嗜熱菌Thermus thermophilus海藻糖合成酶之蛋白質工程

Abstract

海藻糖(trehalose)是由兩分子葡萄糖以α-1,1-糖苷鍵連接而成的非還原性雙糖，能作為生物體內能量儲存與碳源的型式，以及在惡劣環境下，用以穩定生物膜與防止蛋白質變性的保護因子。因其具有許多特殊的物理及化學性質，海藻糖目前已廣泛應用於食品、化妝品及醫藥等工業。海藻糖合成酶(trehalose synthase，TS)可直接將麥芽糖異構化(isomerization)成海藻糖。由於反應只需要單一酵素且原料便宜，具有應用在工業上生產海藻糖的潛力。然而TS催化的反應為可逆反應，亦能將海藻糖轉化成麥芽糖，同時，TS具有不可逆的麥芽糖水解副反應，此水解反應會隨溫度上升而增加，而且副反應產物葡萄糖對TS活性具有抑制作用。因此，逆反應及副反應兩者都會導致海藻糖產量下降，若能降低TS的逆反應速率，以及增進熱穩定性，使水解反應降低，則可以提升海藻糖轉化率，使TS更適於工業應用。Thermus thermophilus海藻糖合成酶(TtTS)是目前已知的一種嗜熱性TS，具有工業應用優勢，但基因重組之TtTS於高溫(65℃)時轉化率僅52%，產量不高，因此本研究期望藉由蛋白質工程技術提高TtTS反應速率與轉化率。提升反應速率方面使用兩種策略，一種為給予TS偏好的受質alpha麥芽糖為正反應作用的主要受質，結果顯示，TS於含有較多alpha麥芽糖的反應中，反應速率提升，但轉化率沒有改變。另一種方法是利用偶合葡萄糖異構酶(Glucose isomerase，GI)，將TS副產物葡萄糖轉化成果糖，來減少副產物的抑制效果，結果顯示，適量的GI能提升TS反應速率，然而對於最終的轉化率仍沒有影響。在提升TtTS轉化率方面，根據模擬的嵌合麥芽糖與海藻糖之TtTS三級結構，分析酵素與受質的結合情況，預測出與麥芽糖無明顯結合力，並且不具有TS家族保守性的胺基酸有Phe141、Phe163、Ile140、Asn244，搭配本研究所建立的高通量TS純化暨活性篩選系統，分別進行四個位點之定位飽和突變，以及多點飽和突變，此外，亦進行TtTS全基因的隨機突變，進行酵素定向演化。結果顯示，篩選足夠數量的單位點定位飽和突變庫後，未發現高於原TtTS轉化效率之突變株，而多點飽和突變與隨機突變庫中各挑了1000個突變株，亦尚未篩選到轉化率提升之突變株。經定序發現，當Phe141突變為Leu141且Phe163突變為Val163時，具有協同作用，能維持轉化率與原TtTS相當，此結果顯示，多點飽和突變的策略非常有機會能組合出轉化率提升的突變酵素。然而，多點飽和突變與隨機突變需要篩選龐大數量之突變株，研發更高效率的篩選系統將有助於得到轉化率提升之突變株。Trehalose is a non-reducing disaccharide, formed by two glucose units with an α-1,1-glycosidic linkage. It has many important physiological functions such as carbon source and energy storage,a protectant of protein and lipid against various environmental stresses. Trehalose has been widely applied in foods, cosmetics and pharmaceuticals industries. Trehalose synthase (TS) can reversibly catalyze the intramolecular transglucosylation of maltose to trehalose. Since the reaction requires only a single enzymetic step and inexpensive substrate, TS has the potential for producing trehalose in industry.However, the use of TS in industry still faces the problem of low conversion rate due to the reversed reaction and the irreversible side-reaction of maltose hydrolysis. Especially, the maltose hydrolysis activity increases when the reaction temperature rises and the product glucose inhibits the TS activity. Therefore, enhancing the forward isomerization activity and reducing the side reaction activity may improving the conversion rate of TS. The thermophilic Thermus thermophilus trehalose synthase (TtTS) is a promising enzyme for the trehalose production. However, at the optimal temperature (65 ℃), the trehlaose conversion rate of the recombinant TtTS is only 52%. In this study, we applied the protein engineering to enhance catalytic activity and the trehalose conversion rate of TtTS. Two appproaches were performed to improving the catalytic activity of TtTS. One is providing TS-prefered α-maltose anomer as major substrate. The results indicated that the initial reaction rate was higher when high concentrations of α-form maltose were used as substrates. However, the conversion rates were not affected. The other one is coupling TS with glucose isomerase(GI) which converts and thus removes the side-reaction product glucose into fructose to reduce the product inhibition. The results indicated that adequate amount and early addition of GI overwhelmed the glucose inhibition and enhanced the TtTS reaction rate, while the conversion rate was not affected. To improve the conversion rate of TtTS, we modeled the tertiary structures of TtTS and TtTS-maltose complexes and analyzed the residues located in close contact with maltose. Ile140, Phe141, Phe163 and Asn244 were found not unique in TS family features and showed no significant interactions with maltose. These 4 residues were chosen for site-specific saturation and multiple-sites saturation mutagenesis. In addition, random mutagenesis was also performed on whole TtTS gene, By using high-throughput TS screening system, 200 mutants for site-specific saturation and 1000 mutants for multiple-sites saturation and random mutagenesis, respectively, have been screened. The results indicated that no mutants with improving conversion rate were identified. By DNA sequencing, the combination of Phe141Leu and Phe163Val showed similar conversion rate to that of the wild type and implicated that simultaneous multiple mutations in a random manner possibly result in improved mutants due to the synergistic effects. In general, large-amount and labor-intensive screening are necessary for multiple-sites saturation and random mutagenesis. Some other high-throughput screen system should be established to screen the mutant library for the beneficial combination of mutations in the future.