染斑海兔 Aplysia juliana 纖維酶之功能及基因序列分析

Abstract

海洋藻類富含醣類或油滴，這些物質含有高能量，在生質能源的開發上有與陸生植物一較高下的潛力。大型藻類如昆布、石花菜等除了主要成分鹿角菜膠（carrageenan）一類多醣類之外，也含有纖維素。這些纖維素不像陸生植物的纖維素混有木質素，較容易使其轉化成葡萄糖。由於收成藻類與分離纖維素的過程都可能使鹽度提高，若要減免淡化鹽度的步驟，工業上需要處於高鹽環境下仍有高活性的纖維酶來提高分解纖維素的效率。由於潮間帶生活的無脊椎動物能適應潮池中因照射陽光而逐漸提高溫度且濃縮的海水。因此，這些動物可能具有適合在高溫及高鹽環境下分解纖維素的纖維酶。在本研究中， 15種在台灣東北部潮間帶常見的無脊椎動物經纖維酶活性篩選後，發現染斑海兔（Aplysia juliana）的消化液在攝氏55度的篩選條件下具有最高的纖維酶活性。進一步的實驗發現染斑海兔的纖維酶相較於黑麴菌Aspergillus niger與木黴Trichoderma viride兩種商品化的纖維酶，在高鹽度的時候依然能保持一定的活性。目前已從染斑海兔身上取得了一段1163鹼基對的纖維酶基因cDNA片段，此基因會在海兔的食道、嗉囊、砂囊與胃表現。基因序列分析的結果顯示此一纖維素應歸屬於醣水解酵素第九群（glycosyl hydrolase family 9），且它主要參與催化的氨基酸序列保守。這些資料顯示我們發現的這個酵素為海兔內生的酵素。相信它將可用於分解藻類纖維素製造生質酒精。

Marine algae have high content of energy rich carbohydrates or oil and possess comparable potential for bioenergy production like terrestrial plants. Macroalgae, namely seaweeds, contained cellulose without lignin are much easier to be converted into bioethanol than lignocellulose from terrestrial plants. However, the process to harvest seaweed and to isolate cellulose might increase the salinity of the cellulose product. To avoid the fresh water wash step, cellulases that have high activity in high salinity condition are required to breakdown the cellulose efficiently. Because invertebrates inhabited intertidal zone have to adapt the concentration changes of seawater during the low tide, they are potential source of cellulase suitable for digesting seaweed cellulose. Recently, 15 marine invertebrates, which are common species at the northeastern coast in Taiwan, have been screened for their cellulase activities. The digestive juice of sea hare Aplysia juliana showed the highest cellulase activity at 55℃. Furthermore, A. juliana digestive juice could tolerate higher salt concentration than the cellulases from two fungi, Aspergillus niger or Trichoderma viride. An 1163bp cDNA partial sequence of A. juliana cellulase gene (AjCel) has been cloned and the gene expression was identified in esophagus, crop, gizzard and stomach. The result of sequence analysis showed the cellulase of A. juliana belongs to glycosyl hydrolase family 9 (GHF-9) and the catalytic residue is conserved through evolution. These data suggest that A. juliana cellulase is an endogenous cellulase which has high potential for bioethanol production in the future.