and the addition of acids and bases, resulting in unwanted byproducts and a need for improvement. In this research, we utilized genetically engineered nitrilase for IDA production, The NitA gene fragment,tagged with six His-tags,was cloned into E.coli BL21 for expression using recombinant protein methods. After successful expression,bacteria were cultured, disrupted by sonication to break the cell walls, and the crude enzyme was purified using immobilized metal affinity chromatography. The purified nitrilase, with a molecular weight of 43 kDa, was confirmed by SDS-PAGE.Enzyme activity against iminodiacetonitrile (IDAN) was tested using the NADH spectrophotometric method. Conversion of 50 mM IDAN resulted in an absorbance of 0.601, yielding 35.6 mM IDA with a conversion rate of 71.2%. Through experimental found that Terrific Broth (TB), providing more carbon and nitrogen sources, significantly increased OD600 within 12 hours, making it suitable for producing nitrilase via immobilized-metal affinity chromatography purification. Additionally, gold nanoparticles with an average size of 19.36 nm were synthesized via a wet chemical method using sodium citrate to reduce chloroauric acid. When mixed with IDAN solution, the color of the gold nanoparticles rapidly changed from purple-red to transparent and colorless. However, no color change occurred when mixed with IDA solution, allowing this property to detect unreacted IDAN in the product. The color change in the presence of IDAN was attributed to nanoparticle aggregation,which altered the surface charge environment, shifting the color from purple-red to blue-gray and eventually to transparent, with the formation of black aggregates. Spectrophotometric analysis and Tauc plot revealed that the maximum absorption wavelength shifted from 520 nm to 640 nm, and the bandgap energy changed from 2.537 eV to 3.085 eV, explaining the observed color transition from purple-red to blue-gray and finally to transparent.
Appears in Collections:	[National Central University Department of Chemical & Materials Engineering] Electronic Thesis & Dissertation

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