Pichia pastoris 生產 Candida rugosa 脂肪酶第三型的純化與性質分析及其在廢報紙脫墨上之應用

Abstract

廢紙回收利用具有確保資源、節約能源、延長纖維資源使用年限及減少污染與CO2 排放等重大意義。尤其台灣地狹人稠，教育普及，紙張消耗甚巨，廢紙之回收再利用益顯重要。本研究乃探討中研院植物暨微生物學研究所蕭介夫教授研究室構築之 Pichia pastoris 轉形株所生產 Candida rugosa 脂肪酶第三型 (CRL3) 在印刷廢報紙脫墨上之應用。經由硫酸銨分劃法、膠體過濾法及疏水性層析法可成功獲得純化之 CRL3，酵素性質分析結果顯示在 pH 5~8，37oC~60oC 的範圍內，酵素活性皆保持穩定，接著針對國內最大宗之新聞廢紙，使用黑白印刷報紙製備紙漿，比較化學及酵素脫墨之脫墨效率，建立具體可行之酵素脫墨流程。當以化學脫墨劑，Sinopol 964H (polyoxyethylene nonylphenyl ether)，於 50 mL 離心管，在 pH 11.5, 50oC, 150 rpm 下震盪 16 小時可測得每克紙漿 (乾重) 內含化學可脫油墨量為 5 mg，以此作為每克紙漿之油墨含量，三小時之脫墨效率可達 86%，在紙漿 (乾重) 與脫墨劑為 10:1 之最適條件下，脫墨速率為 2.4 mg/g．hr。比較 CRL3 之粗酵素液、純化酵素及市售商品脂肪酶 (Sigma) 之脫墨效果，在 pH 7 與 60oC 之最適反應條件下，脫墨效率分別為化學脫墨之 55%、54% 及 73%，每單位酵素活性之脫墨速率分別為 13 μg/g．hr、13 μg/g．hr 及 18 μg/g．hr；或每毫升培養液之脫墨速率為 2.7 mg/g．hr。結果顯示 CRL3 每毫升培養液之脫墨速率，已相當於使用化學脫墨劑之脫墨效果，且無須使用純化後酵素，故在工業應用上極具發展潛力。

Recycling of waste paper has great significance, including economizing energy, reducing pollution and CO2 exhaustion, etc. Because of the high education rate and large paper consumption, recycling of waste paper is fairly important in the especially populated Taiwan. The objective of this study is to explore Candida rugosa lipase 3 (CRL3), produced by recombinant Pichia pastoris, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, in deinking effect of waste newspaper. By ammonium sulfate fractionation, gel filtration and hydrophobic interaction chromatography, simple band of CRL3 on a SDS-PAGE was obtained. Purified enzyme is stable at pH 5~8 and 37oC~60oC. Then comparing the deinking efficiency of chemical and enzymatic method for domestic black and white waste newspaper, our goal is to build the optimal enzymatic deinking procedure. The results show that when chemical agent 964 and pulp react at pH 11.5, 50oC and shake for 16 hours at 150 rpm, the maximum chemical deinking quantity is 5 mg per dry weight and the deinking efficiency can reach to 86 % in 3 hours. Under the optimal condition of mixing pulp and chemical agent in the ratio of 10:1, the deinking rate is 2.4 mg/g．hr. Compared to CRL3 crude extract, purified CRL3 and commercial lipase (Sigma), under the optimal condition of pH 7 and 60oC, the deinking efficiency are 55%, 54% and 73% of chemical deinking, respectively. Moreover, the deinking rate is 13 μg/g．hr, 13 μg/g．hr and 18 μg/g．hr per unit, respectively; or 2.7 mg/g．hr per milliliter culture medium.