Research On Separation And Purification Of B.cereus MBL13-U Collagenase And Its Mechanism For Degradation Of Bovine Bone Collagen

The type I collagen in bovine bones are rich,but they are wasted because they are not used properly.Therefore,in this study,B.cereus MBL13-U was used to prepare the crude collagenase by fermentation.We through a series of separation and purification technology to have the collagenase for specific bovine-bone collagen degradation(CSCD),the CSCD was used to degrading bovine bone collagen,and established the kinetic models of degradation processing,discussed the mechanism of the degradation of bovine bone collagen,laying the theoretical foundation for the deep processing of bovine bones in the future.The main results were:(1)The optimization of collagenase was prepared by fermentation of B.cereus MBL13-U.The influence of inoculation amount,reaction temperature,pH and reaction time on the activity of collagenase were studied by single factor experiment.On the basis of these experiments,the quaternary orthogonal rotation test was carried out with collagenase activity as the index.The results showed that the optimal conditions were as follows: the inoculation amount 4 mL /100 mL,reaction temperature 35 ?,pH 6.4,reaction time 46 h.The enzymatic activity of collagenase was 92.31 U/mL in this conditions.(2)The isolation,purification and characterization of collagenase.The CSCD was obtained by(NH4)2SO4 precipitation,DEAE-Sepharose Fast Flow ion exchange chromatography,Sephadex G-100 gel chromatography,its molecular weight was about 52 k Da,the purification reached 42.85 times.The optimal temperature of CSCD was 50 ?,the thermal stability was good in low temperature environment below 50 ?;the optimal p H was 6.5,when the pH at 5.5-7.5,CSCD was able to degrade bovine type I collagen,and CSCD had the most significant effect on the degradation of bovine achilles tendon type I collagen compared with other proteases.(3)The optimization of enzymatic hydrolysis of bovine bone collagen by CSCD.The influence of reaction temperature,the amount of crude bovine bone collagen,the amount of CSCD,reaction time and p H on degree of hydrolysis were studied by single factor experiment.On the basis of these experiments,the five-element quadratic orthogonal rotation test was carried out with degree of hydrolysis as the index.The results showed that the optimal conditions were as follows: reaction temperature 46 ?,the amount of crude bovine bone collagen 5.14 g /100 mL,the amount of CSCD 0.42 g/100 mL,reaction time 6 h,pH 6.5,the degree of hydrolysis was 31.21% in this optimal conditions.(4)The establishment of kinetic models in the degradation processing of bovine bone collagen.Based on the Michaelis-Menten equation,the models of degradation rate and hydrolysis degree were constructed by the effect of CSCD and crude bovine bone collagen adding amount on the degree of hydrolysis during the degradation process,and the inactivation constant K4 of CSCD was 67.1157 h-1.The experimental value was consistent with the model fitting value,and the model was effective.(5)Preliminary Study on the mechanism of CSCD degradation of bovine bone collagen.The structure of collagen and collagen peptides were characterized by free amino acid,UV spectroscopy,fluorescence spectroscopy,differential calorimetry,infrared spectroscopy and scanning electron microscopy.The results showed that CSCD disrupted the triple helix structure of bovine bone collagen,exposing more amino acid residues on the surface,and releasing a large number of amino acids.With the progress of degradation,the structure of bovine bone collagen gradually opened,the molecular surface C=O,CONH2,COOH gradually increased,collagen peptides distribution of the chromophore also changed,the hydrophobic group in the molecule was released,also the proline residue and the lysine residue were free,the thermal stability of the collagen peptides were enhanced.The-NH2+-on peptide chains of collagen peptides mutual repulsion gradually weakened,the hydrophobic amino acid residues increased,the intramolecular and intermolecular hydrogen bonding effect were also increased.The N-H stretching vibration in the collagen peptides formed a association with the hydrogen bond,and the degradation promoted the production of the ?-corner,at the same time,the surface molecular structure of the crude bovine bone collagen were broken and made it became loose.