Abstract
D-allulose is an epimer of D-fructose at the C-3 position. With similar sweetness to sucrose and a low-calorie profile, D-allulose has been considered a promising functional sweetener. D-psicose 3-epimerase (DPEase; EC 5.1.3.30) catalyses the synthesis of D-allulose from D-fructose. Immobilised enzymes are becoming increasingly popular because of their better stability and reusability. However, immobilised DPEase generally exhibits less activity or poses difficulty in separation. This study aimed to obtain immobilised DPEase with high catalytic activity, stability, and ease of separation from the reaction solution. In this study, DPEase was immobilised on an amino-epoxide support, ReliZyme HFA403/M (HFA), in four steps (ion exchange, covalent binding, glutaraldehyde crosslinking, and blocking). Glycine-blocked (four-step immobilisation) and unblocked (three-step immobilisation) immobilised DPEase exhibited activities of 103.5 and 138.8 U/g support, respectively, but contained equal amounts of protein. After incubation at 60 °C for 2 h, the residual activity of free enzyme decreased to 12.5%, but the activities of unblocked and blocked DPEase remained at 40.9% and 52.3%, respectively. Immobilisation also altered the substrate specificity of the enzyme, catalysing L-sorbose to L-tagatose and D-tagatose to D-sorbose. Overall, the immobilised DPEase with intense multipoint attachment, especially glycine-blocked DPEase, showed better properties than the free form, providing a superior potential for D-allulose biosynthesis.
Highlights
D-allulose is a rare sugar with a molecular formula of C6H12O6
The chemical composition and functional groups of immobilised D-psicose 3-epimerase (DPEase) was analysed on a Fourier-transform infrared spectroscopy (FT-IR) (IS10, Nicolet Instrument Corp., Waltham, MA, USA)
DPEase was immobilised in four steps on amino-epoxide resin HFA (Figure 1), which contains secondary amines that dissociate the hydroxyl ions into water and show weak alkalinity
Summary
D-allulose ( known as D-psicose) is a rare sugar with a molecular formula of C6H12O6. The first step is the physical adsorption of the protein on the carrier to facilitate the second step of covalent binding between the enzyme and epoxy groups of the resin. Under such conditions, several carriers have been designed to exhibit hydrophobicity. After achieving the most suitable immobilisation conditions for each step, the enzymatic properties of the free enzyme, glycine-blocked enzyme, and unblocked enzyme were examined To our knowledge, this is the first report on the immobilisation of DPEase on amino-epoxide support. This study aimed to obtain immobilised DPEase with remarkable multipoint attachment and to provide insights into the production of D-allulose
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