Cloning,Expression And Immobilization Of Arylsulfatase,and Its Application In Desulfatation Of Sulfated Polysaccharides

In the present work,arylsulfatase was cloned into pET-28a and expressed in Escherichia coli BL21?DE3?,and then the recombinant enzyme was characterized.Subsequently,the technical conditions of sulfate hydrolysis of the crude polysaccharides by recombinant arylsulfatase were optimized.Eventually,recombinant arylsulfatase was immobilized onto carboxyl-functionalized Fe₃O₄ nanoparticles?CMNPs?by cross-linking with glutaraldehyde.The physical and chemical properties of the immobilized arylsulfatase were investigated as well.The gene?987 bp?encoding a putative arylsulfatase from Pseudoalteromonas carrageenovora was amplified by PCR,cloned into pET-28a vector and expressed in Escherichia coli BL21?DE3?.The open reading frame of this gene encoded 328 amino acid residues.The target protein was characterized using the synthetic substrate of phenolic ester,p-nitrophenyl sulfate after it was purified to homogeneity using affinity chromatography.The purified recombinant enzyme presented a molecular mass of 35.1 kDa by SDS-PAGE.Gel filtration chromatography suggested that arylsulfatase was a dimer and molecular mass was 68.6 kDa.The enzyme had a specific activity of 26.7 U/mg with the optimal temperature and pH at 50°C and 7.0,respectively.The K_m,V_max and k_cat values of the recombinant enzyme were determined to be 0.65mmol/L,19.99?mol/?mg·min?and 114.27 s^-1,respectively.Considerably less activity was reached after incubated by Zn²⁺?Cu²⁺?Al³⁺and Fe³⁺.Except SDS,the enzyme showed resistance in some degrees toward other tested inhibitors and detergents.Recombinant arylsulfatase was immobilized onto CMNPs by covalent binding with glutaraldehyde.The best process parameters of arylsulfatase immobilization by cross-linking on CMNPs were carried out at 4°C,1.0%glutaraldehyde cross-linker concentration for 3 h,0.7 U free arylsulfatase after 3 h of immobilization with 10 mg of CMNPs.Under the optimized conditions,immobilized enzyme activity and enzyme recovery rate was 4.43 U/mg and 35.2%,respectively.CMNPs and arylsulfatase-CMNPs exhibited excellent dispersibility.Vibration sample magnetometry measurements indicated that the immobilization with arylsulfatase did not influence the magnetic separation of CMNPs.Successful binding was commfirmed using Fourier transform infrared.The result of X-ray diffraction suggested that the cross-linking with arylsulfatase process did not cause the phase change of CMNPs.The amount of free arylsulfatase bound to the surface of CMNPs was 5.65%.The average grain diameter of arylsulfatase-CMNPs was significantly higher than the CMNPs when both they were suspended in deionized water.Free and immobilized enzyme exhibited same temperature-optima at 50°C,but had different pH-optima at pH 7.0 and 7.5,respectively.Thermal and pH stability of the immobilized enzyme was enhanced.K_m values obtained for free and immobilized arylsulfatase were 0.65 mmol/L and0.89 mmol/L,respectively.V_max values changed from 19.99 to 22.47?mol/?mg·min?after immobilization.After 8 cycles for catalytic hydrolysis of p-NPS,the immobilized arylsulfatase conjugate still maintained more than 60%of its initial activity.After stored at 4°C for 5 days,the immobilized arylsulfatase retained 50%of its initial activity.Sulfate hydrolysis of the crude polysaccharides from Gracilaria lemaneiformis by recombinant arylsulfatase were optimized by single factor experiments based on the sulfate content and sulfate removal rate.The optimum process parameters were obtained as substrate concentration of 5 g/L,initial pH of 7.0,enzyme dosage of 108.14 U,enzymatic hydrolysis temperature of 40°C,oscillation rate of 120 times/min and enzymatic hydrolysis duration of 120min.Under the optimum conditions,the gel strength of the products increased by 2.1 folds,and78.4%of the sulfate in the crude polysaccharides had been removed,as well as the solidification temperature and melting temperature were 38.4°C and 91.3°C,respectively.