Enzyme Loading Capacity Research Articles

Stability improvement of alpha-amylase entrapped in kappa-carrageenan beads: Physicochemical characterization and optimization using composite index

Purpose This work examines the influence of various process parameters on α-amylase entrapped in crosslinked κ-carrageenan beads for stability improvement. A three level full factorial design was employed to investigate the effect of three process variables namely κ-carrageenan concentration, potassium chloride concentration and hardening time on % entrapment, time required for 50% ( T 50) and 90% ( T 90) of enzyme release and particle size. Methods The beads were prepared by dropping the κ-carrageenan-containing α-amylase to magnetically stirred potassium chloride solution. The composite index was applied to optimize the process under study. ‘In vitro’ enzyme release profile of the beads was fitted to various release kinetics models to understand the release mechanism. Topographical characterization was carried out by SEM and entrapment was confirmed by FTIR and DSC. Stability testing according to the ICH guidelines for zone III and IV was carried out. Results With the use of ionotropic gelation method, a polymeric matrix prepared by 3.5% (w/v) κ-carrageenan, 0.7 M potassium chloride and hardening time of 30 min resulted in the production of beads characterized by disc shaped with collapsed center, absence of aggregates, % entrapment of 73.79, T 90 of 74.4 min, and composite index of 83.01. Moreover, shelf-life of the enzyme loaded beads was found to increase up to 3.53 years compared to 0.99 year of the conventional formulation. Conclusions It can be inferred that the proposed method can be used to prepare α-amylase loaded κ-carrageenan beads for stability improvement. Also the proper selection of rate-controlling carrageenan concentration and its interactive potential for crosslinking is important and will determine the overall size and shape of beads, the duration and pattern of dissolution profiles and enzyme loading capacity.

Screen-printed bienzymatic sensor based on sol–gel immobilized Nippostrongylus brasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant

Here, we describe the development of a bi-enzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and opens the way for a highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation, which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) was immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 microg/L for paraoxon and 10 microg/L for parathion, which is according to EC regulations the highest tolerable pesticide concentration in infant food.

Immobilisation studies and catalytic properties of microbial lipase onto styrene–divinylbenzene copolymer

Microbial lipase from Candida rugosa was immobilised by physical adsorption onto styrene–divinylbenzene (STYDVB) copolymer. Two different coupling media were evaluated: aqueous solution (sodium phosphate buffer) and organic solvent (heptane). The choice of the immobilisation medium had a large effect on the hydrolytic and synthetic lipase activities. Better results were found when the coupling procedure was performed in the presence of heptane. The effects of enzyme loading and support capacity regeneration were also studied. Both, free and immobilised lipases were characterised by determining the activity profile as a function of pH, temperature and thermal stability. For the immobilised lipase, operational and storage stabilities were also determined. Operational stability tests indicated that a small enzyme deactivation occurs after 12 consecutive batches of 24 h each. Such results revealed good potential for recycling under non-aqueous system.

Immobilization studies of an industrial penicillin acylase preparation on a silica carrier.

Penicillin acylase (EC 3.5.1.11) was immobilized by covalent binding on a silica gel carrier activated by silanization. High immobilization yields, operational stability, enzyme loading and support reutilization capacity were obtained. The effects of several variables on the activation of the support and on the immobilization method were studied. Other supports and immobilization methods were assessed for the immobilization of penicillin acylase and compared with the basic process used. Activity versus temperature, pH, buffer molarity and penicillin concentration profiles were determined and compared for the free, crosslinked and covalently-bound silica carrier penicillin acylase preparations. The hydrolysis performance of the latter preparation was assessed in a batch basket reactor and the time course of the hydrolysis reaction modelled by a two parameter model equation. The operational stabilities of penicillin acylase coupled covalently to the silica gel support and immobilized by a crosslinking procedure using glutaraldehyde were also compared.