Purification And Immobilization Of BphC For Biodegradation Of Aromatic Compounds

The purification and characterization of 2,3-dihydroxybiphenyl 1,2-dioxygenase(BphC) from Dyella ginsengisoli LA-4,immobilization methods of recombinant BphC and the immobilization conditions of sodium alginate as carrier to be optimized are described in this report.A novel dioxygenase(BphC) from Dyella ginsengisoli LA-4 could be purified and characteristics of the BphC including enzyme kinetics,effects of pH,temperature,metal ions and inhibitors on enzyme activity are investigated.The results show that the native BphC enzyme with the molecular weight of approximately 34±1kDa is a novel Fe(â…¡)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase with N-terminal sequence:Ser-Val-Lys-Asn-Leu-Gly -Tyr-Met-Gly-Phe-Ser-Val-Lys-Asp-Val.The purify times were 13.82,and the activity recovery was 8.76%.The purified native BphC_LA-4 could oxidize the substrates such as 2,3-dihydroxybiphenyl,3-methylcatechol,4-methylcatechol,catechol and 4-chlorocatechol, whose specific activities are 95.20,14.37,0.15,3.64 and 0.53U/mg respectively.Among the five test substrates 2,3-dihydroxybiphenyl with the lowest Km has the highest affinity for BphC_LA-4.The optimal pH value of BphC_LA-4 is 8.0 for 2,3-dihydroxybiphenyl,and the enzyme respectively indicates its maximal activity at 20,30 and 20℃for 2,3-dihydroxybiphenyl,3-methylcatechol and 4-methylcatechol.Some metal ions and inhibitors(ascorbic acid,SDS and H₂O₂) are able to inhibit the activity of BphC enzyme.The recombinant BphC could be successfully immobilized on activated carbon,strongly basic anion exchange resin,sodium alginate,polyvinyl alcohol,and ZnO sol-gel matrix.The highest activity of immobilized recombinant BphC is under the following order:sodium alginate as carrier＞ZnO sol-gel matrix as carrier＞polyvinyl alcohol as carrier.The degradation rates of benzoic acid and aniline could be greatly improved by adding immobilized BphC of activated carbon as carrier,but activated carbon adsorption is main function for the improvement of degradation rate.The degradation rate of benzoic acid could be greatly improved by adding BphC immobilized with strongly basic anion exchange resin, but resin adsorption is main function for the improvement of degradation rate.This immobilized BphC has little effect on aniline degradation rate.The immobilization conditions of recombinant BphC with sodium alginate as carrier are optimized by response surface methodology in four main influence factors:the concentration of sodium alginate,the concentration of CaCl₂,the amount of enzyme and immobilization time.Results show that there is not simple linear relationship between the degradation rate of biphenyl and the four main influence factors.Equation is fitted by analysis module,and the model is significant with acceptable errors.Fitting values of the model accord with actual values.The optimal solution is obtained by Optimization Numerical Solution:sodium alginate 3.68%,CaCl₂ 3.72%,the amount of enzyme 1.08mL and immobilization time 5h.The fitting degradation rate of biphenyl is 99.27%,which accords with the actual value(99%).