【摘要】：香草醛素有“食品香料之王”的美譽(yù),廣泛在食品、煙草、飲料、酒類、化工、醫(yī)藥和各類化妝用品等行業(yè)內(nèi)使用,是一種性能比較穩(wěn)定、香味純正獨(dú)特、留香持久的優(yōu)良香料和食品添加劑。通過微生物法轉(zhuǎn)化底物生成的香草醛即是生物香草醛,有著化學(xué)合成香草醛無法比擬的優(yōu)點(diǎn),符合歐盟和美國等的食品安全標(biāo)準(zhǔn),反應(yīng)條件溫和,對(duì)環(huán)境污染程度小。異丁香酚單加氧酶是轉(zhuǎn)化異丁香酚生成香草醛的唯一關(guān)鍵酶,此酶存在底物抑制和產(chǎn)物抑制以及酶純化困難等問題。為解決此類問題,本論文通過對(duì)異丁香酚單加氧酶進(jìn)行改造,結(jié)合具有兩親性自組裝短肽和自切割功能特性的Mxe內(nèi)含肽,用于目標(biāo)蛋白的表達(dá)和制備;通過米曼模式研究其重組蛋白酶動(dòng)力學(xué)特性。采用CLEAs技術(shù)探究固定化的異丁香酚單加氧酶的轉(zhuǎn)化能力。本論文的方法和主要結(jié)論如下:(1)利用基因工程技術(shù)構(gòu)建重組質(zhì)粒pET(30a)-IEM-Mxe-ELK16,結(jié)合具有兩親性的自組裝短肽和具有介導(dǎo)自切割特性的內(nèi)含肽,誘導(dǎo)表達(dá)重組蛋白。經(jīng)SDS-PAGE分析:融合蛋白的分子量在75 kDa左右,與實(shí)驗(yàn)預(yù)期相符合,說明重組質(zhì)粒構(gòu)建成功。通過分析重組前后酶動(dòng)力學(xué)數(shù)據(jù)可得:IEM-Mxe-ELK16的Vmax、Km和Kcat分別是原始IEM的11.3、11.7和7.2倍,說明重組質(zhì)粒pET(30a)-IEM-Mxe-ELK16酶與底物異丁香酚結(jié)合效率降低,因此可在一定程度上解除底物抑制,使得最大反應(yīng)速率增加。條件優(yōu)化結(jié)果為當(dāng)發(fā)酵培養(yǎng)細(xì)胞OD600為0.7左右時(shí),加入IPTG至終濃度0.2 mg/L,200rpm,30℃誘導(dǎo)8 h,離心收集細(xì)胞,加入9 mL 0.05 mol/L甘氨酸-氫氧化鈉緩沖溶液(pH10.5)配制成100 g/L的細(xì)胞懸濁液,150 mmol/L異丁香酚,1 m L DMSO,25℃,200rpm轉(zhuǎn)化48 h,所獲得香草醛濃度最高可達(dá)1.80 g/L。(2)對(duì)Mxe內(nèi)含肽介導(dǎo)的自切割體系進(jìn)行了初步的優(yōu)化,其最佳自切割條件為:4℃,切割緩沖液(20 mmol/L Tris-HCl,0.5 mol/L NaCl,1 mmol/L EDTA二鈉,pH 8.5)重懸10 OD/m L的大腸桿菌裂解液沉淀,切割24 h。(3)通過CLEAs技術(shù)對(duì)異丁香酚單加氧酶融合蛋白進(jìn)行了初步的固定化研究,制備IEM-Mxe-ELK16交聯(lián)酶的最佳交聯(lián)時(shí)間和交聯(lián)劑濃度為100 mmol/L戊二醛交聯(lián)2h;交聯(lián)酶在50℃-100℃的范圍內(nèi)的具有良好的熱穩(wěn)定性,反復(fù)利用7次之后相對(duì)酶活保留50%以上;交聯(lián)酶的最佳轉(zhuǎn)化條件為:0.025 mol/L碳酸鈉-氫氧化鈉緩沖溶液(pH10.5),10%(v/v)DMSO,100 mmol/L異丁香酚,30℃,200 rpm,轉(zhuǎn)化60 h,所得香草醛濃度為0.96 g/L。
[Abstract]:Vanillin is widely used in food, tobacco, beverage, wine, chemical industry, medicine and various kinds of cosmetics and other industries because of its good reputation as "king of food and spices". It is a kind of stable performance, pure and unique fragrance, and has been widely used in the fields of food, tobacco, beverage, wine, chemical industry, medicine and all kinds of cosmetics. A long-lasting bouquet of spices and food additives. Vanillin produced by microbial transformation of substrate is biovanillin, which has the advantage of chemical synthesis of vanillin, which accords with the food safety standards of European Union and USA. The reaction conditions are mild and the pollution to the environment is small. Isoeugenol monooxygenase is the only key enzyme to convert isoeugenol to vanillin. This enzyme has many problems such as substrate inhibition and product inhibition as well as difficulty in enzyme purification. In order to solve this problem, the isoeugenol monooxygenase was modified to combine the amphiphilic self-assembled short peptide and the self-cleavage function of Mxe to express and prepare the target protein. The kinetic characteristics of recombinant protease were studied by Miman model. The conversion ability of immobilized isoeugenol monooxygenase was studied by CLEAs technique. The methods and main conclusions of this paper are as follows: (1) the recombinant plasmid pET (30a)-IEM-Mxe-ELK16, was constructed by using genetic engineering technology to bind the amphiphilic self-assembled short peptide and the endopeptide which mediates the self-cleavage. The recombinant protein was induced to express. SDS-PAGE analysis showed that the molecular weight of the fusion protein was about 75 kDa, which was consistent with the expectation of the experiment, indicating that the recombinant plasmid was successfully constructed. The enzyme kinetic data before and after recombination showed that the Vmax,Km and Kcat of IEM-Mxe-ELK16 were 11.3, 11.7 and 7.2 times higher than those of the original IEM, respectively, indicating that the binding efficiency of recombinant plasmid pET _ (30a)-IEM-Mxe-ELK16 to isoeugenol was lower than that of the original plasmid pET _ (30a)-IEM-Mxe-ELK16. Therefore, the substrate inhibition can be relieved to a certain extent, and the maximum reaction rate can be increased. The optimal conditions were as follows: when the OD600 of the cultured cells was about 0.7, the cells were induced at 0.2 mg/L,200rpm,30 鈩，

本文編號(hào)：2465978