【作者】 汪春蕾；

【导师】 赵敏；

【作者基本信息】 东北林业大学 ， 森林生物工程， 2010， 博士

【摘要】 本文从五常林区和凉水国家级自然保护区的森林土壤中筛选出一株具有高漆酶活性的细菌菌株,通过形态学特征、生理生化特性及16S rDNA分析,鉴定该细菌属于枯草芽孢杆菌Bacillus subtilis,定名为Bacillus subtilis WD23。该菌株的产芽孢率与其细菌漆酶酶活成正相关,说明该芽孢杆菌的外壁蛋白具有漆酶活性,可能来源于芽孢外壁组成蛋白CotA。使用B. subtilis WD23芽孢对偶氮和蒽醌类染料进行脱色试验,在24h内脱色率能达到50-90%,用其固定化的芽孢处理造纸黑液,处理7d色度下降24.9%,COD下降31.2%。利用GenBank上公布的B. subtilis CotA基因序列设计上、下游引物,克隆B. subtilis WD23的CotA基因,并转化Escherich coli BL21(DE3)感受态细胞,用IPTG(异丙基-β-D-硫代半乳糖苷)诱导其CotA蛋白表达,分子量约为65 kDa,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于其芽孢外衣蛋白CotA。采用特异的His标签纯化试剂盒对CotA蛋白进行纯化。CotA漆酶对RBBR和刚果红的脱色率在87%以上,对靛红和结晶紫的脱色率也达55%以上。本文的主要研究内容和结果如下：(1)利用铜离子作为筛选剂,从凉水国家级自然保护区的土壤中筛选出一株具有高漆酶活性的菌株。其菌落在含0.2 mmol/LCu2+的培养基上呈灰白色而在不含Cu2+的培养基上呈浅红色。该菌株革兰氏染色反应呈阳性,菌体为短杆状,1-2μm长,能形成芽孢,具有鞭毛和运动性,不能利用木糖和阿拉伯糖这两种典型的枯草芽孢杆菌能利用的碳源。该菌株的16S rDNA片段长度为1513bp,在GenBank中的登录序号为EU780682。由构建的系统发育树分析,该菌株与枯草芽孢杆菌Bacillus subtilis的同源性为100%。综合其形态学特征、生理生化特征及16S rDNA分析,鉴定该细菌属于Bacillus subtilis,将此菌株定名为Bacillus subtilis WD23.(2)通过对B. subtilis WD23的生物学特性研究,得出其最适生长温度是30℃,最适生长pH为7.0,具有一定的抗盐、抗铜、抗紫外线和抗H2O2的能力。(3)以丁香醛连氮作为底物,测定菌株WD23细菌芽孢漆酶的最适反应温度为60℃,最适反应pH为6.8。B. subtilis WD23芽孢漆酶具有很好的热稳定性及pH稳定性,在80℃高温下,其活性半衰期为2.5h,在pH9.0,60℃的条件下,其活性半衰期达10d。EDTA、甲醇和Zn2+等化学物质都强烈抑制细菌芽孢漆酶的活性,而Cu2+、Fe2+和Mg2+却可以增强细菌芽孢漆酶的活性。B. subtilis WD23的芽孢漆酶在24h之内使RBBR和茜素红脱色率达90%以上,刚果红、甲基橙和结晶紫的脱色率也达50%以上,显示了本研究所采用的细菌芽孢漆酶在染料降解上具有很大的应用潜力。(4)固定化芽孢漆酶最适反应温度为70℃。最适反应pH为6.8。固定化芽孢漆酶贮存于30℃,pH 6.8的封闭容器内,其活性半衰期t1/2超过7个月。固定酶的米氏常数Km较非固定化酶的米氏常数Km小。(5)采用固定式填充床装置,利用固定化微胶囊处理造纸黑液,固定化菌株WD23的芽孢漆酶处理黑液的COD去除率为31.19%,固定化真菌新月弯胞霉处理黑液的COD去除率45.86%,二者联合处理黑液的COD去除率却能达到为61.31%,说明共代谢作用更有利于处理像造纸黑液这样的难处理的复杂污染废水。(6)以B. subtilis WD23总DNA为模板,扩增得到1558 bp CotA基因,通过BLAST比对,其与枯草芽孢杆菌的CotA基因同源性很高,达到99%,可见克隆的序列为CotA基因。(7)构建重组表达载体pET22b/CotA,测序得到CotA基因序列共有1542 bp,在GenBank数据库中的登陆号为GQ184294,其氨基酸序列在GenBank数据库中的登陆号为ACS44284。用阳性重组子的质粒转化表达菌株E. coli BL21(DE3),得到了CotA基因的异源表达菌株。用IPTG诱导CotA基因表达,经SDS-PAGE检测到表达的CotA蛋白在粗提液中,用丁香醛连氮作底物,测定粗提液的漆酶活性达到1700 U/mL,表明菌株WD23的漆酶活性来源其芽孢CotA蛋白。为了提高漆酶的产量,优化了漆酶诱导表达条件,结果表明：诱导剂IPTG的浓度为1.0 mmol/L,加入IPTG时菌液的OD600值为1.0,诱导温度为25℃和诱导时间为15h漆酶的产量最高。(8)利用BioEdit软件对CotA氨基酸的亲疏水性进行分析,其亲水性比较强的,表明其易于表达。利用BLAST软件对B. subtilis WD23的CotA蛋白的保守结构分析,其含有铜氧化酶超家族。用Clust X软件将B. subtilis WD23的CotA氨基酸序列与一些典型的细菌和真菌漆酶进行比对,结果表明其与真菌的漆酶氨基酸序列之间的相似度非常低,而与细菌漆酶如E.coli cueO、嗜热栖热菌、薰衣草链霉菌和抗生素链霉菌的漆酶之间的相似度也比较低,但与1GSK_A (B.subtilis CotA)的同源性很高。(9)采用特异的His标签纯化试剂盒对CotA蛋白进行纯化的效果比较好,经Native-PAGE,纯化的CotA蛋白在pH5.0条件下被0.1%丁香醛连氮染成红色。以丁香醛连氮为底物测定CotA漆酶的最适反应温度为45℃,最适反应pH为7.2。pH7.2条件下,80℃时CotA漆酶活性半衰期t1/2为0.5h。在最适反应温度45℃下,pH9.0时CotA漆酶活性半衰期t1/2达到8h。CotA蛋白的Km值小于芽孢漆酶的Km值。CotA漆酶对RBBR和刚果红的脱色率在87%以上,对靛红和结晶紫的脱色率也达55%以上,此CotA漆酶在短时间内对染料能够有效地脱色,能够成为有潜力的工业用酶。更多还原

【Abstract】 A novel strain showing a high degree of laccase activity was screened and purified from soil of WuChang forest and Liangshui National Nature Reserve in Heilongjiang. The strain was identified as Bacillus subtilis WD23 by the morphological, physiological, biochemical characteristics and the comparative analysis of 16S rDNA sequence. It was deduced that the laccases were bound to B. subtilis WD23 spores because the laccase activities correlated well with the rate of production of spores. The laccase activity might come from the spore coat protein CotA. The spore laccase could decolorize azo and anthraquinone dyes with 50% to 90% efficiency in 24 h. The immobiled spore laccases could decolorize black pulping liquor with 24.9% efficiency and decrease COD by 31.2% in 7 days. The CotA gene was cloned by upstream and downstream primers designed according to B. subtilis CotA gene on GenBank. CotA gene was transformed to Escherich coli BL21(DE3) competent cell successfully. The CotA protein, showing a induction band of approximately 65 kDa, was expressed by IPTG (Isopropylβ-D-1-thiogalactopyranoside) induction method. The laccase activity of the extraction of the fermentation broth was 1700 U/mL. The result suggested that the laccase activity derived from the spore coat protein CotA. The CotA protein was purified by the special His Microspin Purification kit. The CotA laccases could decolorize Remazol brilliant blue R (RBBR) and Congo red with 90% and Isatin and Crystal Violet with 50%. The main results summarized as follows:(1) A novel strain exhibiting a high degree of laccase activity was screened and purified from forest soil of Liangshui National Nature Reserve in Heilongjiang with Cu2+ as the enrichment culture reagent. The strain formed pinkish colonies on LB agar plate and formed offwhite colonies on LB agar supplemented with 0.2 mmol/L Cu2+. The strain was a Gram-positive, spore forming, rod shaped,1-2 um long, having flagella and motile bacterium. This strain was unable to utilize xylose and gum sugar, while the classical strains of Bacillus subtilis were able to do according to Bergey’s Manual.Amplification and sequencing of 16S rRNA gene was performed. The strain yielded a PCR product of 1513 bp for 16S rRNA gene. The 16S rDNA sequence was submitted to the NCBI databases under the accession number EU780682. The comparative analysis of DNA sequence with available database from GenBank searched with BLAST showed that the strain was close to the members of Bacillus subtilis. The highest sequence similarity (100%) and phylogeny based on Clustal X 1.81 software indicated that it was a strain of B. subtilis. The strain was identified as Bacillus subtilis WD23 by the morphological, physiological, biochemical characteristics and the comparative analysis of 16S rDNA sequence. (2) The optimum pH of B. subtilis WD23 was 7.0 and the optimum temperature was observed at 30℃. The strain WD23 showed strength of tolerance to NaCl and copper. It exhibited ability of tolerance to ultraviolet radiation and hydrogen peroxide.(3) Using syringaldazine as the substrate to determine the spore laccase activity of the strain WD23, the optimum temperature of the spore laccase was 60℃and the optimum pH was 6.8. The spore laccases exhibited a higher thermal stability and pH-stabilities. The temperature half-life of the laccases was 2.5 h at 80℃and the pH half-life was 10 days at pH 9.0,60℃. The spore laccases were strongly inhibited by EDTA, methyl alcohol and Zn2+, however were activated by Cu2+, Fe2+ and Mg2+. The spore laccases could decolorize Remazol brilliant blue R (RBBR) and Alizarin Red with 90% and Congo Red, Methyl Orange and Crystal Violet with 50%.(4) The optimum pH and temperature of immobilized spore laccase were 6.8 and 70℃respectively. The pH half-life was more than 7 months at pH 6.8,30℃. The Km of the immobilized spore laccases was less than that of the mobile spore laccases.(5) Using immobiled laccases treatment of papermaking black liquid in a fixed packed-bed reactor, the immobiled spore laccases could decrease COD by 31.19%, and the immobiled Curvularia lunata could decrease COD by 45.86%, while both the immobiled spore laccase and the Curvularia lunata could decrease COD by 61.31%. It showed that cometabolism was propitious to processing biorecalcitrance wastewater.(6) CotA gene was cloned using the genomic DNA of B. subtilis WD23 as the template, 45.7℃as the annealing temperature determined by gradient PCR. Sequencing result showed that the gene was made of 1558 bp. The gene had high homology with four sequences those were CotA genes of Bacillus subtilis by BLAST alignment. It showed that the gene cloned was CotA gene.(7) CotA gene and expression vector pET-22b(+) digested respectively by BamH I and Hind III were linked by T4 DNA ligase. Recombinant expression vector pET22b/CotA was obtained by transforming the linked production to E. coli DH5a competent cells. The CotA gene was composed of 1542 bp. The CotA gene was submitted to the NCBI databases under the accession number GQ184294. The sequence showed amount of similarity of 99% to B. subtilis CotA gene of AB007638. The CotA heterogenous expression strain was obtained by transforming positive recombinant plasmid pET22b/CotA to E. coli BL21(DE3).The CotA protein was expressed by IPTG induction method and released by permeation pressure crushing cell measure. The CotA protein, showing an induction band of approximately 65 kDa, was detected in the extraction by SDS-PAGE. The laccase activity of the extraction of the fermentation broth was 1700 U/mL. The result suggested that the laccase activity derived from the spore coat protein CotA. The condition of induction expression was optimized as follows:the concentration of IPTG was 1.0 mmol/L, the OD600 of the culture broth was 1.0 when IPTG was added, the induction temperature was 25℃and the induction time was 15 h.(8) The amino acid hydrophobicity analysis of CotA protein of B. subtilis WD23 showed that its hydrophilicity was stronger, but its hydrophobicity was weaker by BioEdit software. The result showed that the CotA protein was easy to be expressed.The conserved domains of the CotA was Cu-oxidase superfamily by BLAST. The Alignment result of deduced amino acid sequence of CotA of B. subtilis WD23 and the sequences of other bacterial and fungus laccases with Clustal X program domenstrated that the CotA had few similarity to fungus laccases and some bacterial laccases such as E.coli CueO, Thermus thermophilus laccase, Streptomyces lavendulae laccase and S. antibioticus laccase, but had high similarity to the amino acid sequence of B. subtilis CotA of 1GSK_A.(9) The purification result of the CotA protein was good by the special His Microspin Purification kit. The CotA protein was stained red by syringaldazine after Native-PAGE.Using syringaldazine as the substrate to determine the CotA laccase activity, the optimum temperature was 45℃and the optimum pH was 7.2. The temperature half-life of the CotA laccases was 0.5 h at 80℃. The pH half-life was 8 h at pH 9.0.The Km of the CotA laccases was less than that of spore laccases. The CotA laccases could decolorize Remazol brilliant blue R (RBBR) and Congo red with 87% and Isatin and Crystal Violet with 55%. The result indicated that the CotA laccase had the potential to be industrial enzyme.更多还原