一般寡醣無法被人體代謝吸收，但是具有增進消化作用、促進益菌增生、影響營養素吸收及促進細胞訊息傳遞，目前巿面上的寡糖包括了異麥芽醣、異麥芽三醣、果寡醣、乳寡醣等多種。近年來許多研究發現透明質酸寡醣具有特殊生理作用，但是尚未大量生產使用。本論文探討利用基因工程方法在E. coli中大量表現鏈球菌噬菌體之透明質酸降解酶，並製備具金屬親和性磁性微粒親和純化並固定化透明質酸降解酶，進行降解透明質酸製備透明質酸寡醣。
透明質酸(hyaluronic acid，HA)源自於動物組織及微生物筴膜，從動物組織萃取透明質酸的程序繁雜且耗時，從原生鏈球菌發酵雖可產出透明質酸且產量大及成本低，但是所得產品存有致病因子的風險，本論文第二章探討利用基因重組技術，將原生菌種Streptococcus zooepidemicus的透明質酸合成酶(HA synthase)基因(hasA)及尿嘧啶雙磷酸葡萄醣去氫酶(UDP-glucuronic acid dehydrogenase)基因(hasB)於E. coli中表現生產透明質酸，以解決鏈球菌病原菌生產透明質酸所產生之感染風險。僅帶有重組hasA基因之質體的E. coli BL21(DE3)時即可合成出透明質酸。當表現hasB時有助於菌體的生長，同時表現hasA及hasB這兩個基因時，透明質酸產量為單獨表現hasA的2倍，但其產量遠低於S. zooepidemicus原生菌。
不論從動物組織或微生物莢膜中萃取透明質酸的過程需要進行多次的乙醇或季胺鹽錯合物沈澱回收透明質酸，但是沈澱物分離需要以膜過濾或離心的方式進行較耗時且消耗資源。本論文第三章製備帶正電荷性的磁性微粒，利用靜電引力回收帶負電荷之透明質酸，在所施之磁場下可輕易分離吸附透明質酸的磁性微粒與溶液。經氨基丙基三乙氧基矽烷(APTMS)修飾的磁性微粒(NH2-M)無法將吸附的透明質酸完全脫附，但是經幾丁聚醣(chitosan)處理的磁性微粒(Chitosan-M)的透明質酸脫附量則可達吸附量的92.5 %，利用Chiotsan-M直接回收發酵液中透明質酸，則回收率不及8 %，且Chitosan-M的透明質酸吸附量僅達理論值的一半，原因在於發酵液中存在其他帶電荷的物質與幾丁聚醣或透明質酸作用形成了遮蔽效應導致透明質酸與Chitosan-M無法有效作用。
本論文第四章利用E coli表現重組噬菌體透明質酸裂解酶(HA lyase)，HA lyase置於pH4 ~ 8的環境1小時活性維持100 %，置於溫度20 ~ 30 oC的活性也維持在100 %，在溫度35 oC放置1小時活性則剩下20 %，當溫度愈高失活速度愈快；HA lyase可存放於含有500 mM imidazole、500 mM NaCl，pH6，10 mM的磷酸鹽緩衝溶液中在溫度-20 oC、4 oC及室溫(20 oC)長達一個月，活性仍維持在95 %以上。由HA lyase裂解透明質酸所得產物中不飽和鍵含量可計算出產物透明質酸寡醣的分子量平均約2950 kDa，而由MALDI-TOF質譜儀分析所得的分子量主要在1300 kDa左右。此HA lyase具有對透明質酸專一性及所產生之透明質酸裂解產物具有不飽和鍵的特性，已成功可應用於透明質酸定量分析上。
論文的第五章探討製備固定金屬親和磁性微粒(Immobilized metal affinity magnetite，IMAM)直接從含細胞破脆片粗蛋白質溶液中回收及固定化HA lyase，不論使用市售的IMAC用的膠體或自製IMAM固定化HA lyase，其活性皆降至游離態的10 %以下，主要是因為固定化後HA lyase的活性中心被包埋在內部造成立體障礙不易與透明質酸作用，另外固定化HA lyase在pH 5及pH 6帶負電荷與透明質酸產生斥力導致固定化後的活性下降。HA lyase直接固定化於IMAM微粒經重覆使用於透明質酸寡醣之製備，其活性隨著使用次數而降低，在第三次批式反應以後，活性達穩定，推測HA lyase與IMAM之間非親和性作用，在第一次與第二次反應後，HA layse從IMAM上脫落，導致活性下降。

Oligosaccharides such as isomaltoses isomaltotrises, fructooligosaccharides and galactooligosaccharideshave been used as supplementary nutrition fool. Clinical studies have show that administering several oligosaccharides can increase the number of friendly bacteria such as Bifidobacteria and Lactobacillus species in the colon while simultaneously reducing the population of harmful bacteria. Recently, oligomers of hyaluronic acid (Oligo-HA) have been found to specific beneficial biological activity. In order to further test its efficacy, an efficient oilgo-HA preparation method has to be developed. In this study, the hyaluronic acid (HA) lyase of Streptococcus pyogenes bacteriophage H4489A was expression in Escherichia coli, not only purified from crude extract directly by by immobilized metal affinity magnetite (IMAM) but also immobilized on IMAM for oligo-HA preparation.
HA is a linear, unbranched polysaccharide made of alternating N-acetyl-Dglucosamine and D-glucuronic acid. HA is commercially obtained from rooster combs and certain attenuated strains of group C Streptococcus which synthesize this compound naturally as part of their outer capsule. However, these are less-than-ideal sources. All rooster comb-based HA products carry warnings directed to those who are allergic to avian products, while Streptococci can be difficult or expensive to ferment, are challenging to genetically manipulate, and have the potential to produce exotoxins. Therefore, the hasA gene and hasB gene from Streptococcus zooepidemicus, which encodes the enzyme hyaluronan synthase and UDP-gluronic acid dehydrogenase respectively, was cloned and expressed in E. coli in order to develop a new and safer HA producing strain. However, due to the very different membrane structure, the production of HA in the recombinant Gram (-) E. coli was about thousand fold less that in Gram (+) S. zooepidemicus.
Usually HA isolation from the crude HA extract involves with quaternary ammonium compound such as cetyl pyridinium chloride (CPC) or ethanol precipitation. Instead of using filtration or centrifugation to recover the precipitate, submicron size magnetite modified with various positively charged functional groups was prepared to recover HA through electrostatic interaction under magnetic field from fermentation broth. Chitosan-magnetite rather than NH2-magnetite has demonstrated its HA adsorption ability and can achieve 92.5 % elution yield, the purified HA is free from proteins contamination.
Unlike most bacterial HA lyase, HA lyase of Streptococcus pyogenes bacteriophage specifically cleaved HA to unsaturated oligosaccharides which has an optimum absorption at 232 nm. The limiting absorbance showed linearity in the range of concentrations. Based on this fact, a specific, simple, easy to apply, low cost, and fast enough method was developed for routine determination of HA concentration of a microbial HA production process.
HA lyase of Streptococcus pyogenes bacteriophage was employed to prepare oligo-HA. The gene of this phage enzyme was over-expressed in E. coli. Metal chelating ligands were immobilized metal affinity magnetite (IMAM). This IMAM micro-particle was employed to directly recover the recombinant HA lyase from the unclarified crude extract. The HA lyase specifically adsorbed on IMAM was directly used for oligo-HA preparation. The one-step purification-immobilization of HA lyase in reduced the inevitable losses of enzymatic activity during HA lyase purificartion and immobilization. However, the activity of HA lyase immobilized by either IMAM or Pharmacia’s IMAC gel reduced to 10 % of the free HA lyase. The mass transfer resistance between the immobilized HA lyase and the high molecular weight substrate HA contributed to the low activity. On the other hand, the stability of this immobilized HA satisfactory since its activity maintained at the same level after the 2nd repeated used.

淩沛學、侯麗君、賀豔麗、郭學平、楊曉紅、張天民著，透明質酸，中國輕工業出版社
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