麥芽糖轉葡萄糖基酶修飾樹薯澱粉之分子結構與理化性質

Abstract

麥芽糖轉葡萄糖基酶 (EC 2.4.1.25; amylomaltase; AMase) 是一轉移酶，可改變澱粉分子結構，並影響澱粉理化性質及食品應用性。本研究以AMase為工具，生產不同分子結構的酵素修飾澱粉，探討澱粉分子結構對理化性質的影響。自嗜熱菌 Thermus thermophilus (T. thermophilus) 分離AMase基因，以pET-20b(+) 載體接合後轉形至E. coli BL21 (DE3)，經過70oC熱處理30分鐘，即可取得高純度酵素，適合澱粉及酵素工業使用，進一步再以鎳親和性層析管柱純化，可得到更高純度的AMase，適合學術上對酵素作用模式的探討。1% (w/w) 的樹薯澱粉以不同酵素活性 (E/S, enzyme/substrate ratio) 及修飾時間，改變澱粉分子結構及回凝特性，生產出不同分子量、鏈長分布的澱粉，並解析分子量及鏈長對回凝特性、冷凍安定性的影響。AMase修飾澱粉在短時間作用下，分子量會改變，並受酵素活性分成兩個途徑。高酵素活性 (≥ 2 U/g) 下，分子量先增加再下降，低酵素活性 (≤ 1 U/g) 下，分子量會隨修飾時間增加而增加，直鏈澱粉含量及鏈長皆會下降，表示直鏈澱粉被AMase水解後轉移到支鏈澱粉，使支鏈澱粉鏈長變長。支鏈澱粉鏈長DP 9–25會被增長為DP > 25，高活性修飾以增加DP 26–75的鏈為主，低活性修飾以增加DP 35–55的鏈為主，表示短鏈A鏈及B1鏈會增長成B2鏈、B3鏈，甚至B4鏈。AMase修飾後，澱粉-碘錯合物的最大吸收波長會往短波長移動，與直鏈澱粉的減少與支鏈澱粉鏈長提高有關，更多的碘與支鏈澱粉錯合。高活性及長時間的AMase修飾，會使長期回凝程度提高。AMase修飾後，會使冷凍解凍的離水率降低、抗凍性提高，低直鏈澱粉含量有較佳抗凍性，但分子量的下降會使抗凍性變差。支鏈澱粉長短鏈比例 (L/S) 可作為分子結構指標，與長期回凝呈高度正相關。透過多種結構的AMase修飾澱粉，(E/S)2t可作為一預測AMase作用模式的指標，以49.2為分界，區分成以歧化活性 (disproportionation) 主導的分子量提高，或者是水解活性主導的分子量降低，可於操作前事先預測終產品的分子量，依需求調控，酵素活性及修飾時間。其中，10 U/g修飾10分鐘具有最佳的產業應用性，僅10分鐘修飾即可達到高分子量及良好的冷凍解凍安定性。綜合上述，本研究以L/S建立澱粉分子結構及理化特性的關聯性，並以 (E/S)2t建立酵素活性及修飾時間對分子結構的影響，並了解AMase的作用模式。

Amylomaltase (EC 2.4.1.25, AMase) is a transferase, which can transfer α-1,4-linked glucosyl units from one glucan to another. Properties and applications of starch influenced by its structure, which changed by AMase. This study was to explore the relationship between molecular structure and physicochemical properties of starches using AMase. AMase from Thermus thermophilus (T. thermophilus) was overexpressed by E. coli BL21 (DE3) constructed in pET-20b(+). High purity of AMase was prepared after heating at 70oC for 30 min for starch and enzyme industry. For the academic research on the action mode of enzyme, AMase could be further purified by nickel affinity column chromatography with higher purity. 1% (w/w) of cassava starch was modified with AMase at different enzyme/substrate ratios (E/S) and reaction times to explore the structural changes and the retrogradation properties. The amylomaltase-treated starches (ATS) were produced with different molecular weight and chain length distribution, and their effects on the retrogradation property and frezze-thaw stability were studied. ATS showed an increase in weight-average molecular weight (Mw) at a short reaction time, and the action mode of AMase revealed two trends dependent on E/S. At high E/S (≥ 2 U/g), Mw increased initially and then decreased with time. At low E/S (≤ 1 U/g), Mw increased slightly with time. Amylose was hydrolyzed, its content and degree of polymerization (DP) were reduced, and the hydrolyzed segments were transferred to amylopectin, thereby extending the chain length by AMase reaction. Some DP 9–25 amylopectin chains were elongated to DP > 25, resulting in the increase in DP 26–75 at high E/S and in DP 35–55 at low E/S; the short chains (A or B1 chains) of amylopectin were extended to long chains (B2, B3, and B4 chains). The maximum absorption wavelength (λmax) of starch-iodine complex was shifted to shorter wavelength indicating that more iodine reacted with longer amylopectin chains due to the reduction of amylose and extending of amylopectin chains. More iodine molecules were complxed with amylopectin chains. AMase treatment resulted in a severe long-term retrogradation at high E/S along with long reaction time. After AMase treatment, the syneresis after freeze-thaw stability were reduced indicating the higher frozen stability. Low amylose content showed better stability; however, the reduction of molecular weight made the stability worse. The long-to-short chain ratio in amylopectin (L/S), a parameter of molecular structure, was positively correlated with long-term retrogradation. (E/S)2t was also proposed as a parameter to determine whether the domination of AMase action mode was disproportionation or hydrolysis, with a critical value of 49.2. The desired molecular weight of end products could be predicted and manipulated by E/S and reaction time. For industrial utility, the starch with higher molecular weight and better freeze-thaw stability were treated at 10 U/g for 10 min. Overall, A new insight into the structural characteristics and physicochemical properties of ATS was revealed through L/S, E/S, and reaction time, as well as an understanding of the action mode of AMase.