Dextransucrase entrapment as an efficient alternative for increased recycling efficiency of free enzyme within agar-agar film matrix

Abstract

Dextransucrase (DS), the extracellular enzyme is of immense industrial importance, due to ability to produce dextran and oligosaccharides (OS). Worldwide interest in OS has been increasing, since they have been accorded the prebiotic status. However, the industrial application of DS for OS synthesis is limited, due to low yield of enzyme production and its low catalytic activity. Hence, there is a great interest for development of new technologies that can provide improved performance of biocatalyst. Enzyme immobilization technology is considered to be a crucial step for cheaper and more efficient usage of DS. Entrapment is one of the widely investigated immobilization methods, where enzymes are enclosed or confined within the polymer matrix without altering their native structure, developing bioreactors for commercial applications. Different matrices such as polyacryl-amide gel, alginate beads and agar–agar have been used for the entrapment of different enzymes and among them agar–agar is a biocompatible, non-toxic and strong solidifying agent for immobilization of various enzymes. In this work, the entrapment of DS was initiated by different quantity (1:9, 1:4 and 1:1) of dialyzed enzyme into agar- agar solution. Agar solution was prepared in distilled water by vigorous shaking at 100°C, autoclaved and was allowed to cool to 40–45°C. Afterwards, enzyme was incorporated and mixed thoroughly. This mixture was immediately poured into a clean Petri plate and left to solidify at room temperature. Polymer films with and without immobilized DS were analyzed in terms of enzyme activity and reusability and mechanical properties (tensile strength, elongation at break and elastic modulus). In order to remove un-entrapped enzyme the films were washed with double deionized water and sodium acetate buffer (pH 5.4) three times prior to enzymatic assay. For quantitative analysis of samples for OS production a Dionex Ultimate 3000 HPLC system was used. Results showed that maximum immobilization yield (74.11%) was achieved by use of 2 % agar and (1:9) enzyme: agar ratio. HPLC analysis confirmed the similar trend of OS formation in immobilized samples compared to free enzyme. The lowest tested fraction of enzyme immobilized into polymer matrix (1:9) improved tensile strength of films in comparison with control film, whereas higher concentration of enzyme led to decrease in mechanical resistance of films. Scanning electron microscopy of agar films before and after DS entrapment revealed significant morphological change on the matrix surface. Considering the economic feasibility, the entrapped DS indicated imperative recycling efficiency up to six reaction cycles. The results of this study revealed that an easily available and inexpensive matrix could be successfully employed for DS immobilization and OS production.