發(fā)布時間：2018-03-29 06:07

  本文選題：亞硝基胍　切入點：突變　出處：《東北林業(yè)大學》2017年碩士論文

【摘要】：本文通過化學誘變的方法即用亞硝基胍(NTG)處理疣孢漆斑菌孢子,使其產生隨機突變。通過96孔板高通量篩選的方法,篩選出了兩株產耐熱性能好的漆酶的菌株,命名為MF-01和MF-02,并對其及其產生的漆酶進行了生理指標檢測,結果顯示,兩株突變株與未突變株比較發(fā)酵液中的酶活性,pH以及菌體生長量并沒有突出變化,發(fā)酵液pH均為下降后上升,菌體生長量為線上升后下降。對兩個菌株和未突變株的漆酶進行純化,得到單一純蛋白。MF-01的反應最適pH值為3.0,pH穩(wěn)定性最好的時候在pH3.0-3.5。MF-02的反應最適pH值為4.0,pH穩(wěn)定性最好時候在3.5-4.0,而未突變株的兩值分別在pH4.5和pH4-5之間。說明兩個突變株更耐酸性,穩(wěn)定性更好。在熱穩(wěn)定性和最適溫度的實驗中,突變株MF-01在30-50℃時有相對較高的酶活,但MF-02與未突變株最適溫度的差異不是十分明顯。當溫度上升至70-90℃,酶活均下降至50%以下,但MF-01的酶活性仍然高于MF-02和未突變菌株。同樣在50%的相對酶活條件下,突變株漆酶放置的時間更長。經過分析,兩株菌株產生的漆酶均有較好的穩(wěn)定性,但其中一株MF-01的性能更加突出,并且能夠穩(wěn)定遺傳,分析是突變劑使其產漆酶的基因序列發(fā)生了變化。為了驗證這一猜想,實驗用RACE的方法對未突變菌株的產漆酶基因進行了克隆,得到了產漆酶基因的編碼區(qū)全部序列,并以此基因設計引物,對突變菌株的產漆酶基因編碼區(qū)進行克隆,對兩段基因對比發(fā)現,在編碼區(qū)的第1522 bp左右的位置,有堿基發(fā)生了變,由GCT變成了GAT,致使其編碼的氨基酸由丙氨酸變成了天冬氨酸,突變并沒有對漆酶的銅中心造成結構變化。銅中心是一個相對保守的區(qū)域,活性中心的銅離子位點就位于這個由1個半胱氨酸和10個組氨酸殘基構成的配體結構中。丙氨酸為中性氨基酸,而轉變?yōu)樘於彼岷?變成了帶負電荷的酸性氨基酸,組氨酸屬于帶正電荷堿性氨基酸;分析這兩種氨基酸可能產生了分子間作用力,使漆酶蛋白分子更加穩(wěn)定,但漆酶不僅由氨基酸組成,還有糖基的參與,分析穩(wěn)定性的變化還有可能是誘變使糖基化過程發(fā)生了某些變化,本文僅在漆酶基因水平上做了基礎性研究。
[Abstract]:In this paper, two laccase producing strains with good heat tolerance were screened by chemical mutagenesis method, I. e., treatment of spores with nitrosoguanidine (NTG) and random mutation. MF-01 and MF-02 were named, and the physiological indexes of them and their laccase production were detected. The results showed that there was no significant change in enzyme activity and pH and cell growth between the two mutant strains compared with the non-mutant strains. The pH of fermentation broth decreased and then increased, and the growth of bacteria increased linearly, then decreased. The laccase of two strains and non-mutant strains were purified. The results showed that the optimum pH value of single protein 路MF-01 was 3.0g and the optimum pH value of pH3.0-3.5.MF-02 was 4.0U, while the two values of unmutated strains were between pH4.5 and pH4-5, which indicated that the two mutants were more acid tolerant. In the experiment of thermal stability and optimum temperature, the mutant MF-01 had relatively high enzyme activity at 30-50 鈩，

本文編號：1679863