Silk fibroin hydroxyapatite composite thermal stabilisation of carbonic anhydrase

Abstract

Event Abstract Back to Event Silk fibroin hydroxyapatite composite thermal stabilisation of carbonic anhydrase Zishuai Zhang1, Joao J. Lopes1, Benedetto Marelli2, Forenzo F. Omenetto2, David D. Kaplan2, Jake E. Barralet1 and Geraldine Merle1 1 McGill University, Dentistry, Canada 2 Tufts University, Engineering, United States Enzyme immobilization has been widely developed as a method to maximise operational and thermal stability and minimise the cost by reuse[1]. Several approaches have been employed for enzyme immobilization, like adsorption, covalent binding, affinity immobilization and entrapment et al. However, the immobilized enzyme always has less catalytic activity due to the loss of active sites[2], which hampered its practical application. Carbonic anhydrase (CA), a zinc metalloenzyme which captures and transforms CO2 to protons and bicarbonate ions, was used as model enzyme[3]. Here we developed a novel technique to immobilise enzyme without altering its active conformation, preventing desorption and allowing its reuse1. Enzyme was adsorbed and entrapped during the self-assembly of hydroxyapatite via ultrasonic bonding and silk fibroin was deposited to prevent enzyme from desorption. This macromolecule was further assembled into organized crystalline domains of beta sheets and less organized more flexible domains[4] to create mechanically strong barrier very resistant to changes in temperature and moisture[5]. Briefly, HA nanoparticles were prepared by precipitation of a mixture of calcium nitrate tetrahydrate with ammonium phosphate dibasic. After HA nanoparticles were mixed with Na3PO4 solution and CA and subjected to an ultrasonic treatment[6]. In parallel, B.mori silkworm cocoons were boiled in Na2CO3 solution to remove sericin, then fibers were extracted and dried[7]. The fibers were dissolved into LiBr solution, and after 2 days at room temperature, fibroin solution was centrifuged. Silk fibroin layers were deposed on the surface of the enzymatic microparticles by alternating incubations cycles in 5wt% silk solution and ethanol solutions. Beside the determination of the physico-chemical properties of the microparticle such as specific surface area and morphology, enzymatic activity and stability measurements were performed. By applying ultrasound the initial HA nanoparticles formed mechanically robust HA microparticles (Figure 1A) that were separated from the unbounded nanoparticles by washing and sieving. As observed by SEM, the HA microparticles had a linear dimension of ~75-125μm (Figure 1A) and a nanoporosity of less than 100nm (Figure 1B). After consecutive heat treatments of one hour at temperatures increasing from 50°C to 140°C (Figure 2), the free enzyme degraded at 50°C with no catalytic activity measureable whereas the enzyme simply adsorbed on HA exhibited a gradual loss of activity with 30% of remaining activity at 90°C. However the thermal stability was much greater when the silk was incorporated with 80% of its initial activity retained up to 110°C. To better characterize this thermal stability, we also recorded the enzymatic activity in an amine solvent, MDEA at 80˚C. The enzymatic activity remained unchanged up to 3 days, decreased to 45% and then remained stable for 3 weeks. In summary, silk fibroin hydroxyapatite nano-composite showed a remarkable operational, storage and thermal stability, with enzymatic activity almost unchanged after a one hour’s treatment at 110°C and the assembly retained 45% of its initial activity after 3 weeks of continuous heating at 80°C in an amine solution. This thermal stability was excellent compared with described CA immobilization systems and indicates that silk fibroin may limit thermally induced enzyme conformation changes and prevent desorption.