Clone And Heterologous Expression Of CotA Gene Of Spore Coat Protein Pocessing Bacterial Laccase Activity

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.