【摘要】：脂肪酶是一種既可催化水解反應(yīng)又可催化合成反應(yīng)的生物催化劑。因微生物脂肪酶的產(chǎn)量高,便于基因操作,生產(chǎn)無(wú)季節(jié)波動(dòng)等原因,比植物和動(dòng)物來(lái)源的脂肪酶的應(yīng)用更為廣泛。本研究依靠羅丹明B橄欖油初篩培養(yǎng)基從5份土樣中篩選到16株細(xì)菌和16株真菌。通過(guò)測(cè)定發(fā)酵液酶活和專(zhuān)一性,對(duì)菌株進(jìn)行復(fù)篩,并利用SHERLOCK(?)全自動(dòng)微生物鑒定系統(tǒng)、26-28S rDNA或ITS序列測(cè)定等方法進(jìn)行鑒定。復(fù)篩出7株產(chǎn)酶較優(yōu)菌,其中Pseudomonas sp. B1-1、 Acinetobacter sp. B1-2、Acinetobacter sp. B5-1、Trichosporon sp. F1-2為sn-1(3)位專(zhuān)一性,Staphylococcus sp. B2-1、Acinetobacter sp. B3-8和Galactomyces candidum F1-1無(wú)專(zhuān)一性。所有分離菌中T. sp. F1-2的酶活力最高,因此選為重點(diǎn)研究對(duì)象。系統(tǒng)發(fā)育分析顯示其與T. cacaoliposimilis和T. laibachii的進(jìn)化關(guān)系最近。研究發(fā)現(xiàn),T. sp. F1-2的胞外比酶活高于胞內(nèi),所以更有利于純化。通過(guò)50%飽和度的硫酸銨沉淀、8000-14000 Da透析和DEAE-sepharose FF弱陰離子交換柱層析(pH 8.3)的純化步驟,將T. sp F1-2脂肪酶純化了3.96倍,比酶活達(dá)到223.13 U/mmg,經(jīng)過(guò)SDS-PAGE分析,測(cè)得其分子量為32.6 kDa。本論文詳細(xì)研究了T. spF1-2脂肪酶的性質(zhì)。該酶能長(zhǎng)期耐受的最高溫度為45℃,短期最適反應(yīng)溫度為50℃,其最適保藏pH范圍為7.0～9.0,最適反應(yīng)pH為8.0。該酶具有sn-1(3)位專(zhuān)一性。其對(duì)底物的鏈長(zhǎng)也具有明顯的選擇性,最優(yōu)的底物是辛酸酯；該酶在Na+、K+、Ca2+、Mg2+和M112+等金屬離子中酶活穩(wěn)定性較好,Zn2+是其最強(qiáng)的酶活抑制劑。該酶對(duì)各種類(lèi)型的表面活性劑都很敏感,但對(duì)非離子型表面活性劑的耐受性稍好于陰離子表面活性劑。該酶對(duì)多種有機(jī)溶劑都表現(xiàn)出非常突出的耐受性,乙醚、二氯甲烷、甲苯和正己烷還能一定程度上提升其酶活,其對(duì)強(qiáng)極性試劑丙三醇、二甲基亞砜和甲醇的高耐受性在脂肪酶中較少見(jiàn)。深入研究了兩種誘導(dǎo)方式,即外加誘導(dǎo)油和自身合成油脂分別為產(chǎn)酶誘導(dǎo)物。當(dāng)添加誘導(dǎo)油發(fā)酵時(shí),菌體會(huì)轉(zhuǎn)運(yùn)誘導(dǎo)油到胞內(nèi),形成球狀的脂質(zhì)體,并且在轉(zhuǎn)運(yùn)前先將誘導(dǎo)油乳化成極小的油滴,以便于轉(zhuǎn)運(yùn)。低濃度葡萄糖發(fā)酵時(shí),由于第一碳源很快耗盡,轉(zhuǎn)運(yùn)在發(fā)酵24 h時(shí)已經(jīng)很顯著。高濃度葡萄糖發(fā)酵時(shí),雖然并不缺乏葡萄糖,也會(huì)在稍晚于低濃度發(fā)酵一段時(shí)間后開(kāi)始快速轉(zhuǎn)運(yùn)油脂,形成大量脂質(zhì)體,這很可能便是高糖發(fā)酵時(shí)產(chǎn)酶仍維持高水平的原因。經(jīng)過(guò)脂肪酸組成分析確認(rèn),誘導(dǎo)油發(fā)酵時(shí),胞內(nèi)脂質(zhì)體的脂肪酸組成與誘導(dǎo)油一致,確系來(lái)自胞外轉(zhuǎn)運(yùn),這與葡萄糖的初始濃度無(wú)關(guān)。當(dāng)不添加誘導(dǎo)油發(fā)酵時(shí),該菌仍會(huì)通過(guò)轉(zhuǎn)化葡萄糖形成較多的脂質(zhì)體。由于合成脂類(lèi)需要脂肪酶參與,且合成后積累在體內(nèi)又成了自產(chǎn)的誘導(dǎo)油,進(jìn)一步促進(jìn)脂肪酶的生成,因此,即使不添加誘導(dǎo)油,該菌也能生產(chǎn)一定量的脂肪酶。該菌自產(chǎn)的脂類(lèi)主要含五種脂肪酸：肉豆蔻酸、棕櫚酸、硬脂酸、油酸和亞油酸。降低發(fā)酵培養(yǎng)基中的氮源濃度,有利于油脂積累,且會(huì)影響脂肪酸的組成,使得飽和脂肪酸的含量增加,不飽和脂肪酸的含量減少。對(duì)比兩種誘導(dǎo)途徑,直接添加誘導(dǎo)油發(fā)酵產(chǎn)酶的效率要高于自身產(chǎn)油誘導(dǎo)的效率,且采用前種發(fā)酵時(shí),從種子培養(yǎng)就開(kāi)始添加誘導(dǎo)油會(huì)顯著提高產(chǎn)酶量,而對(duì)于后種發(fā)酵,種子培養(yǎng)基中添加油并不會(huì)提高胞外產(chǎn)酶。生產(chǎn)能力偏低是限制T. sp F1-2實(shí)現(xiàn)工業(yè)化生產(chǎn)的主要原因。于是利用常壓室溫等離子體對(duì)野生菌進(jìn)行了誘變處理,并建立了96孔板培養(yǎng)結(jié)合對(duì)硝基苯酚棕櫚酸酯法測(cè)定酶活力的高通量篩選方法,實(shí)現(xiàn)了60個(gè)突變菌株的初篩。以酶活力為篩選指標(biāo)時(shí),突變率和正突變率分別為51.7%和28.3%。8株初篩菌株的搖瓶發(fā)酵結(jié)果顯示,A13和A5的產(chǎn)酶提高最顯著,培養(yǎng)96h后分別比野生菌增加2.64倍和1.54倍,且兩個(gè)突變菌株的遺傳穩(wěn)定性良好。進(jìn)一步的對(duì)比研究發(fā)現(xiàn),突變菌株A13相較野生菌的最大優(yōu)勢(shì)在于提前24 h便能達(dá)到最高產(chǎn)酶量。本論文還利用商業(yè)化酶制劑研究了脂肪酶的兩種重要特性,位置專(zhuān)一性和�；w移。脂肪酶的位置專(zhuān)一性在結(jié)構(gòu)酯的合成和油脂改性中具有重要意義。由于水體系和非水體系的差異,傳統(tǒng)的水解判定法得出的專(zhuān)一性與該酶在合成反應(yīng)中的表現(xiàn)可能并不一致。于是建立了利用月桂酸和山茶油的酸解反應(yīng)來(lái)直接評(píng)估酶在無(wú)溶劑體系中位置專(zhuān)一性的方法。利用此方法,Lipozyme RM IM、L02、L03和L04被鑒定為sn-1(3)位專(zhuān)一性,L01為弱專(zhuān)一性,Novozym 435近似無(wú)專(zhuān)一性。通過(guò)替換酶的底物,模型反應(yīng)的可預(yù)測(cè)性得到驗(yàn)證。根據(jù)酸解法和水解法結(jié)果的對(duì)比分析,兩種條件下酶的位置專(zhuān)一性通常是相同的,除了易受到溶劑體系影響的Novozym 435。因此,新方法能夠避免水解判定結(jié)果在合成反應(yīng)中應(yīng)用的局限性。除了酸解反應(yīng)模型,還建立了一個(gè)酯交換反應(yīng)模型來(lái)研究�；w移的影響因素。模型反應(yīng)的底物為等摩爾量的三月桂酸甘油酯和1,3-棕櫚酸-2-油酸甘油酯,三種固定化脂肪酶參與了此反應(yīng)。通過(guò)測(cè)定甘油三酯的組成和脂肪酸的分布來(lái)檢測(cè)sn-1(3)位的酯交換和sn-2位的�；w移。固定化于聚丙烯的Rhizopus oryzae脂肪酶表現(xiàn)出非常嚴(yán)格的sn-1(3)位專(zhuān)一性,2位發(fā)生的改變非常小。而固定化于二氧化硅的Thermomyces lanuginosus脂肪酶(Lipozyme(?) TLIM)能在24 h內(nèi)完成完全的隨機(jī)化。固定化于聚丙烯的T. lanuginosus脂肪酶能催化2位發(fā)生中等程度的改變。因此,T. lanuginosus脂肪酶和二氧化硅會(huì)促進(jìn)脂肪酸分布的隨機(jī)化,而R. oryzae脂肪酶和聚丙烯則不會(huì)。高水分活度促進(jìn)水解因此會(huì)增加不完整甘油酯的濃度,但同時(shí)也會(huì)抑制這些中間產(chǎn)物的的酰基遷移,最后的結(jié)果是,當(dāng)酯交換率的程度相同時(shí),不同水分活度下2位的�；w移沒(méi)有顯著差異,而低水分活度的主要優(yōu)勢(shì)是能保證甘油三酯的產(chǎn)量。
[Abstract]:Lipase is a kind of biocatalyst which can catalyze both hydrolysis and synthesis reactions. Microbial lipase is more widely used than plant and animal lipase because of its high yield, easy gene manipulation and no seasonal fluctuation in production. This study relied on Rhodamine B olive oil primary screening medium to screen five soil samples. Sixteen strains of bacteria and 16 fungi were screened by determining enzyme activity and specificity of fermentation broth, and identified by SHERLOCK (?) automatic microbial identification system, 26-28S rDNA or ITS sequencing. Seven strains of bacteria with better enzyme production were screened out, including Pseudomonas sp.B1-1, Acinetobacter sp.B1-2, Acinetobacter sp.B5-1, Tri. Chosporon sp.F1-2 is sn-1(3) site specific, Staphylococcus sp.B2-1, Acinetobacter sp.B3-8 and Galactomyces candidum F1-1 are not. T.sp.F1-2 has the highest enzyme activity in all isolates, so it is selected as the key research object. Phylogenetic analysis shows that it has the closest evolutionary relationship with T.caoliposimilis and T.baclaihii. T.sp.F1-2 lipase was purified by 50% ammonium sulfate precipitation, 8000-14000 Da dialysis and DEAE-sepharose FF weak anion exchange column chromatography (pH 8.3). The specific activity of T.sp.F1-2 lipase was 3.96 times and 223.13 U/mmg, respectively. The molecular weight of T.spF1-2 lipase was 32.6 kDa.The properties of T.spF1-2 lipase were studied in detail in this paper.The optimum temperature for long-term tolerance of T.spF1-2 lipase was 45, the optimum reaction temperature was 50, the optimum preservation pH ranged from 7.0 to 9.0, and the optimum reaction pH was 8.0. Zinc 2+ is the strongest inhibitor of the enzyme activity. The enzyme is sensitive to various types of surfactants, but has a slightly better tolerance to nonionic surfactants than anionic surfactants. The enzyme exhibits good stability in various organic solvents. Ethyl ether, dichloromethane, toluene and n-hexane can also enhance their enzyme activity to a certain extent, and their high tolerance to strong polar reagents glycerol, dimethyl sulfoxide and methanol is rare in lipase. Two induction methods, i.e. external induction oil and self-synthesized oil, are studied in depth, which are enzyme-producing inducers respectively. In low concentration glucose fermentation, the first carbon source is quickly exhausted, and the translocation is already significant at 24 h of fermentation. Lack of glucose also causes the rapid translocation of lipids and the formation of a large number of liposomes after a period of fermentation at a later time than at a lower concentration, which may be the reason why enzyme production remains high during high glucose fermentation. This is not related to the initial concentration of glucose. When induced oil is not added, the bacteria can still form more liposomes by converting glucose. Because lipase is involved in the synthesis of lipids and accumulated in the body after synthesis, it becomes a self-produced inducing oil, which further promotes the production of lipase, even without the addition of inducing oil. The lipids produced by the bacteria mainly contain five kinds of fatty acids: myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid. Reducing the concentration of nitrogen source in the fermentation medium is beneficial to the accumulation of lipids, and will affect the composition of fatty acids, which will increase the content of saturated fatty acids and decrease the content of unsaturated fatty acids. Compared with the two induction pathways, the efficiency of enzyme production by direct addition of induction oil was higher than that by self-induction. Adding induction oil from the beginning of seed culture could significantly increase the enzyme production by using pre-seed fermentation, but for post-seed fermentation, adding oil to seed medium would not increase the extracellular enzyme production. The main reason for industrialized production of T.sp F1-2 was that the wild bacteria were mutagenized by atmospheric pressure room temperature plasma and a high throughput screening method was established by 96-well plate culture combined with p-nitrophenol palmitate method for the determination of enzyme activity. The results of shaking flask fermentation showed that the enzyme production of A13 and A5 increased by 2.64 times and 1.54 times respectively after 96 h culture, and the genetic stability of the two mutant strains was good. Two important properties of lipase, location specificity and acyl migration, were studied by commercial enzyme preparations. The location specificity of lipase is very important in the synthesis of structural esters and the modification of lipids. The specificity of Lipozyme RM IM, L02, L03 and L04 was identified as sn-1(3) site specificity, L01 as weak specificity, Novozym 435 as position specificity by acid hydrolysis of lauric acid and Camellia oil. The predictability of the model reaction was validated by substituting the substrates of the enzymes. According to the comparative analysis of the results of acid hydrolysis and hydrolysis, the site specificity of the enzymes under the two conditions is usually the same, except for Novozym 435, which is susceptible to the influence of the solvent system. Limitations of application. In addition to the acidolysis model, a transesterification model was established to study the factors affecting the migration of acyl groups. The substrate of the model reaction was equal molar amounts of glycerol laurate and 1,3-palmitic acid-2-oleic acid glycerol ester. Three immobilized lipases participated in the reaction. The composition of triglycerides was determined. Fatty acid distribution was used to detect the transesterification of sn-1(3) and the migration of acyl groups at sn-2. The immobilized polypropylene-based Rhizopus oryzae lipase showed very strict sn-1(3) site specificity, with very small changes in the two sites. The immobilized silica-based Thermomyces lanuginosus lipase (Lipozyme (?) TLIM) could be completed within 24 hours. Complete randomization. T. lanuginosus lipase immobilized on polypropylene catalyzes moderate changes in the two sites. Therefore, T. lanuginosus lipase and silica promote randomization of fatty acid distribution, while R. oryzae lipase and polypropylene do not. High water activity promotes hydrolysis and therefore increases the concentration of incomplete glycerides. But it also inhibits the acyl migration of these intermediates. The final result is that when the degree of transesterification is the same, there is no significant difference between the two acyl migration under different water activity, and the main advantage of low water activity is to ensure the yield of triglycerides.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：TQ925;Q814

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