| In the waster of tanning and electroplating industry, chromium is the heavy metal hazard which is more difficult to deal with. Cr(Ⅵ) is the second-largest heavy metal pollutants after Lead contamination.It has a strong biological toxic and serious affection for agriculture, flora and fauna, human body and ecological environment. Cr(Ⅵ) reducing bacteria can reduce toxicity and pollution by restore Cr(Ⅵ). In this article, we explored in detailed for the restore characteristics and mechanism of reducing bacteria.The Cr(Ⅵ) reduction bacteria C-2 was isolated from activated sludge and it was Bacillus thuringiensis by morphological, physiological and biochemical and 16S rDNA identified. It can tolerant 250mg/L Cr(Ⅵ) and had better reducing capacity at pH 7.2. And C-2 strain could tolerate 2.6 mg/L Ag+,22 mg/L Hg2+,242 mg/L Cu2+, 205 mg/L Zn2+,1000 mg/L Co2+,4900mg/L Mn2+.The reduction of Cr(Ⅵ) in the determination of different factors showed that the conditions of 37℃ and pH9.0 were good for C-2 strain to reduce the Cr(Ⅵ). And xylose, fructose, corn cake powder, malic acid, succinic acid, citric acid could promote reduction rate, but glucose, maltose, sucrose, NH4Cl, urea had the opposite effect. Tthe inoculum size increased, the reduction time faster. The ions of Cu2+、 Fe2+、Ca2+ had an active role for reduction, the Co2+、Zn2+ had bad influence. The results also showed that the resting cells reduce Cr(Ⅵ) was faster than the penetration of cells, this phenomenon indicated that the permeability of cells on the reduction of Cr(Ⅵ) had a very important role.The total chromium had not significant changed throughout whole restore process, and remnant of Cr(Ⅵ) and C-2 bacteria’s growth had negative correlation.The study of crude enzyme solution of Cr(Ⅵ) reductase result showed that the optimum pH and temperature were pH7.0 and 37℃. The different temperature and pH had different affection for the stability of the reductase. At 50℃,65℃, pH4.0 condition, the reductase had worst stability, lost more than 80% activity. Co2+, Cu2+, Fe2+ and DTT, NADH could increase the reductase catalytic efficiency, while Hg2+, Ag+, Mn7+, Tween 80, Tween 20, TritonX-100, EGTA, EDTA and SDS had different inhibition. Fe2+、Mn2+ had more positive influence than Fe3+、Mn7+ indicated that the higher electric of the same metal ion, the more inhibition forenzyme activity; The Km and Vmax of Cr(VI) reductase was 692.13 μmol/L and 23.585 nmol/(min·mg).The sequencing result showed that the length of the reduce gene was 735bp. The best inducing conditions for recombinant strain was 30℃,150r/min,0.2mmol/L final IPTG concentration and 5h induction time. The crude enzyme of recombinant strain reduced Cr(Ⅵ) was more quickly than non-recombinant strain, and NADH could promote reduction. The 150mmol/L of imidazole was the best elution concentration for target protein which had reduction activity.The bioinformatics analysis of Cr(VI) reductase gene result showed that GC content was 38%, the pI and molecular weight were 5.79 and 27.79KDa. The positive charge amino acids, negative charge amino acids, hydrophobic amino acids and hydrophilic amino acids were 12.7%,16%,38.5%,61.5%, respectively. The target protein was hydrophobin and belonged to nitroreductase family, this protein had 75% chance in cytoplasm. There were two casein kinase Ⅱ phosphorylation sites, three N-terminal fourteen alkylation sites, two protein kinase C phosphorylation sites in it. The secondary structure of protein might be caused by 51.64% a-helix,10.66% exten-ded chain,37.70% random coil, and there was no signal peptide and transmembrane helix. The result of homologous sequence analysis displayed that the highest homolo-gy protein which existed in B.t. BGSC 4AW1 strain was 97.5%, and the lowest homology protein was NfsA protein, only 35.8%. |
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