一株产漆酶细菌的分离、鉴定及对造纸黑液处理的初步研究

Isolation and identification of a bacterium strain exhibiting laccase activity and its treatment to papermaking black liquid

利用铜离子作为筛选剂,从凉水国家级自然保护区的土样中分离纯化出一株具有高漆酶活性的细菌。通过形态学观察、生理生化特性测定及16S rDNA序列分析,鉴定其为枯草芽孢杆菌(Bacillus subtilis),命名为B.subtilisWD23。根据GenBank上公布的Bacillus subtilis cotA基因序列设计引物,克隆B.subtilis WD23的cotA基因,并采用pET--22b(+)/E.coli BL21(DE3)系统进行诱导表达,测定工程菌株发酵液的粗提液的漆酶活性达到1700U/mL,表明该菌株漆酶活性来源其芽孢。用固定化的B.subtilis WD23芽孢处理造纸黑液,处理7d色度下降24.9%,化学需氧量(COD)下降31.2%。

A novel bacterium strain exhibiting high laecase activity was separated and purified from the forest soil of the Liangshui National Nature Reserve in Heilongjiang Province with Cu^2＋ as the enrichment culture reagent. The strain was identified as Bacillus subtilis WD23 by morphological observation, physiological and biochemical tests and 16S rDNA sequence analysis. The eotA gene was cloned by primers designed aceording to B. subtilis cotA gene sequence on the GenBank. The cotA gene was inducibly expressed by the system of pET-22b（ ＋ ）/E. coli BL21 （DE3）. The laccase activity of the culture extraction was 1 700 U/mL. The result suggests that the lacease activity is derived from the spore coat protein. The immobilized spore laccase could decolorize black pulping liquid with a decoloration rate of 24.9% and a COD reduetion by 31.2% after seven days of treatment.