【摘要】：目前對漆酶的研究主要集中在白腐真菌漆酶上,但隨著對細菌漆酶研究的增多,結(jié)果發(fā)現(xiàn),細菌漆酶與白腐真菌漆酶相比而言,細菌漆酶具有耐高溫、pH耐受度高、來源豐富、容易在工業(yè)宿主生物中表達便于實現(xiàn)大規(guī)模工業(yè)化生產(chǎn)等優(yōu)點,由芽孢桿菌產(chǎn)生的芽孢漆酶更具有最適溫度高、熱穩(wěn)定性強、pH適用范圍寬,堿性耐受度高等特點,使其具有廣泛的應用前景。(1)芽孢桿菌C3-41產(chǎn)漆酶性質(zhì)的初步探究結(jié)果顯示,常用漆酶檢測體系中,pH4時0.1mol/L磷酸氫二鈉-檸檬酸緩沖溶液1.8mL、0.6mM/L ABTS 0.2mL以及1mL漆酶樣品所組成的3mL緩沖體系為該芽孢漆酶的最佳檢測體系。三種處理方式不同的樣品的漆酶活性和蛋白含量的比較即可得知球形芽孢桿菌C3-41產(chǎn)生漆酶的主要部位是芽孢,且該芽孢漆酶為胞內(nèi)酶。(2)芽孢桿菌C3-41芽孢漆酶的酶學性質(zhì)研究顯示,pH對芽孢漆酶活性影響比較明顯,當pH為4時芽孢漆酶活性最大;該芽孢漆酶的溫度穩(wěn)定性較好,在較高的溫度的下酶活仍然維持在較高的水平上,其最佳溫度高達50℃;在體系中金屬離子終濃度為0.2mmol/L時,Fe2+、Fe3+均對漆酶活性具有抑制,Cu2+、Zn2+、Ba2+、Ca2+對漆酶活性均具有促進作用,其中Ca2+的促進作用最明顯;添加工作濃度的DTT、β-巰基乙醇、EDTA、PMSF四種常用蛋白酶抑制劑發(fā)現(xiàn),DTT和β-巰基乙醇對其酶活的抑制作用較明顯,PMSF的抑制作用最微弱。(3)芽孢桿菌C3-41芽孢漆酶對活性艷藍KN-R、活性艷紅X-3B、孔雀綠三種染料的脫色特性研究顯示,在染料濃度不超過2g/L情況下,該芽孢漆酶對活性艷藍的脫色率隨染料濃度增大而增大,最大濃度時脫色率為87.8%;對活性艷紅的脫色率隨染料濃度增大而減小,最大濃度時脫色率為72.8%;對孔雀綠的脫色率隨染料濃度變化不大,除濃度達到2g/L時脫色率為82.2%外,其他濃度脫色率均大于86%。(4)芽孢漆酶與純化漆酶相比,芽孢漆酶的溫度耐受度和pH耐受度更高本論文研究結(jié)果表明,球形芽孢桿菌C3-41芽孢漆酶對染料脫色、漆酶酶學特性以及以及工程應用中降低成本方面均有較好的利用價值。
[Abstract]:At present, the research on laccase is mainly focused on white rot fungus laccase, but with the increase of bacterial laccase, the results show that bacterial laccase has high temperature resistance, pH tolerance and abundant sources compared with white rot fungus laccase. It is easy to express in industrial host organisms to realize large-scale industrial production. The spore laccase produced by Bacillus has the characteristics of high optimum temperature, strong thermal stability, wide application range of pH and high alkaline tolerance. It has a wide range of application prospects. (1) the preliminary study on the properties of laccase produced by Bacillus C3341 shows that 0.1mol/L disodium hydrogen phosphate citric acid buffer solution 1.8 mL in common laccase detection system, The 3mL buffer system composed of 0.6mM/L ABTS 0.2mL and 1mL laccase samples is the best detection system for the spore laccase. By comparing the laccase activity and protein content of the samples with different treatment methods, it can be found that the main part of laccase produced by Bacillus globularis C _ 3 / 41 is spores. The spore laccase was intracellular enzyme. (2) the enzyme properties of Bacillus C3o41 spore laccase showed that pH had obvious effect on spore laccase activity, and when pH was 4, spore laccase activity was the highest. The temperature stability of the spore laccase is good, and the enzyme activity is still maintained at a high level at higher temperature, and the optimum temperature is as high as 50 鈩，

本文編號：2487706