後熟過程中香蕉果膠酯酶抑制劑活性變化與其對蔬果果膠酯酶之抑制機制

Abstract

香蕉是台灣最重要的水果之一，是一種很典型的更年性水果，果 實質地會隨著後熟作用的進行而軟化，其中最主要先經果膠酯酶 （Pectinesterase，PE）作用，再經聚半乳糖醛酸酶（polygalacturonase， PG）及纖維素酶（Cellulase）的作用達到軟化的目地。已有多位學者 發現，在數種蔬果植物體中具有抑制果膠酯酶的物質存在，稱之果膠 酯酶抑制劑（Pectinesterase inhibitor，PEI）。本實驗嘗試多種方法來 純化香蕉中PEI，探討其性狀及其對PE 之抑制作用機制。本實驗採 用未催熟的綠香蕉進行溫度變化（10℃組、室溫組、10℃後續室溫組） 貯存實驗，探討香蕉中果膠酵素及果膠酯酶抑制劑的活性變化。PE 在室溫貯存下第14 天始測得活性，於第16 天逹到最高活性，而低溫 貯存組（10℃）PE 活性到第18 天皆沒有被測到，而PEI 活性趨勢則 與PE 活性相反，與日遞減。另外，貯存實驗中的總酚含量變化不大， 故酚類含量與PE 活性消長並無相關性。後熟的香蕉具有PAL（pectate lyase）活性，經再酯化CL-AIS 親和性膠體層析後得知其有兩種同功 酶。利用Sepharose CNBr gel 連接（conjugate）PE 的親和性層析純化 香蕉中PEI，未催熟綠香蕉（PCI 1，Peel Color Index【香蕉果皮顏色 指數PCI 1-7】）萃出液經親和層析純化後，分析發現其PEI 為多醣類， PCI 3 的香蕉萃出液經親和層析純化後，再搭配Western blotting 及免 疫（兔血清抗PE 抗體）染色，可顯示出一條PEI 的專一性蛋白質色 帶，其分子量23.1kDa。推測香蕉中會抑制PE 的物質不只一種，且 隨成熟度的不同而會有不同的抑制物質被合成（蛋白質）或被降解（多 醣類）。PEI 對citrus、香蕉PE 抑制機制屬於競爭型抑制。利用貯存 實驗中未催熟的綠香蕉之第0 天凍乾PEI 粉末作0-100µg/ml 不同濃 度稀釋進行ELISA 和PEI 活性檢測，將PEI 濃度分別對ELISA 吸光 值和PEI 活性作出兩條迴歸曲線（檢量線），再取貯存實驗中各貯存 條件的凍乾PEI 粉末進利ELISA，得到的ELISA 吸光值帶入兩條檢 量線，可得到各貯存條件下的PEI 活性。由結果得知PEI 活性及含量 會隨著貯存時間進行而降低且受溫度（10℃，室溫）影響，低溫下 PEI 活性除第18 天外，仍維持高活性而室溫第18 天PEI 活性降至接 近0。

Banana, one of important fruits in economic in Taiwan, is a very typical climacteric fruit. It softens during ripening due to the first pectinesterase (PE) reaction and the subsequent polygalacturonase (PG) and cellulose actions. Many substances exhibiting PE inhibitory activity, PE inhibitor (PEI), in fruits and vegetables have been reported. In the present study, isolation and purification of banana PEI and its inhibition mechanisms on PEs were conducted using affinity gel chromatographies, ELISA, SDS-PAGE, and western blotting. Green banana with a peel color index (PCI) 1 were divided into low-temperature (10°C) storage group (for up to 18 days) and low-temperature room temperature (25 ± 2 °C) storage group (for 18 days first then 7 days at room temperature) to investigate the changes in PE and PEI activities. In room temperature storage group, PE activity was undetectable until on the14th day and it reached a maximum on the 16th day. In low-temperature storage group, PE activity was not detected and PEI activity decreased with the increasing storage days throughout the 18-day storage. PEI activity remains high during low-temperature storage for up to 18 days, while it was almost undetectable during room-temperature storage for the same period of time. Total phenol content in banana changed slightly during storage so as to be considered irrelevant to the PE activity change during ripening process. Pectate lyase (PAL) activity was detected in ripe banana. From which, two isozymes were isolated by high-methoxylated cross-linked alcohol insoluble-substance (CL-AIS) affinity column. PEI from water extract of green banana (PCI 1) by Sepharose CN-PE affinity chromatography was determined to be a polysaccharide, while PEI from extract of PCI 3 banana by the same chromatographic method was one single protein band with a molecular weight is 23.2 kDa with the aid of Western blotting analysis. It revealed that PEI in banana was in various forms and displayed PE inhibitory activity in different IV ripening processes. Competitive inhibition of banana PEI on both banana and citrus PEs were determined. Crude PEI powder from PCI 1 green banana was diluted to be 0-100µg/mL to detect and construct the calibration curve of ELISA value versus PEI activity to evaluate the PEI activity change during banana ripening. Results showed that PEI activity and concentration decreased with increasing storage period of time. Storage factors such as temperature strongly influenced the PEI activity.