本文選題：產(chǎn)酸克雷伯氏菌 + 賴氨酸脫羧酶��； 參考：《江南大學(xué)》2016年博士論文

【摘要】：戊二胺是一種重要的平臺化合物,可以合成高附加值、性能優(yōu)異的新型聚氨酯、新型聚酰胺等材料。用微生物發(fā)酵法生成戊二胺代謝途徑復(fù)雜、產(chǎn)率低難以工業(yè)化。酶法制備戊二胺過程簡單、不需要復(fù)雜的代謝調(diào)控、沒有副產(chǎn)物的累積、避免了戊二胺對微生物細(xì)胞的毒害作用,但是賴氨酸脫羧酶的酶活低、堿性環(huán)境下p H穩(wěn)定性差,是工業(yè)化開發(fā)過程中存在的技術(shù)瓶頸。目前大多研究者對多物種賴氨酸脫羧酶的生物信息學(xué)分析仍不夠充分,僅對目的基因表達(dá)的局部環(huán)節(jié)進(jìn)行考察和研究。本研究選擇在自然界可以過量積累戊二胺的產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶作為研究對象,運用分子生物學(xué)手段對目的基因表達(dá)的轉(zhuǎn)錄過程和翻譯過程進(jìn)行系統(tǒng)調(diào)控,并對酶分子的結(jié)構(gòu)基因進(jìn)行定向改造:包括順式作用元件對基因表達(dá)的影響,特別是對基因轉(zhuǎn)錄起始的調(diào)控、翻譯過程對表達(dá)的影響如密碼子偏愛性、SD序列等調(diào)控元件的優(yōu)化,通過分子定向進(jìn)化構(gòu)造新的酶蛋白。通過這些手段提高賴氨酸脫羧酶的表達(dá)量、活性以及在堿性環(huán)境下的穩(wěn)定性,并對構(gòu)造的新型賴氨酸脫羧酶催化合成戊二胺的過程進(jìn)行研究。大幅度提高了賴氨酸脫羧酶催化合成戊二胺的效率,為生物法戊二胺的產(chǎn)業(yè)化奠定基礎(chǔ),主要研究內(nèi)容和結(jié)果如下:(1)首次對產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶進(jìn)行了異源表達(dá),并對其基因的密碼子和啟動子進(jìn)行了優(yōu)化,顯著提高了賴氨酸脫羧酶的表達(dá)量。對產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶基因ldc進(jìn)行擴(kuò)增,構(gòu)建了p UC18-KOldc質(zhì)粒,轉(zhuǎn)入Escherichia coli K12 MG1655構(gòu)建了重組菌E.coli LN18,實現(xiàn)了產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶的異源表達(dá)。密碼子優(yōu)化將產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶基因的GC含量從54%降低到49%,基因序列與野生型的賴氨酸脫羧酶基因序列相比有81.9%的相似性。密碼子優(yōu)化的菌株E.coli LN20發(fā)酵液中戊二胺的含量達(dá)到6.6 g/L發(fā)酵液,比出發(fā)菌株E.coli LN18提高了50%,比過表達(dá)大腸桿菌賴氨酸脫羧酶的E.coli LN35相比提高了16%,這說明密碼子優(yōu)化顯著提高了產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶在大腸桿菌的表達(dá)效率,也說明產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶的活力比大腸桿菌賴氨酸脫羧酶活力高。構(gòu)建了不同啟動子的重組菌株,其中E.coli LN24合成的戊二胺濃度最高達(dá)到7.6 g/L發(fā)酵液,說明Ptac啟動子更有利于產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶的表達(dá)。(2)基于高通量篩選的產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶基因的RBS(核糖體結(jié)合位點)序列優(yōu)化、分子定向進(jìn)化和相關(guān)生物信息學(xué)統(tǒng)計分析。建立了突變株高通量篩選方法,利用quickchange PCR對RBS的序列進(jìn)行突變,并對1000株突變株進(jìn)行篩選得到合成戊二胺水平最高的菌株E.coli LN1103,是E.coli LN18合成戊二胺水平的2.3倍,通過RBS強(qiáng)度計算器分析和可溶性蛋白表達(dá)量分析,說明核糖體結(jié)合位點序列優(yōu)化可以增強(qiáng)賴氨酸脫羧酶m RNA的翻譯效率。通過易錯PCR對產(chǎn)酸克雷伯氏菌賴氨酸脫羧酶進(jìn)行隨機(jī)突變、進(jìn)行分子定向進(jìn)化。從1000株突變株中篩選得到5株合成戊二胺水平明顯高于對照的重組菌。最優(yōu)菌株E.coli LN3014合成的戊二胺水平達(dá)到11.2 g/L發(fā)酵液,是E.coli LN18合成戊二胺水平的2.5倍。(3)經(jīng)過分子定向進(jìn)化的賴氨酸脫羧酶具備了獨特的酶學(xué)特性,在堿性條件下保持高效催化性和穩(wěn)定性。構(gòu)造的賴氨酸脫羧酶(E.coli LN3014)在p H值8.0下保持36h,相對酶活力保留85%,在堿性環(huán)境下表現(xiàn)出很高的穩(wěn)定性。而大腸桿菌的賴氨酸脫羧酶Cad A在p H 8.0沒有活力。當(dāng)p H值從6.0提高到8.0,野生型的賴氨酸脫羧酶(E.coli LN18)的最大反應(yīng)速度從1.67 mmol/min下降到0.65 mmol/min,下降幅度為60%;而經(jīng)過分子定向進(jìn)化的賴氨酸脫羧酶(E.coli LN3014)在p H 8.0時的最大反應(yīng)速度和p H6.0時相比,僅下降了7.0%。從酶的米氏常數(shù)來看,賴氨酸脫羧酶的點突變并不影響蛋白與底物L(fēng)-賴氨酸的結(jié)合。說明分子定向進(jìn)化改造后的E.coli LN18賴氨酸脫羧酶在堿性條件下具備高效催化性和穩(wěn)定性,可建立酶法合成戊二胺的新工藝,使之具備工業(yè)實施可行性。(4)對重組菌E.coli LN3014在搖瓶和10 L罐規(guī)模進(jìn)行了發(fā)酵產(chǎn)酶特性研究。重組菌產(chǎn)酶過程屬于生長關(guān)聯(lián)型,隨著微生物的生長,酶活不斷提高。通過正交實驗優(yōu)化了搖瓶發(fā)酵條件,E.coli LN3014發(fā)酵液的賴氨酸脫羧酶活為128 U/g發(fā)酵液,比未優(yōu)化時提高了16%。進(jìn)一步考察了重組菌E.coli LN3014在10 L罐的分批發(fā)酵和補料分批發(fā)酵工藝。補料分批發(fā)酵工藝采用p H-Stat的控制策略,流加葡萄糖和氨水,E.coli LN3014的菌體密度OD600可以達(dá)到82,酶活力達(dá)到240 U/g發(fā)酵液,是搖瓶發(fā)酵結(jié)果的1.9倍,實現(xiàn)了E.coli LN3014的高密度培養(yǎng),并顯著高于文獻(xiàn)報道的酶活。(5)對重組菌細(xì)胞催化生成戊二胺的過程參數(shù)進(jìn)行了考察和優(yōu)化,建立了不調(diào)節(jié)p H值就能高效合成戊二胺的反應(yīng)體系。產(chǎn)物戊二胺積累過程中導(dǎo)致的p H上升使酶失活,是酶法制備戊二胺的技術(shù)瓶頸之一。對反應(yīng)體系中的底物濃度、細(xì)胞濃度、輔酶添加量等影響因素進(jìn)行了系統(tǒng)考察,經(jīng)過優(yōu)化,戊二胺生成速率為887mmol/(g-cell·h)。所用的催化劑和催化時間大大節(jié)約,催化效率有了顯著提高,而且整個反應(yīng)過程中不調(diào)節(jié)p H,減少了反應(yīng)體系的無機(jī)鹽含量,對后續(xù)的純化和產(chǎn)品質(zhì)量都有很好的幫助,克服了酶法合成戊二胺催化效率低、反應(yīng)過程中p H升高導(dǎo)致賴氨酸脫羧酶易失活的技術(shù)瓶頸。選用聚乙烯醇包埋法固定化E.coli LN3014細(xì)胞,固定化細(xì)胞可以重復(fù)利用30次進(jìn)行催化反應(yīng),底物到產(chǎn)物的轉(zhuǎn)化率沒有明顯下降,固定化細(xì)胞賴氨酸脫羧酶最適p H向堿性區(qū)域發(fā)生了遷移,在4℃下,固定化細(xì)胞賴氨酸脫羧酶活半衰期為30天,是游離細(xì)胞的2倍。
[Abstract]:Amyl two amine is an important platform compound, which can synthesize high added value, new polyurethane with excellent performance and a new type of polyamides. The metabolic pathway of amyl two amine is complicated by microbial fermentation, and the yield is low and difficult to industrialize. The process of preparing amyl two amine by enzyme method is simple, no need for complex metabolic regulation, no accumulation of by-products, and avoidance of the accumulation of by-products. The toxic effect of amyl two amine on microbial cells is avoided, but the enzyme activity of lysine decarboxylase is low and the stability of P H in alkaline environment is poor. It is a technical bottleneck in the process of industrial development. At present, most researchers are still not sufficient for the bioinformatics analysis of the multi species lysine decarboxylase, only the local link of the target gene expression is entered. In this study, the study selected Klebsiella acid decarboxylase, which can accumulate amyl amyl amines in nature, as the research object, systematically regulates the transcriptional process and translation process of the target gene expression by molecular biological means, and redirects the structure genes of the enzyme molecules: including CIS action. The effect of components on gene expression, especially the regulation of gene transcription initiation, the effect of translation process on expression, such as codon bias, SD sequence and other regulatory elements, to construct new enzyme proteins through molecular directed evolution. Through these methods, the expression of lysine decarboxylase, activity and stability in alkaline environment are improved. The process of synthesis of amyl two amine by new lysine decarboxylase was studied. The efficiency of lysine decarboxylase catalyzed synthesis of amyl amyl two amine was greatly improved, and the basis for the industrialization of amyl two amines was laid. The main contents and results were as follows: (1) a heterologous table was first made to the lysine decarboxylase of Klebsiella acid. The codon and promoter of the gene were optimized and the expression of lysine decarboxylase was significantly improved. The P UC18-KOldc plasmid was constructed by amplification of the lysine decarboxylase gene LDC of Klebsiella acid, and Escherichia coli K12 MG1655 was transferred to the recombinant bacteria E.coli LN18 to realize Klebsiella lysine production. The GC content of Klebsiella lysine decarboxylase gene was reduced from 54% to 49% by codon optimization, and the gene sequence was similar to that of the wild type lysine decarboxylase gene sequence. The content of amyl two amine in the fermentation broth of the codon E.coli LN20 reached 6.6 g/L fermentation broth. The strain E.coli LN18 increased by 50%, increased by 16% compared to the E.coli LN35 that overexpressed the lysine decarboxylase, which indicated that codon optimization significantly improved the expression efficiency of Klebsiella lysine decarboxylase in Escherichia coli, and also indicated that Klebsiella lysine decarboxylase was more active than lysine in Escherichia coli. The carboxyenzyme activity was high. The recombinant strains of different promoters were constructed, in which the concentration of amyl two amines synthesized by E.coli LN24 reached up to 7.6 g/L fermentation broth, indicating that Ptac promoter was more beneficial to the expression of Klebsiella lysine decarboxylase. (2) RBS (ribosome junction) based on high throughput screening of Klebsiella lysine decarboxylase gene. Sequence optimization, molecular directed evolution and related bioinformatics analysis. A high throughput screening method for mutant strains was established. The sequence of RBS was mutated with quickchange PCR, and 1000 strains of mutant strains were screened to get the highest level of amyl two amine, E.coli LN1103, which was 2.3 of the level of the synthesis of amyl two amine in E.coli LN18. RBS strength calculator analysis and analysis of soluble protein expression showed that the optimization of ribosome binding site sequence could enhance the translation efficiency of lysine decarboxylase m RNA. By random mutation of Klebsiella acid decarboxylase by error PCR, molecular directed evolution was carried out. 1000 strains of mutant strains were screened from 5 strains. The level of synthetic amyl two amine was significantly higher than that of the control recombinant. The optimal strain E.coli LN3014 synthesis of amyl amyl two amine reached 11.2 g/L fermented liquid, which was 2.5 times the level of E.coli LN18 synthesis amyl two amine. (3) the lysine decarboxylase after molecular directed evolution has a unique characteristic of enzymology and maintains high catalytic activity and stability under alkaline conditions. The lysine decarboxylase (E.coli LN3014) maintained 36h under P H value 8, the relative enzyme activity remained 85%, and was highly stable in the alkaline environment. The lysine decarboxylase Cad A of the Escherichia coli was not active in P H 8. When p H was increased from 6 to 8, the maximum reaction rate of the wild type lysine decarboxylase (E.coli) From 1.67 mmol/min to 0.65 mmol/min, the decrease was 60%; while the maximum reaction rate of lysine decarboxylase (E.coli LN3014) by molecular directed evolution at P H 8 was compared with P H6.0, only the decrease of 7.0%. from the enzyme's Michaelis constant, the point mutation of lysine decarboxylase did not affect the binding of protein and substrate L- lysine. It is indicated that E.coli LN18 lysine decarboxylase has high catalytic activity and stability under basic conditions. It can establish a new process of enzymatic synthesis of amyl amyl amyl amine, which has the feasibility of industrial implementation. (4) the enzyme production of recombinant strain E.coli LN3014 in the scale of shake flask and 10 L tank was studied. The process belongs to the growth correlation type. With the growth of microorganism, the enzyme activity is continuously improved. The conditions of the shake flask fermentation are optimized by orthogonal experiment. The lysine decarboxylase of E.coli LN3014 fermentation liquid is 128 U/g fermented liquid, and 16%. is further improved by the optimization of the batch fermentation of the recombinant strain E.coli LN3014 in the 10 L cans and the batch batch fermentation. The P H-Stat control strategy was adopted in the feeding batch fermentation process, with glucose and ammonia water added, the density OD600 of E.coli LN3014 could reach 82, the enzyme activity reached 240 U/g fermentation liquid, which was 1.9 times the result of shake flask fermentation, which realized the high density culture of E.coli LN3014, and was significantly higher than the reported enzyme activity. (5) the recombinant bacteria cells were catalyzed. The process parameters for the formation of amyl two amine were investigated and optimized. The reaction system of amyl two amine could be synthesized without adjusting the P H value. The increase of P H caused by the accumulation of amyl amyl amine made the enzyme inactivated, which was one of the technical bottlenecks in the preparation of amyl amines by enzyme method. The concentration of substrate, cell concentration, and the addition of coenzyme in the reaction system were taken as a bottleneck. The reaction factor was systematically investigated. After optimization, the formation rate of amyl amyl amine was 887mmol/ (G-cell. H). The catalyst and the catalytic time were greatly saved, the catalytic efficiency was greatly improved, and the P H was not regulated in the whole reaction process. The inorganic salt content of the reaction system was reduced, and the subsequent purification and product quality were well helped. The catalytic efficiency of the enzyme synthesis of amyl two amine is low and the increase of P H in the reaction process leads to the technical bottleneck of lysine decarboxylase. Immobilized E.coli LN3014 cells with polyvinyl alcohol and immobilized cells can reuse 30 times for catalytic reaction. The conversion rate of substrate to the product is not obviously decreased, and the immobilized cell Reys on the immobilized cells. The optimum P H was migrated to the alkaline region. At 4, the half-life of lysine decarboxylase of immobilized cells was 30 days, which was 2 times of that of free cells.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：TQ925;Q55

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李林，黃長福，蔣國英;一株α－乙酰乳酸脫羧酶產(chǎn)生菌株的篩選及初步鑒定[J];新疆大學(xué)學(xué)報(自然科學(xué)版);1996年03期

2 曹茂新;洪楓;朱利民;;草酸脫羧酶及其應(yīng)用[J];中國生物工程雜志;2005年S1期

3 鄭向麗;林陳水;;重組L-天冬氨酸-β-脫羧酶融合蛋白復(fù)性條件的研究[J];氨基酸和生物資源;2012年01期

4 韋成昱;林日輝;梁躍;龍寒;yそ鴆，

本文編號：1956870