【摘要】：漆酶(苯二醇:氧氧化還原酶,EC 1.10.3.2)是一種含銅的多酚氧化酶,是藍(lán)色多銅氧化酶家族的重要成員,由于其具有催化多種底物的氧化能力,已經(jīng)引起食品、造紙、環(huán)保、紡織及生物傳感器等各種工業(yè)應(yīng)用的重點(diǎn)關(guān)注。為了能滿足漆酶日益增長的市場需求,本論文開展了變色栓菌產(chǎn)漆酶發(fā)酵過程優(yōu)化的研究,建立了優(yōu)化漆酶發(fā)酵的玉米漿及法尼醇添加策略,并進(jìn)一步在通氣攪拌式發(fā)酵罐中進(jìn)行了變色栓菌漆酶發(fā)酵優(yōu)化策略的放大實(shí)驗(yàn),為后續(xù)漆酶的工業(yè)化生產(chǎn)奠定基礎(chǔ)。玉米漿的添加明顯促進(jìn)了變色栓菌發(fā)酵產(chǎn)漆酶,在優(yōu)化的玉米漿添加濃度為20 g L-1的條件下,發(fā)酵液漆酶活力在第5天達(dá)到633.3 UL-1,比對照提高了 96.3%。玉米漿中的酚類物質(zhì)經(jīng)鑒別有5種,分別是香草酸、咖啡酸、對香豆酸、阿魏酸和芥子酸,其中咖啡酸及對香豆酸對變色栓菌的產(chǎn)酶沒有影響,香草酸及阿魏酸是有效的產(chǎn)酶促進(jìn)劑,芥子酸能抑制變色栓菌的產(chǎn)酶,而香草酸和阿魏酸二者具有共同促進(jìn)漆酶生產(chǎn)的作用。添加真菌信號分子法尼醇具有顯著促進(jìn)變色栓菌發(fā)酵產(chǎn)漆酶的作用,在發(fā)酵開始時添加60 μM的法尼醇,發(fā)酵周期6天,發(fā)酵液漆酶活力達(dá)到629.3 UL-1,比對照提高92.3%。對菌絲形態(tài)觀察后發(fā)現(xiàn),添加法尼醇能形成許多超支化的結(jié)構(gòu)。法尼醇的添加能引起變色栓菌胞內(nèi)H202含量的增加,第1天時胞內(nèi)H202含量是對照的2.7倍,H202含量的增加引起細(xì)胞防御機(jī)制的啟動,漆酶在細(xì)胞抵御氧化脅迫中起著關(guān)鍵的作用。對變色栓菌漆酶lac基因及rhoA基因進(jìn)行定量分析,添加法尼醇后lac基因表達(dá)量比對照要高,第3天時lac基因表達(dá)量最高是對照的2.26倍,基因表達(dá)量的增加提高了漆酶的合成能力;與菌絲形態(tài)有關(guān)的rhoA基因表達(dá)量也因法尼醇的添加而提高,第3天時最高rhoA表達(dá)量是對照的4.4倍,rhoA基因的超高表達(dá)導(dǎo)致菌絲的超支化,從而促進(jìn)了漆酶的分泌,采用法尼醇的優(yōu)化策略,在5 L和2000 L通氣攪拌式發(fā)酵罐中完成變色栓菌發(fā)酵產(chǎn)漆酶的放大。在5 L發(fā)酵罐中法尼醇的優(yōu)化變色栓菌發(fā)酵漆酶水平為704.5 U L-1,比未加法尼醇的提高105.6%,比搖瓶中的產(chǎn)量629.3 U L-1高11.9%。在2000 L發(fā)酵罐中法尼醇優(yōu)化變色栓菌發(fā)酵漆酶水平為543.6 U L-1,比未加法尼醇的對照組提高53%。發(fā)酵結(jié)束,發(fā)酵液經(jīng)過濾濃縮可制備粗酶液。經(jīng)鹽析-柱層析分離純化及磁性分離均能制得純酶,但磁性分離更具優(yōu)勢及工業(yè)應(yīng)用的價值。漆酶純酶的最適反應(yīng)pH為4.0,最適反應(yīng)溫度25℃,在40℃仍然具有較高活性,10 mM的Cu2+能夠提高漆酶活性10%。漆酶能夠降解多種酚類物質(zhì),其降解最適pH條件為4.0-5.0,而最適反應(yīng)溫度:4-甲氧基酚和2,4-二氯酚為35℃,苯酚為25℃。漆酶能夠降解多種染料,所降解染料的最適降解pH和溫度為5.0和40℃,染料脫色速率的大小主要受染料結(jié)構(gòu)及性狀的影響。
[Abstract]:Laccase (Phenediol: oxygen Oxidoreductase, EC 1.10.3.2) is a copper-containing polyphenol oxidase and an important member of the blue polycopper oxidase family. Paper, environmental protection, textile and biosensor and other industrial applications focus on. In order to meet the increasing market demand of laccase, this paper studied the optimization of laccase fermentation process by Color-changing suppository bacteria, and established the strategy of cornmilk and farnesol adding to optimize laccase fermentation. Furthermore, the optimization strategy of laccase fermentation of Color-changing suppository was carried out in the aerated agitation fermenter, which laid a foundation for the subsequent industrial production of laccase. The addition of corn pulp significantly promoted the laccase production by Color-changing Thrombidium. The laccase activity of the fermentation broth reached 633.3 UL-1, on the 5th day, and the laccase activity of the fermentation broth increased by 96.3g 路L ~ (-1) compared with the control at the optimized concentration of 20 g 路L ~ (-1). The phenolic substances in corn pulp were identified as vanillic acid, caffeic acid, p-coumaric acid, ferulic acid and erucic acid. Vanillic acid and ferulic acid are effective promoters for producing laccase. Erucic acid can inhibit the production of laccase, while vanillic acid and ferulic acid can promote laccase production together. The addition of fungous signal molecule farnesol could significantly promote the laccase production of Color-changing Thrombium. At the beginning of fermentation, 60 渭 M farnesol was added. The fermentation period was 6 days, and the laccase activity of the fermentation broth reached 629.3 UL-1,. 92.3% higher than the control. After observation of mycelium morphology, it was found that many hyperbranched structures could be formed by adding farnesol. The addition of farnesol could increase the intracellular H202 content of Colotropic suppositories. On the first day, the intracellular H202 content was 2.7 times higher than that of the control, and the increase of H202 content led to the initiation of the cell defense mechanism. Laccase plays a key role in cell resistance to oxidative stress. The quantitative analysis of laccase lac gene and rhoA gene in Color-changing suppositories showed that the expression of lac gene was higher than that of the control, and the highest expression of lac gene was 2.26 times higher than that of the control on the 3rd day. The ability of laccase synthesis was improved with the increase of gene expression. The expression of rhoA gene related to mycelium morphology was also increased by the addition of farnesol. The highest expression of rhoA was 4.4 times higher than that of the control on the third day. The hyperbranched expression of rhoA gene led to hyphal hyperbranching, which promoted the secretion of laccase. The fermentation of laccase was amplified in 5 L and 2000 L aerated agitation fermenter with farnesol. The optimal laccase level of farnesol in 5 L fermenter was 704.5 U L -1, 105.6% higher than that without farnesol, and 11.9 U L ~ (-1) higher than that of shaking flask (629.3 U L ~ (-1). In a 2000 L fermenter, the laccase level of fermentative laccase of the strain was 543.6 U L -1, which was 53% higher than that of the control group without farnesol. At the end of fermentation, the fermentation broth can be filtrated and concentrated to produce the crude enzyme solution. The purified enzyme can be obtained by salting-column chromatography and magnetic separation, but magnetic separation has more advantages and industrial application value. The optimum reaction temperature of laccase was 4.0, and the optimum reaction temperature was 25 鈩，

本文編號：2326095