For immobilization of enzymes natural polymers
For immobilization of enzymes, natural polymers depict several outstanding features as support. These materials are inert, non-toxic, biodegradable and biocompatible. As well as, they can be treated with different functional groups easily by chemical reactions that occur under gentle conditions in absence of impurities. Also, the industrial applications of these carriers are justified due to their low prices and great availability , . The recent growth in the use of carriers based on cellulose, as the cheapest natural polymer on the earth, for enzyme immobilization was noticeable because of their ideal properties like hydrophilicity, renewability, and low contamination risk to the environment. Cellulose could be plant-based or synthesized by algae, tunicates, and some bacteria. Additionally, three hydroxyl groups exist in every monomeric unit (glucose) of cellulose chemical structure with the potential of making covalent bonds with amino acids of biocatalysts. So, this polymer has a good chemical reactivity and could be applied reasonably for enzyme immobilization , , . But, the direct interaction between hydroxyl groups of cellulose and enzyme is not strong enough. So, in order to improve the binding efficiency, the surface of this matrix should be modified by employing chemical coupling agents to create more appropriate functional groups on its surface. Hence, enzyme immobilization will take place by stable and firm multipoint covalent linkages between activated support and amino S63845 residues of biocatalyst (NH2, COOH, SH) which are frequently involved in covalent coupling. The cellulosic support which is treated by amino group would be capable to interact covalently with carboxyl group of amino acids like aspartic acid or glutamic acid. Also, if the chemical treatment introduces aldehyde, carboxyl or epoxy group onto the matrix, the enzyme molecule will attach to cellulose through N-terminus (amine group of lysine) . Several activating agents such as cyanogen bromide, cyanuric chloride, epichlorhydrine, and organic sulfonyl chlorides were used for modification of the cellulose hydroxyl groups by diversified methods in previous studies , . The bisoxiranes like 1, 4-butanediol diglycidyl ether (BTDE), as the chemicals which contain two epoxy functional groups, could be applied for activation of cellulose-based carriers under a mild condition. The surface treated matrices by the free reactive epoxy groups are capable to form strong covalent bond with the enzyme's amino groups . Also, 1,1′-carbonyldiimidazole (CDI) is an active carbonylating reagent which contains two acyl imidazole leaving groups that are capable to convert the free hydroxyl groups on the cellulose surface into cyclic imidazolyl-carbamate groups. In the following, with the reaction of these groups with N-nucleophiles on enzymes, the relatively stable N-alkyl carbamates can be formed. As the CDI agent is sensitive to hydrolysis, the support modification should be conducted under nonaqueous conditions like in dry acetone . The enzymes which are linked to the supports via the spacer arms would have more degree of mobility and as a result, they will represent higher activity relative to the biocatalysts which are attached directly . As well as, the amount of loading capacity and the stability of the immobilized biocatalyst would improve noticeably by this technique . In this study, the plant cellulose powder was modified using two various techniques. In the first method, BTDE and in another CDI was used as the chemical coupling agent. Afterwards, the OPH enzyme from Flavobacterium ATCC 27551 was immobilized on any of activated support by covalent attachment. The identified effective parameters on the preparation of immobilized enzyme in both methods were analyzed, and the optimal conditions were found. The processes for OPH immobilization are shown in the Fig. 1.