Improvement of Lysozyme Activity Determination Method and Its Application in Recombinant Human Lysozyme Quality Standard Establishment

KeyWord:recombinant human lysozyme  turbidimetry  double line method  Lowry method

Abstract:

      A turbidimetric method which could verge on a homogeneous reaction system was obtained.The method was applied in study of recombinant human lysozyme activity,and the related operation procedures and quality standards were established.In this research,microscopic counting method was used for the quality control of substrate bacteria liquid.The mixing manner of enzyme and bacteria liquid was improved,so as the amount of enzyme used for detection.¡°Double line method¡± was adopted to optimize the reaction time interval for activity detection.Also,the content of recombinant human lysozyme was detected accurately.By means of microscope count method,the bacteria number in reaction substrate was kept at about 1.1¡Á108/mL,and the concentration of the reaction substrate was 0.3 mg/mL.To approach the homogeneous system,the reaction liquid of lysozyme and bacteria was mixed by sucking-expelling method in the sample pool.The optimum amount of lysozyme was 3 ¦Ìg,the optimum reaction time was confirmed as 0-60 s.According to the operation procedure of enzymatic activity detection,the standard for activity of recombinant human lysozyme was confirmed as 45 000-65 000 U/mg,the RSD of intra-day and inter-day were both less than 5%.Lowry method was chosen for the content determination of recombinant human lysozyme,and the protein content was £¨1.0¡À0.1£© mg/mL.Stability of the optimized method was improved obviously,also,the operation was convenient and the detection result was reliable.Furthermore,the optimized method was verified and accepted by three research institutes and enterprises.The quality standards and operational procedures were established with important practical reference value.