Study On ?-Carrageenase Of Pseudoalteromonas Carrageenovora ASY5 And Its Immobilization

Carrageenases are a group of glycoside hydrolase that break down ?-1,4 bonds among carrageenans into oligosaccharides which have a variety of biological activities.In the present work,first of all,?-carrageenase was purified from supernatant culture of Pseudoalteromonas carrageenovora ASY5 and it was indentified by Peptide Mass Finger print.Then homology modeling through SWISS-MODEL was used to analysis the space structure of ?-carrageenase.Secondly,enzymatic properties were investigated to analysis the difference of catalytic activity from different resources.Finally,?-carrageenase from Pseudoalteromonas carrageenovora ASY5 was immobilized onto carboxyl-functionalized magnetic nanoparticles with glutaraldehyde as cross-linking agent.The immobilization conditions were further optimized,and the characterizations and degradation product of the immobilized ?-carrageenase were investigated.The ?-carrageenase was purified by?NH₄?₂SO₄ precipitation,anion-exchange chromatography?DEAE Sepharose Fast Flow?and gel filtration chromatography?SuperdexTM 75 10/300 GL?from supernatant culture of Pseudoalteromonas carrageenovora ASY5.According to the alone band on the sodium dodecyl sulfatepolyacrylamide gel electrophoresis?silver nitrate staining?,the molecular mass of the purified enzyme was estimated to be 30 kDa.Enzyme was purified to homogeneity with a specific activity of 2678.9 U/mg,a purification fold of 10.9 and a yield of 20.1%.It was indentified as kappa-carrageenase from Pseudoalteromonas carrageenovora by PMF and the 3D structure of the enzyme was successfully obtained by homology modeling with SWISS-MODEL.Properties of ?-carrageenase were investigated to analyze the difference of catalytic activity from different resource.The optimal temperature and pH were 60 °C and 7.5,respectively.It was stable at pH 7.0–9.0 and below 50 °C.The enzyme could specifity convert ?-carrageenan.The Km and Vmax values of the enzyme for ?-carrageenan were 2.28 mg/m L and 147.06 ?mol/?min·mg?,respectively,at 60 °C and pH 7.5.The enzyme was significantly stimulated by Na⁺,K⁺,Ca²⁺,Mg²⁺,Al³⁺.The enzyme was inhibited strongly by Ag⁺,Zn²⁺,Cd²⁺ and SDS.The properties of this ?-carragennase were different from the reported enzyme.The ?-carrageenase was immobilized onto magnetic iron oxide nanoparticles using glutaraldehyde as the coupling agent,and the bonding was verified by Fourier transform infrared spectroscopy.The optimal conditions were 2.5%?w/v?glutaraldehyde,13.9 U ?-carrageenase for 20 mg of magnetic nanoparticles,a 2-h cross-linking time,and a 2-h immobilization time at 25 °C.Under these conditions,the activity of the immobilized enzyme and the enzyme recovery rate were 326.0 U/g carriers and 46.9%,respectively.The mean diameter of the immobilized ?-carrageenase was 15 nm.Space between particles was reduced and structure more closely after immobilized.It also exhibited distinct magnetic response and superparamagnetic properties.The change of elemental and weight afer immobilized were verified by elemental analysis and thermal gravimetric analysis.Zeta potential and particle size analysis of the immobilized ?-carrageenase showed that enzyme aggregates colloidal particles stable exist in deionized water.The properties studied of immobilized ?-carrageenase showed that the optimal temperature and pH were 55 °C and 7.5,respectively.It maintained more than 80% activity at 50–65 °C and stability at the range of pH 5–9.The immobilized ?-carrageenase retained more than 40% of the original activity after being used 4 times.The half life time of immobilized enzyme at 4 °C was 20.6 d and that of free enzyme was 17.4 d.The kinetic constant value?Km?of immobilized ?-carrageenase was 22.5 mg/m L.The immobilized ?-carrageenase was used to convert ?-carrageenan and hydrolysis products were identified as disaccharides and tetrasaccharides by electrospray ionization time-of-flight mass spectrometry?ESI-TOF-MS?.The results indicated that the immobilized ?-carrageenase can be used in the preparation of carrageenan oligosaccharides.