SR 11302 mg Although the immobilization by histidine

Although the immobilization by histidine tag exhibits effectiveness in oriented enzyme immobilization, it still faces some challenges [31]. On one hand, some unnecessary metal-binding proteins that interfere with the combination between the target protein and the supports or the additional interactions between the immobilized protein and the affinity support may have a restriction on this immobilization method [[22], [32]]. On the other hand, the supports should firmly bind metal ions due to the undesired release of metals to the reaction solution in the immobilized process, which may affect the enzyme stability and contaminate the product. Depending on the above points, the current research on this methodology has two types of the requirements [33]. Firstly, the availability of necessary histidine on the surface of the enzyme is important. As it is well-known, the number of histidine groups on the enzyme is limited. In the past few years, the effort of the genetic engineering modification of enzyme catalysts had realized the production of histidine tags that consisted of six consecutive histidine residues by adding at N or C-terminal of the protein far away the active site. Secondly, in many cases, the commonly used supports do not meet the requirements for this method. The choice of supporting material is essential. This is why the goal of the present research is to synthesize high affinity immobilization carriers that can recognize the tagged enzyme. Adenylate cyclase (AC) is an important metabolic catalyst because it maintains important physiological functions of organisms by catalyzing the conversion of SR 11302 mg to cyclic adenosine-3′, 5′- monophosphate (cAMP). The level of cAMP in the second messenger of target cells can be regulated by the changes of the activity of AC, thereby affecting cell metabolism and cell behavior [34]. The research on cAMP has attracted more attention due to its various pharmaceutical values such as relaxing smooth muscle, promoting nerve regeneration and regulating the balance of blood glucose [[35], [36]]. Hence, with the help of the early genetic engineering modification of enzyme catalysts, we develop a simple and efficient method for immobilization of his-tagged AC, a kind of model enzyme, to produce cAMP. First, we prepared iminodiacetic acid (IDA)-Ni2+ particles to separate and immobilize his-tagged AC. Then AC was immobilized on the surface through the combination of imidazole and Ni2+ to form IDA-Ni2+-AC. The immobilization conditions, such as the added amount of AC, the incubating time, pH and temperature were investigated. The optimal pH, temperature and the reaction stability were investigated. Storage stabilities and the reusability experiments were also evaluated. To this date, the formation of IDA-Ni2+-AC to immobilized AC has not been reported in the literature. Therefore, the proposed method of obtaining immobilized AC will provide a new direction for producing cAMP.
Materials and methods
Results and discussion
Conclusion Based on the combination of IDA and Ni2+, a new procedure for AC immobilization was developed by utilizing the specific affinity between his-tagged protein and Ni2+. The synthesized IDA-Ni2+ carriers exhibited skeleton pores of 50 nm and specific surface areas of 45.8 m2/g, displaying an improvement on the AC purification and immobilization. The immobilized AC exhibited obvious advantages over the free AC, in terms of high temperature resistance, favorable storage stability and high catalytic efficiency. The activity of the immobilized enzyme was higher than that of the free enzyme after incubation at different temperatures during the test. After storing it for seven days, the immobilized enzyme retained a residual enzymatic activity of approximately 98%, while the free enzyme lost half of its activity. The maximum conversion rate of cAMP by using immobilized enzyme was higher than that of the free enzyme. The methodology and strategy we propose here can provide an effective way to orientation immobilization of enzymes for chemical biosynthesis.