Immobilization of a thermostable, fungal recombinant chitinase on biocompatible chitosan beads and the properties of the immobilized enzyme

Abstract

A recombinant, thermostable fungal chitinase from the thermophilic fungus, Thermomyces lanuginosus, was immobilized on glutaraldehyde cross‐linked chitosan beads, and the properties of the immobilized chitinase were studied. The enzyme was found to be almost completely immobilized in 6 H under shaking condition at 30 °C. The immobilized enzyme exhibited much wider pH optimum and was more stable at alkaline pH values as compared with the soluble enzyme. Both the forms of the enzyme were optimally active at 60 °C and stable at 50 °C for 3 H, and after 3 H, the activity of the soluble enzyme declined sharply, whereas the immobilized chitinase was stable up to 6 H without any significant loss in the activity. KM and Vmax values of the immobilized enzyme were 1.18 mM and 445.7 μmol/Min/mg of protein, respectively. The immobilized enzyme was stable at least for 1 month at 4 °C without any significant loss in the activity.

Current Perspectives on Chitinolytic Enzymes and Their Agro-Industrial Applications

“…Different activators and cross-linking agents, such as 3-aminopropyl triethoxylsilane (APTES), cyanogen bromide, etc., are popularly used for the preparation of immobilized enzymes. Glutaraldehyde cross-linked chitosan beads were used to immobilize a recombinant, thermostable fungal chitinase from Thermomyces lanuginosus , which further increased the stability of the immobilized chitinase, as compared to the free enzyme [ 100 ]. Similarly, the stability and catalytic activity of the purified recombinant Chit36 enzyme were improved by immobilization on Ca 2+ cross-linked alginate beads and Ca 2+ cross-linked alginate–sepiolite nanocomposite beads [ 101 ].…”

Production and characterization of chitooligosaccharides by the fungal chitinase Chit42 immobilized on magnetic nanoparticles and chitosan beads: selectivity, specificity and improved operational utility

“… 26 Chitosan beads and GA have also been used to immobilize chitinases such as that from Streptomyces griseus and Paenibacillus illinoisensis , with an immobilization yield of about 42%, 19 and a thermostable enzyme from Thermomyces lanuginosus with almost 100% of immobilization yield but without data of the activity recovery reported. 20 In addition, different polymers have also been previously used to immobilize chitinolytic enzymes. Among them, hydroxypropyl methylcellulose acetate succinate for a commercial chitinase from Serratia marcescens , with an immobilization yield of 78% and recovery activity of 41% 27 and k-carrageenan-alginate gel for chitinase from Aspergillus awamori with immobilization and recovery activity yields of 93% and 77%, respectively.…”

“…11,[14][15][16][17][18] In contrast, most chitinases previously immobilized using both types of supports lost their activity aer being reused in consecutive cycles or no data concerning production and characterization of COS were reported. [19][20][21] Chitinase Chit42 from the fungus Trichoderma harzianum was previously expressed in the yeast Pichia pastoris with about 3 g L À1 of the protein produced in this heterologous system. This fungal exo-chitinase of the family GH18 hydrolyzed chitin oligomers with a minimal degree of polymerization (DP) of 3 units, with chitobiose being the main hydrolysis product.…”

Construction of an Immobilized Enzyme Membrane Reactor for Efficient and Sustainable Conversion of Ionic Liquid/Ultrasound-Pretreated Chitin

“…Depending on the different interaction types between chitinases and the carriers, the published immobilization methods could be basically classified into four categories: covalent, cross linking, adsorption, and entrapment. Accordingly, the research studies in the covalent category were most extensive, and the covalent carriers varied from organic polymers to inorganic materials, such as hydroxypropyl methylcellulose acetate succinate (AS-L), , chitin, chitosan beads, , carrageenan-alginate/guar gum gel beads, , and polyurethane with copper oxide nanoparticles (PU/nCuO) . Besides, covalent immobilizations of chitinase on chitosan beads or magnetic nanoparticles were combined with the cross-linking agent glutaraldehyde (GA) and genipin (Gpn). , Adsorption of chitinase on zinc oxide (ZnO) nanoparticle and entrapment of chitinase in Ca 2+ -cross-linked alginate/sepiolite nanocomposite have also been reported. − Despite the fact that many efforts have been performed into immobilizing chitinase, some further exploration on chitinase immobilization based on its specific characteristics and applications are still highly essential.…”