Hydrolysis of pullulan by entrapped pullulanase in Ca/alginate beads

Abstract

Pullulanase from Klebsiella pneumoniae was entrapped into calcium alginate beads. Its activity was estimated by the determination of number-average molar masses using two different methods: a colorimetric assay of reducing ends (REs) and a size-exclusion chromatography/multiangle light scattering/differential refractive index. The second method also provided weight-average molar masses of hydrolyzed pullulan and the quantity of maltotriose (DP3) and its multiples (DP6 and DP9) produced by the enzymatic treatment. The alginate beads showed a good retention of the loaded pullulanase (30%), and the system showed a downturn of hydrolysis kinetics in comparison with free pullulanase due to the limiting access of substrate-enzyme. On the contrary with the results obtained from free enzyme hydrolysis, for which a large distribution of pullulan fragments is observed during the treatment, the immobilized enzyme system has evidenced, during the enzymatic treatment, the coexistence of native or only slightly degraded pullulan chains together with maltotriose units. Complete hydrolysis of pullulan chains was achieved once diffused into the gel.

Covalent immobilization of pullulanase on alginate and study of its hydrolysis of pullulan

“…The number and weight molar masses were determined by coupling on‐line a size exclusion chromatograph (SEC) with a multiangle light scattering system (MALS) and a differential refractive index detector (DRI). The principle of this method was already described in previous works . The carrier LiNO 3 at 0.1 mol L −1 was filtered through a 0.1 µm filter unit (Millipore, USA), degassed (DGU‐20A3 Shimadzu, Japan), and eluted at a 0.5 mL min −1 flow rate (LC10Ai Shimadzu, Japan).…”

“…The principle of this method was already described in previous works. 7 The carrier LiNO 3 at 0.1 mol L 21 was filtered through a 0.1 mm filter unit (Millipore, USA), degassed (DGU-20A3 Shimadzu, Japan), and eluted at a 0.5 mL min 21 flow rate (LC10Ai Shimadzu, Japan). Sample solutions were injected with an automatic injector (SIL-20A Shimadzu, Japan).…”

“…[3][4][5][6] However, it can be classified in two various methods that are physical or chemical retention. The physical retention, largely used in hydrogels systems 7 is based either on steric entrapment or on physical interaction (ionic or hydrophobic). The steric entrapment exploits the size difference between the enzyme and its substrate.…”