本文關(guān)鍵詞：脂肪酶產(chǎn)生菌的篩選鑒定、產(chǎn)酶條件優(yōu)化及酶學性質(zhì)研究　出處：《湖北工業(yè)大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 脂肪酶 銅綠假單胞菌 發(fā)酵工藝優(yōu)化 分離純化 酶學性質(zhì)

【摘要】：脂肪酶(E.C.3.1.1.3),又稱三酰甘油�；饷�。在水相中,能將甘油三脂水解生成甘油一酯、二酯或者直接生成甘油和脂肪酸。在非水相體系中,脂肪酶具有良好的脂化、轉(zhuǎn)脂、醇解和胺解等特性,該特性可使脂肪酶廣泛應(yīng)用在藥物合成、造紙、紡織、食品、化妝品等行業(yè)。本文以橄欖油為唯一碳源選育出了一株高產(chǎn)脂肪酶菌株,優(yōu)化了發(fā)酵產(chǎn)酶工藝條件,研究了酶的分離純化及酶學性質(zhì),并探討了菌株及其所產(chǎn)脂肪酶在生物降解餐館廢水中的應(yīng)用。其主要研究內(nèi)容如下:利用油脂同化平板和三丁酸甘油酯平板從生活廢渣中篩選出了一株產(chǎn)脂肪酶的菌株HFE733,其酶活為1.93 U/mL。該菌株經(jīng)形態(tài)學、生理生化及系統(tǒng)發(fā)育學方法鑒定為銅綠假單胞菌(Pseudomonas aeruginosa)。對出發(fā)菌株HFE733進行紫外及硫酸二乙酯誘變,最終得到最優(yōu)突變菌株D13。該菌株經(jīng)搖瓶發(fā)酵測得脂肪酶酶活為5.03 U/mL,比出發(fā)菌株HFE733的酶活提高了160.62%。并通過遺傳穩(wěn)定性試驗證明,D13菌株有良好的遺傳穩(wěn)定性。將其命名為HFED13。采用單因素試驗確定了HFED13菌株發(fā)酵產(chǎn)脂肪酶的最適發(fā)酵條件為:發(fā)酵溫度30℃,發(fā)酵周期60 h,初始pH 7.0,接種量5%,裝液量50 mL/250 mL,搖床轉(zhuǎn)速220 r/min。通過Plackett-Burman設(shè)計試驗、最陡爬坡試驗、響應(yīng)面法最終得到發(fā)酵培養(yǎng)基的最佳配方為:酵母膏34 g/L、橄欖油5.92 m L/L、蔗糖12.5 g/L、硫酸銅0.4 g/L、硫酸錳0.2 g/L。脂肪酶酶活可達到9.27 U/mL,試驗結(jié)果與預測值基本吻合,且比優(yōu)化前酶活提高了84.29%。通過硫酸銨鹽析、Sephandex G-25凝膠過濾脫鹽、DEAE-cellulose A-52離子交換層析的方法,對HFED13菌株發(fā)酵所產(chǎn)脂肪酶進行了分離純化。經(jīng)SDS-PAGE電泳分析,純化后的脂肪酶為單一蛋白,其分子量為51.02 kDa,純化倍數(shù)為9.97,回收率為31.54%,比酶活為79.25 U/mg。純化的脂肪酶最適pH為7.0~8.5,最適反應(yīng)溫度為40℃,該酶在pH6.5~9.0范圍內(nèi)保持穩(wěn)定。不同的金屬離子和化學試劑對該脂肪酶具有不同的影響。其中,Fe3+、Al3+、β-巰基乙醇、半胱氨酸和二硫蘇糖醇對該酶有一定的促進作用,而Co2+、Cu2+、Tween 80和Triton X-100對該酶有一定的抑制作用,10 mM的十二烷基硫酸鈉完全抑制了該酶的活性。該菌株及其所產(chǎn)脂肪酶對餐館廢水有很好的降解作用,通過廢水和發(fā)酵液的混合處理,經(jīng)過六天的培養(yǎng),廢水中油脂含量和COD(化學需氧量)分別由4296.00 mg/L和11449.42 mg/L下降到195.79 mg/L和1243.97mg/L,下降了95.44%和89.14%,基本達到了排放標準。
[Abstract]:Lipase E.C.3.1.1.3, also known as triacylglycerol hydrolase. In aqueous phase, triglyceride can be hydrolyzed to glycerol monoester. In non-aqueous phase, lipase has good characteristics of lipase, such as lipid, fat, alcoholysis and amination, which make lipase widely used in drug synthesis, papermaking and textile. In this paper, a high yield lipase strain was selected with olive oil as the sole carbon source, the fermentation conditions were optimized, and the purification and properties of the enzyme were studied. The application of the strain and its lipase in biodegradable restaurant wastewater was also discussed. A lipase producing strain HFE733 was isolated from the domestic waste residue by using the oil assimilation plate and the tributyrate triglyceride plate. The enzyme activity was 1.93 U / mL. The strain was morphologically. Physiological, biochemical and phylogenetic methods were used to identify Pseudomonas aeruginosa as Pseudomonas aeruginosa. The original strain HFE733 was mutagenic by UV and diethyl sulfate. The optimal mutant strain D13 was obtained. The lipase activity of this strain was 5.03 U / mL by shaking flask fermentation. The enzyme activity of the original strain HFE733 was 160.62% higher than that of the original strain HFE733, and proved by genetic stability test. D13 strain had good genetic stability. It was named HFED13. The optimum fermentation conditions for lipase production of HFED13 strain were determined by single factor test: fermentation temperature was 30 鈩，

本文編號：1403408