在大腸桿菌胞外表現 Streptomyces 轉麩氨醯胺酶之探討

陳楷婷

doi:10.6845/NCHU.2015.00464

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在大腸桿菌胞外表現 Streptomyces 轉麩氨醯胺酶之探討

Extracellular Expression of Recombinant Streptomyces Transglutaminase in E. coli

陳楷婷 , Masters　　Advisor：楊明德　　

繁體中文 DOI： 10.6845/NCHU.2015.00464

轉麩氨醯胺酶；角質酶；胞外表現； Transglutaminase ； Cutinase ； Extracellular Expression

Abstract │ Reference (227) │ Times Cited (1) │ Altmetrics

Abstract 〈TOP〉

Parallel Abstract 〈TOP〉

Transglutaminase (protein-glutamine:amine γ- glutamyl transferase, EC 2.3.2.13;TGase) is an acyl-transferase enzyme that catalyzes crosslinking between glutamine
residue and a variety of primary amines in proteins. It is currently widely used in food processing, textile and pharmaceutical industry. An efficient production of intracellular soluble recombinant pro-TGases in E. coli have been demonstrated by processing the TGases with exogenous protease. Moreover, the strategy of applying co-expression of individual propeptide and mature TGase genes as polycistronic mRNA in E. coli, active form mature TGase could be obtained without protease digestion. However, efficient secretion of TGase into culture medium without tedious purification steps was consider to be more cost-effective. In this study, the modified Streptomyces netropsis TGase propeptide and its mature TGase were individually fused with PelB signal peptide and coexpressed as individual polypeptide under a single T7 promoter control. The amount of extracellular production of mature TGase by the order of gene expression, fusion of thioredoxin to propeptide, and genes regulated by different promoters, were investigated in this study. Results showed that extracellular production of mature TGase was detected only when the genes were expressed in the order of pro-peptide and
mature TGase, indicating that the order of gene expression has impact on the mature TGase secretion. However, no significant increase in extracellular TGase production by simultaneous expression of propeptide and mature TGase and those constructs with thioredoxin fused to the propeptide was observed. To facilitate extracellular production of mature TGase, cutinase gene from Thermobifida fusca was cloned and co-expressed
with the recombinant TGase genes in E. coli BL21(DE3). Results revealed that T. fusca cutinase as well as the recombinant TGases could be successfully secreted into culture medium. By introducing of exogenous protease genes from S. netropsis into the above host strains, mature S. netropsis TGases could be recovered from intracellular and culture supernatant. After purification by Ni2+-NTA affinity column, 0.35 mg of mature TGase could be obtained from 50 ml culture supernatant and have a specific activity of 18.9 U / mg.

Reference ( 227 ) 〈TOP〉

陳裕成 (2006). Streptomyces 轉麩氨醯胺酶基因選殖分析及利用大腸桿菌細胞生
連結：
謝秀英 (2010). 利用 Corynebacterium glutamicum 生產胞外重組 Streptomyces
連結：
王冠棋 (2012). 在 Corynebacterium glutamicum 表現具活性之胞外重組
連結：
張文政 (2013). 鏈黴菌轉麩氨醯胺酶前胜肽作用之探討，國立中興大學分子生
連結：
洪妙子 (2014). 由 Streptomyces netropsis 選殖活化轉麩氨醯胺酶之金屬蛋白酶
連結：

Times Cited (1) 〈TOP〉

紀科衡(2017)。轉榖氨醯胺酵素(Transglutaminase)基因轉殖至水稻葉綠體之研究。中興大學園藝學系所學位論文。2017。1-189。

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