建構生產透明質酸之大腸桿菌及生產條件之優化

Construction and optimization of Escherichia coli strain for hyaluronic acid production

林星佑(Sing-You Lin)

指導教授 ： 吳立真

國立暨南國際大學/科技學院/應用化學學系/碩士(2018年)

摘要

目前透明質酸傳統生產系統主要是萃取生物結締組織和少數的臍帶與關節滑液。但是由於此系統花費大量的資源以及時間，生產成本高，以及動物體所可能帶來的疾病 例如:禽流感。因此利用微生物生產透明質酸的方式逐漸普及。鏈球菌Streptococcus zooepidemicus 上的HasA基因所造出的透明質酸結合酶(Hyaluronic Synthase)，透過將細胞內環境中的底物UDP-glucuronic acid以及UDP-N-acetylglucosamine合成為透明質酸(Hyaluronic Acid, HA)，再透過膜蛋白將合成的HA推送出至細胞外，因此可透過此種方式大量生產高分子量的HA。然而鏈球菌Streptococcus zooepidemicus 屬於實驗室標準規定之二級細菌，其感染性對於人體以及動物有其危害，造成使用此細菌大量生產HA時在衛生安全上產生疑慮。本論文利用大腸桿菌Escherichia coli進行基因工程，將HasA基因轉殖至大腸桿菌，利用IPTG的誘導與表達，透過不同時間點，以及IPTG濃度變化進行生產條件的優化，達成HA產量的最佳化。所生產的HA利用乙醇沉澱以及離心分離的方式萃取，最後透過NMR進行鑑定。

關鍵字

馬疫鏈球菌 ； 透明質酸 ； 大腸桿菌

並列摘要

The conventional hyaluronic acid (HA) production system is generally extracted from animal connective tissue and a few from umbilical cord and synovial fluid. However, this system takes a lot of resources and time. Therefore, the use of microorganisms to produce hyaluronic acid becomes popular. A membrane protein, hyaluronic synthase (HAS) on S. zooepidemicus converts UDP-glucuronic acid and UDP-N-acetylglucosamine into HA, and extrudes extracellularly of these high molecular weight polymers. However, S. zooepidemicus is a hazardous second class bacteria, which would cause significant safety concerns. Herein, we constructed a pET28a plasmid vector containing HAS A gene that are expressed in an Escherichia coli system regulated by IPTG. The expression of HAS A gene was subjected to different time points and IPTG concentrations to optimize of the production The extraction of HA was achieved by ethanol precipitation and centrifugal extraction, and the resulted HA was examined by NMR spectrometry.

並列關鍵字

Streptococcus zooepidemicus ； hyaluronic acid ； Escherichia coli

參考文獻

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