Decolorization Of Remazol Brilliant Blue KN-R With A Novel Coupled Perhydrolase- Chemical Oxidation Technology

The Remazol Brilliant Blue KN-R (RBBR) dye solution was decoloured using a novel coupled perhydrolase-chemical oxidation technology. In this work, three research contents were carried out, (1) Perhydrolase was purified and the enzymatic characterization was analyzed; (2) The decolorization process of RBBR was optimized using perhydrolase-chemical oxidation technology; (3) Perhydrolase was immobilized by cross-linked aggregates technology and entrapped by polyvinyl alcohol (PVA). The decolorization effect of the immobilized-perhydrolase was evaluated. The main results were as follows:1. Purification and characterization of perhydrolase. The perhydrolase was purified to homogeneity using HisTrapTM HP chromatography column. The relative molecular weight of the purified perhydrolase is 28 kDa, and the specific activity was 9.03 U/mg. The optimum pH and temperature of perhydrolase were 6.0 and 65℃ respectively. The perhydrolase displayed excellent thermostability in the range of 30-65℃ and the T502h of perhydrolase was 83.4℃. The perhydrolase kept stable in the pH range of 3.0-6.0.2. The decolorization process of RBBR by the coupled perhydrolase-chemical oxidation technology. Based on the results of the single factor test, the optimal decolorization conditions for RBBR were obtained via orthogonal experiment. The optimized decolorization conditions were found to be:20 U per reaction enzyme dosage, the molar ratio of ethyl acetate to hydrogen peroxide at 40:1,80 mg/L dye RBBR concentration, and pH 5.0 in the 5 mL decolorization system. The decolorization of RBBR was 81.11% for 6 h and 91.96% for 24 h under the optimal conditions, respectively. The decolorization rate of RBBR was 84.55% for 24 h under the optimal conditions in 250 mL decolorization system.3. Immobilization of perhydrolase and evaluation of the decolorization effect. The optimum parameters for perhydrolase cross-linked aggegrates preparation (named Per-CLAs) were:the ratio of enzyme to BSA at 16:2,60%(v/v) of precipitant S2,100 mmol/L glutaraldehyde. Under the above parameters, the maximal activity recovery of perhydrolase was about 79.72%. In the following further entrapment of Per-CLAs, the optimum parameters were as follows:10%(w/v) polyvinyl alcohol (PVA) and 50%(w/v) NaNO3. The immobilized perhydrolase could be recycled more than 6 times and kept the same decolorization. The RBBR decolorization of free perhydrolase, perhydrolase CLAs and entrapped-perhydrolase CLAs was 63.72%,95.98%,83.51% in 30 min, respectively.