甘藷塊根糊化過程澱粉磷解酶降解機制探討

The Studies of Proteolytic Degradation Mechanism of L-Form Starch Phosphorylase from Sweet Potato Roots During Gelatinization

鄧慧馨(Hui-Shin Deng)

指導教授 ： 莊榮輝

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.01585

摘要

植物的L型澱粉磷解酶 (L-form starch phosphorylase, L-SP) 在其分子中央，較動物的肝醣磷解脢多出一段由78個胺基酸組成的片段 (L78)。因為組成及結構特殊的關係，L78極易斷裂，造成L-SP分子裂解成兩群分子量相近的片段 (約50 kDa, F50s)。已知甘藷塊根加熱至45oC會使L-SP產生規律之階段性降解，隨著加熱反應時間的增加，抗體Anti-L78無法辨識任何的L-SP，加熱亦造成甘藷切片之糊化現象。此種L-SP降解似乎具有調節性。進一步觀察發現，在加熱糊化初期，糊化區中幾乎不存在完整的L-SP 110 kDa分子，大部分都降解為特定的F50片段。由於產生特定F50片段的現象只在加熱後第16小時觀察得到，第24小時後L-SP會快速降解，因此我們推測加熱產生的糊化初期 (加熱16小時)，會引發專一性L78降解機制；隨著加熱時間延長至24小時後，糊化情形加劇導致胞器破裂，釋放更多蛋白酶，對L-SP產生隨機且快速的降解。本研究取16小時之糊化樣品，經硫酸銨分劃後以膠體過濾法分離，再經蛋白酶活性分析，得知此專一性降解蛋白酶位於20-40% 硫酸銨分劃。目前已知硫酸銨分劃中主要有四個以gelatin為基質的蛋白酶活性，

關鍵字

澱粉磷解酶蛋白酶降解

並列摘要

The L-form starch phosphorylase (L-SP) is abundant in sweet potato (Ipomoea batatas) roots with a higher molecular mass of 110 kDa. In the middle of sweet potato L-SP, a 78-amino acid insert (L78) was found when compared with its counter part in animal cells, glycogen phosphorylase. L78 was susceptible to multiple proteolytic modification because of its unique amino acid sequence and molecular structure. L-SP was cleaved into two groups of fragments having the molecular mass around 50 kDa (F50s) by these modifications. If the slices of sweet potato roots were heated at 45oC, we could observe the change of the mobility of L-SP on native gel electrophoresis. During the heating process, the sweet potato splices were subjected to gelatinization and the controlled proteolytic modification began. At the beginning of gelatinization (16 h heating), the 110 kDa L-SP almost disappeared on the native or SDS gel. Gelatinization might therefore trigger amechanism to cleave L78 specifically. After longer period of incubation (24 h), L-SP was degraded rapidly in an uncontrolled process. A L-SP specific protease might be induced by the controlled heating process which led to the gelatinization of the sweet potato tissue. Using ammonium sulfate fractionation and gel filtration, the protease activities were isolated and partially purified.

並列關鍵字

Starch phosphorylase ； Proteolytic degradation

參考文獻

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