利用酵素工程技術提升海藻糖合成酶的酵素轉化效率

Abstract

海藻糖 (trehalose) 存在於許多生物體當中，除了作為能量的儲存形式之外，在脫水等逆境時穩定蛋白質和生物膜的結構，保護細胞免於環境壓力。在食品、化妝、醫藥業上都有廣泛的應用。海藻糖合成酶 (trehalose synthase，TS) 為可逆催化麥芽糖分子內鍵結轉化成海藻糖的酵素，屬於一種麥芽糖異構酶，同時，TS 亦有水解麥芽糖產生葡萄糖的副反應，為海藻糖的生合成路徑之一。本研究在提升 TS 的轉化效率，並以Deinococcus radiodurans trehalose synthase (DrTS) 為研究對象，利用已知結構之 SI (sucrose isomerase) 為模板模擬出 DrTS 立體三級結構。結構顯示，DrTS 的整體結構與一般的 α-amylase 的結構相似，在中央擁有一個催化的區域，由 (β/α)8 的桶狀結構所組成，以及一個富有 loop 的子區域和兩個反向平行的 β-sheet 區域。藉由將麥芽糖偶合來模擬 DrTS 與受質結合的立體結構，發現距離麥芽糖 5 Å 的周圍有 21 個胺基酸會與受質結合。從 DrTS與 SI 的胺基酸序列與立體結構的比對，發現這 21 個胺基酸當中有 9 個是不相同的，推測這 9 個胺基酸可能造成 DrTS 與 SI 功能上有差異的原因，再與其他 TS 的胺基酸比對後，發現這 9 個不同的胺基酸中，有 3 個胺基酸不同於其他 TS，分別為 Thr154、Phe174 和 Gln254。推測這 3 個胺基酸位置可能是決定不同的 TS 具有不同生化性質或轉化率的原因，本研究利用定點飽和突變與高效率純化暨活性篩選系統針此 3 個胺基酸進行突變，篩選出高海藻糖轉化率突變種，其中在 Thr154 突變庫當中，挑選出兩株活性大於野生型三倍者，經 DNA 定序顯示其 Thr154皆被取代為 Phe。此外，Asn317 被預測可能與參與水解反應的水分子結合，而在 316 位置 DrTS 具有一種與其他 SI 明顯不保守的胺基酸 Arg，推測可能也會影響與水分子的結合，以及 Asn253 被預測可能藉由阻礙活性中心的開口處保護中間產物，避免水分子進入催化中心造成水解。為了減少水解的副反應，因此將親水性的 Asn317 藉由定位突變取代成疏水性的 Phe、Leu 或 Ala，並將帶正電的 Arg316 藉由定位突變取代成不帶電性的 Gly，結果顯示 Asn317 突變成 Phe、Leu 或 Ala 以及 Arg316 突變成 Gly 雖然會使水解副反應減少，然而卻會造成酵素活性降低。將 Asn253 藉由定位突變取代成芳香族的 Phe，結果顯示 Asn253 突變成Phe 會使水解副反應與酵素活性完全喪失。此外利用另兩種策略來改善 DrTS 轉化率。一種是偶合葡萄糖異構酶 (Glucose isomerase，GI)，將 TS 反應副產物葡萄糖轉化成果糖，來減少副產物的抑制效果，結果顯示偶合 GI 能提升 DrTS 在高溫的海藻糖轉化率約 4%。另一種方法是抑制 TS 的逆反應，藉由加入 α-葡萄糖苷酶抑制劑 (validoxylamine A、validamycin A、kanamycin A、acarbose 或 azactidine) 或是海藻糖的類似物 (lactose、lactulose 或 isomaltulose)。結果 kanamycin A 抑制逆反應比抑制正反應多 5% ，validoxylamine A、validamycin A 或 acarbose 對正逆反應的抑制程度相近，然而 azactidine、lactose、lactulose或 isomaltulose 對 DrTS 的正逆反應沒有影響。以上結果可以提供從事 TS 之蛋白質工程的參考。Trehalose, a non-reducing disaccharide, existing in various organisms can serve as energy storage and as a protectant of protein and lipid against various stresses. It has become as an important compound in foods, cosmetics and pharmaceuticals industries. Trehalose synthase (TS) is one of the biosynthesis pathways of trehalose, which reversibly catalyzes the intramolecular transglucosylation (isomerization) of maltose to produce trehalose, also known as maltose isomerase, as well as the side reaction of irreversible hydrolysis of maltose to produce two glucose molecules. However, the structure as well as the enzymatic mechanism of TS has not been determined. Sucrose isomerase (SI) and TS have isomerase activity and belong to the same subfamily of the α-amylase family. Three-dimensional structures of three SIs have been determined. The tertiary structural model of Deinococcus radiodurans trehalose synthase (DrTS) which has been characterized as a cold-active enzyme was built using a SI structure as template. The overall DrTS structure is highly conserved with α-amylase family. It possesses a central catalytic domain formed by a (β/α)8-barrel structure, a loop rich subdomain and two antiparallel β-sheet domains. The DrTS-substrate complex model was also built by docking with its substrate maltose. 21 amino acid residues located in close contact with maltose within the 5 Å cut-off distance have been identified and may play important roles in substrate binding. By amino sequence and three-dimensional structure alignments with the SIs, nine of the 21 residues were identified as different from SIs and may play important roles in the TS function. Among these nine distinct residues, Thr154, Phe174, and Gln254 are not conserved in the TS family from various species. To deduce the three residues are relative with biochemical properties and conversion rate of TS, site-specific saturation mutations of these residues were performed to screen for mutant enzymes which exhibit high isomerization activity. A high-throughput purification and assay system was developed to screen Thr154 random libraries, resulting that 2 coloies with higher activies than the wild type, showing 3-fold increased activity, DNA sequencing showed that Thr154 were altered to Phe. Furthermore, the Asn317 was predicted to interact with the water molecule which may participate in the hydrolysis side-reaction. The Arg316 may influence the catalytic water binding whereas it is non-identical in the SIs. The Asn253 was predicted to be able to prevent hydrolysis by blocking the entrance of the active site pocket. To reduce hydrolysis activity of the side reaction, the hydrophilic Asn317 were altered to hydrophobic Phe, Leu or Ala by site-directed mutagenesis. The non-identical and positively charged of Arg316 to nonpolar Gly may result in reducing hydrolysis side-reaction, and the Asn254 were altered to aromatic Phe.The results showed that the activities of N317A, N317F, N317L and R316G mutants were signicantly decreased, and N253F mutant led to a complete loss in activity. In addition, two approaches were also performed to improve the conversion rate of DrTS. 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 the trehalose conversion rate of GI-coupled DrTS was enhanced. The other one is inhibiting the reversed reaction of TS by adding α-glucosidase inhibitors (validoxylamine A, validamycin A, kanamycin A, acarbose or azactidine), or trehalose analogs (lactose, lactulose or isomaltulose). The results indicated that both the forward and reversed reactions of DrTS were inhibited by validoxylamine A, validamycin A, kanamycin A and acarbose. However, azactidine, lactose, lactulose and isomaltulose almost had no inhibition effect on the forward and reversed reaction of DrTS. In general, the results provide feasible methods to improve the conversion rate of DrTS for industrial application of trehalose production.