石油脱硫菌Gordonia sp.WQ-01激光诱变选育及关键酶DszC克隆表达分析

Laser-Induced Mutation of Biodesulfurization Strain Gordonia sp.WQ-01 and Cloning Expression of Monooxygenase Gene( DszC)in Escherichia coli

利用脱硫菌株Gordonia sp.WQ-01为出发菌株进行激光诱变育种,考察了不同照射时间和输出功率对菌株的正突变率影响,根据DszC酶活检测、序列比对、空间结构分析以及在大肠杆菌的异源表达研究了突变菌株的关键性能。结果发现,在最适宜诱变条件下(激光照射15 min、输出功率20.0 mW)获得脱硫效率提高28%[0.15 mmol/(L·h)]、有效脱硫时间缩短(36 h)的脱硫菌株,该菌株关键酶DszC活性提高了33.3%。此外,突变菌株脱硫基因dszC的核酸和氨基酸序列、二级结构和三级结构均发生了改变,从而导致酶活显著提高。dszC基因在大肠杆菌BL21(DE3)中经IPTG诱导后可以表达出45 kDa大小的DszC蛋白。

Laser-induced mutation was used to get a mutant of biodesulfurization strain Gordonia sp. WQ-01,and the various irradiation time and output power was investigated. Based on the DszC enzyme activity, nucleotide sequence alignment, structure analysis and heterologous expression in Escherichia coli, the relevant desulfurization characteristic was revealed. The results showed that a highefficient desulfurization strain with the efficiency of 0.15 mmol·L-1·h-1 and a reduced desulfurization time （36 h） was achieved under the optimal irradiation condition, which was 15 min and 20.0 mW. Compared with the parent strain, the key enzyme DszC was improved by 33.3%. The nucleotide and amino acid sequence, second and third structure were changed for dszC in the mutant strain, thus resulted in an increase of enzyme activity. The E. coli BL21（DE3） expressing the dszC produced a protein with 45 kDa with IPTG induction.