产耐高温漆酶疣孢漆斑菌的诱变育种及基因分析

发布时间：2018-03-29 06:07

  本文选题：亚硝基胍　切入点：突变　出处：《东北林业大学》2017年硕士论文

【摘要】：本文通过化学诱变的方法即用亚硝基胍(NTG)处理疣孢漆斑菌孢子,使其产生随机突变。通过96孔板高通量筛选的方法,筛选出了两株产耐热性能好的漆酶的菌株,命名为MF-01和MF-02,并对其及其产生的漆酶进行了生理指标检测,结果显示,两株突变株与未突变株比较发酵液中的酶活性,pH以及菌体生长量并没有突出变化,发酵液pH均为下降后上升,菌体生长量为线上升后下降。对两个菌株和未突变株的漆酶进行纯化,得到单一纯蛋白。MF-01的反应最适pH值为3.0,pH稳定性最好的时候在pH3.0-3.5。MF-02的反应最适pH值为4.0,pH稳定性最好时候在3.5-4.0,而未突变株的两值分别在pH4.5和pH4-5之间。说明两个突变株更耐酸性,稳定性更好。在热稳定性和最适温度的实验中,突变株MF-01在30-50℃时有相对较高的酶活,但MF-02与未突变株最适温度的差异不是十分明显。当温度上升至70-90℃,酶活均下降至50%以下,但MF-01的酶活性仍然高于MF-02和未突变菌株。同样在50%的相对酶活条件下,突变株漆酶放置的时间更长。经过分析,两株菌株产生的漆酶均有较好的稳定性,但其中一株MF-01的性能更加突出,并且能够稳定遗传,分析是突变剂使其产漆酶的基因序列发生了变化。为了验证这一猜想,实验用RACE的方法对未突变菌株的产漆酶基因进行了克隆,得到了产漆酶基因的编码区全部序列,并以此基因设计引物,对突变菌株的产漆酶基因编码区进行克隆,对两段基因对比发现,在编码区的第1522 bp左右的位置,有碱基发生了变,由GCT变成了GAT,致使其编码的氨基酸由丙氨酸变成了天冬氨酸,突变并没有对漆酶的铜中心造成结构变化。铜中心是一个相对保守的区域,活性中心的铜离子位点就位于这个由1个半胱氨酸和10个组氨酸残基构成的配体结构中。丙氨酸为中性氨基酸,而转变为天冬氨酸后,变成了带负电荷的酸性氨基酸,组氨酸属于带正电荷碱性氨基酸;分析这两种氨基酸可能产生了分子间作用力,使漆酶蛋白分子更加稳定,但漆酶不仅由氨基酸组成,还有糖基的参与,分析稳定性的变化还有可能是诱变使糖基化过程发生了某些变化,本文仅在漆酶基因水平上做了基础性研究。
[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，

本文编号：1679863