以Paecilomyces nostocoides生產脂解酶之研究及其應用

Abstract

本研究自金門土壤中篩得一株具有基質特異性的脂解酶生產菌 Paecilomyces nostocoides NTU-FC-LP01 (BCRC 34144)。此菌株具有生產單、雙酸甘油酯脂解酶之能力。其最適生產條件為：以 500 mL 錐形瓶盛裝 100 mL 含有 1 %大豆粉、0.5%單硬酯酸甘油酯、0.5%黃豆蛋白水解物、0.1%酵母抽出物、0.5%磷酸氫二鉀、0.5%硫酸銨、0.05%硫酸鎂、0.1%聚乙烯醇，於 27℃下、以120 rpm 培養 60 小時，可得高的脂解酶活性 ( 76 U/mL)。將上述培養所獲得之培養液，經超過濾濃縮 (MWCO: 10kD)，丙酮沈澱區分、CMC 陽離子交換層析，可以得到比活性為 57 U/mg protein 的脂解酶，其純化倍數約為 56 倍，而回收率約為 4.3 %。以 SDS-PAGE 測得此純化酵素之分子量為 31 kDa。酵素性質分析結果顯示，此酵素屬於鹼性脂解酶，最適反應溫度及 pH 分別為40℃及10.0。在基質特異性方面，此純化之脂解酶只能水解單、雙酸甘油酯，無法水解三油酸甘油酯、橄欖油和大豆油等三酸甘油酯，其水解活性依單、雙酸甘油酯類型而異，依次為單油酸甘油酯、單硬酯酸甘油酯及雙油酸甘油酯。此外，本研究直接以 P. nostocoides NTU-FC-LP01 細胞進行催化酯化反應，可以合成單油酸甘油酯，當提升反應溫度至 60℃，細胞催化反應的專一性可以進一步提高。

In this study, a fungus strain was isolated from the soil in the Kinmen area of R.O.C., which could hydrolyze mono- and diacylglycerol but not triacylglycerol. This strain was tentatively identified as Paecilomyces nostocoides, and designated as P. nostocoides NTU-FC-LP01(BCRC 34144). The optimal cultural conditions of P. nostocoides NTU-FC-LP01 for the production of lipase were established, for which the culture to be shaken at 27℃ with 120 rpm for 60 hours in 500 mL flask containing 100 mL of medium consisting of 1.0 % soybean meal, 0.5% monostearin, 0.5% soytone, 0.1% yeast extract, 0.5% K2HPO4, 0.5% (NH4)2SO4, 0.05% MgSO4．7H2O and 0.1% polyvinyl alcohol. The most lipase activity of 76 U/mL was obtained. The crude lipase was purified with ultrafiltration, acetone fractionation, and carboxy methyl cellulose cation exchange column chromatography. A 56-fold purified lipase was obtained with 57 U/mg of specific activity and 4.3% of recovery. The molecular weight of the enzyme was 31 kDa (by SDS-PAGE) and classified as an alkaline lipase. The optimal temperature and pH for the lipase activity were 40℃and pH 10, respectively. The purified enzyme could not hydrolyze triglycerides (i.e. triolein, olive oil and soybeam oil), but it showed hydrolytic activity against mono- and diacylglycerol (diglycerides). Those of hydrolytic activity depended on the type of glycerides, such as monoolein, monostearin and diolein in that order. In addition, P. nostocoides NTU-FC-LP01 cells were able to directly catalyze esterification for the production of monoolein.When the reaction temperature was raised to 60℃, the reaction specificity of the cells was increased.