Immobilization and Characterization of Bacillus Thuringiensis HCB6 Amylase in Calcium Alginate Matrix

Abstract

Free enzyme in solution react with substrates to result in products which cannot be recovered for reuse. These problems can be overcome to a certain extent by the use of enzyme immobilization method. Immobilized enzymes are more robust and more resistant to condition changes. More importantly, the heterogeneous immobilized enzyme systems allow an easy recovery of both enzymes and products, multiple re-uses of enzymes, and continuous operation of enzymatic processes. Entrapment of enzymes in Ca-alginate is one of the simplest methods of immobilization. The aim of this research was to obtain the optimum condition of the making of immobilized amylase beads using a Ca-alginate bead and to determine its characteristics. The optimization of immobilized amylase beads includes variation of sodium alginates and variations of enzyme contact time with CaCl2. The characterization of immobilized amylase includes determination of optimum substrate concentration, optimum pH, and optimum incubation time as well as amylase stability test. Amylase activity was determined by using dinitro salicylic (DNS) method. The results showed that the optimum immobilized amylase obtained at alginate concentrations of 5% (w/v), contact time of 60 minutes and immobilization efficiency of 67.5%. Furthermore, immobilized amylase showed optimum substrate concentration of 1.5-2.5% (w/v), optimum pH of 6, an optimum incubation time of 20 minutes with the activity of 179.8 U/mL. The KM value for free amylase and immobilized amylases were 0.3 mM and 0.12 mM respectively. Vmax value for free amylase and immobilized amylases were 105.3 U/mL and 10.1 U/mL respectively. Immobilized Amylase can be used up to six times with the residual activity of 52.7%. Keywords: Amylase, Ca-alginate, enzyme immobilization ABSTRAKEnzim dalam keadaan bebas (larutan) dapat bereaksi dengan substrat menghasilkan produk kemudian tidak dapat digunakan kembali. Permasalahan ini dapat diatasi dengan metode amobilisasi enzim. Enzim amobil lebih kuat dan lebih tahan terhadap perubahan keadaan. Sistem heterogen enzim amobil memungkinkan kemudahan recovery antara enzim dan produk, pemakaian enzim secara berulang, dan proses enzimatik secara kontinu. Penjebakan enzim dalam kalsium alginat (Ca-alginat) adalah salah satu metode amobilisasi paling sederhana. Penelitian ini bertujuan untuk memperoleh kondisi optimum pembuatan beads amilase amobil menggunakan matrik Ca-alginat, dan mengetahui karakteristiknya. Optimasi pembuatan beads amilase amobil meliputi variasi konsentrasi natrium alginat dan variasi waktu kontak enzim dengan CaCl2. Karakterisasi amilase amobil meliputi penentuan konsentrasi substrat optimum, pH optimum, waktu inkubasi optimum serta uji kestabilan amilase. Aktivitas amilase ditentukan dengan menggunakan metode asam dinitrosalisilat (DNS). Hasil penelitian menunjukkan kondisi optimum pembuatan beads amilase amobil diperoleh pada konsentrasi alginat 5 % (b/v) dan waktu kontak 60 menit dengan nilai efisiensi amobilisasi sebesar 67,5 %. Amilase amobil mempunyai ak...

Stability enhancement of Bacillus subtilis ITBCCB148 originating -amylase by immobilization using chitin

“…This problem can be overcome by enzyme immobilization methods, because immobilization has been acknowledged to offer several improvements such as higher economic beneficial [1]; increased biomolecular stability under various reaction conditions [2]; increased endurance of the enzyme against various application variables, such as temperature, solvents, pH of the reaction system, and the presence of unwanted components (contaminants and impurities) [3][4][5]; easier separation of the enzyme from the product with minimized or free from protein contamination from the product; and reduced enzyme and enzymatic product costs [6]. Moreover, immobilized enzymes allow easy recovery of the enzymes from the products, repeated use of enzymes, and continuous enzymatic processes [7]. In this study, α-amylase (EC.3.2.1.1) was used is as an enzyme that is able to break down starch and glycogen molecules by cutting the glycosidic α-1,4 bonds in starch molecules (carbohydrates) to form shorter carbohydrate molecules [8].…”