A Study On Immobilization Of D-aminoacylase From M.natoriense TNJL143-2

The immobilized enzyme is made by physical or chemical means to be insoluble in water,but still has an enzyme active state,plays a catalytic role in a certain space,and can be used repeatedly and continuously.At present,in the field of enzyme application,immobilization technology has become an important research topic.Compared with free enzyme,immobilized enzyme has the advantages of high stability,convenient recovery,controllable reaction,low cost,and recyclability.In the fields of clinical diagnosis,chemical analysis,environmental protection,and energy development,etc.,immobilized enzymes have shown an irreplaceable role.As an important intermediate,D-amino acid is often used in the synthesis of antibiotics,active peptides,pesticides,and food additives.At present,chemical methods are mainly used for the production of D-amino acids,there are unavoidable shortcomings,such as very severe reaction conditions,great pollution and high cost.However,the production of D-amino acids by biological methods has the characteristics of mild reaction conditions and low pollution.Among those methods,the one for preparing D-amino acid by using D-aminoacylase to specifically hydrolyze N-acyl-Damino acid has a theoretical productivity of 100%,so it has great potential for applications.In this study,for D-aminoacylase derived from M.natoriense TNJL143-2,the first attempt was made to use agarose,mesoporous silica MCM-41,and SBA-15 as carriers,and embed them by entrapment and adsorption methods to perform immobilization.The applicable scope,stability,reusability,and immobilization effect of the prepared immobilized enzymes were tested and analyzed to compare the performance differences between the immobilized enzymes.The results showed that the three immobilized enzymes showed D-aminoacylase activity,and the protein immobilization rates were above 79%.Compared with the free enzyme,the Km value of each immobilized enzyme is significantly increased.Although the pH and temperature conditions of the optimal enzyme reaction are similar to the free enzyme(the optimum pH range for the enzymatic reaction is 6.8 ? 7.2,and the optimum temperature for the enzymatic reaction is 37 ?.),the acid-base and temperature applicable ranges of each immobilized enzyme are greater than the free enzyme.Among them,the immobilized enzyme using SBA-15 as the carrier has the widest range of acid-base applications and has the highest acid-base stability.While the immobilized enzyme using MCM-41 as the carrier has the widest temperature range and has the highest thermal stability.In addition,the retention time of each immobilized enzyme activity is approximately the same as that of free enzymes,and the attenuation of the activity of the immobilized enzymes using two kinds of mesoporous silica as carriers is less than that of the immobilized enzymes using agarose as carriers.On the other hand,the protein loss rate of the immobilized enzyme using mesoporous silica SBA-15 as the carrier was significantly lower than the corresponding conditions under the different pH or temperature enzymatic reactions,storage conditions and reuse processes.The protein loss rate of the other two immobilized enzymes indicates that the SBA-15 vector has more stable protein immobilization.