Maltose Production Using Starch from Cassava Bagasse Catalyzed by Cross-Linked β-Amylase Aggregates

Abstract

Barley β-amylase was immobilized using different techniques. The highest global yield was obtained using the cross-linked enzyme aggregates (CLEA) technique, employing bovine serum albumin (BSA) or soy protein isolate (SPI) as feeder proteins to reduce diffusion problems. The CLEAs produced using BSA or SPI showed 82.7 ± 5.8 and 53.3 ± 2.4% global yield, respectively, and a stabilization effect was observed upon immobilization at neutral pH value, e.g., after 12 h at 55 • C, the free β-amylase is fully inactivated, while CLEAs retained 25 and 15% of activity (using BSA and SPI, respectively). CLEA using SPI was selected because of its easier recovery, being chosen to convert the residual starch contained in cassava bagasse into maltose. This biocatalyst permitted to reach almost 70% of maltose conversion in 4 h using 30.0 g/L bagasse starch solution (Dextrose Equivalent of 15.88) and 1.2 U of biocatalyst per gram of starch at pH 7.0 and 40 • C. After 4 reuses (batches of 12 h) the CLEA using SPI maintained 25.50 ± 0.01% of conversion due to the difficulty of recovering.

Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System

“…The crosslinking mechanism generally involves reactive primary amino groups of the protein (epsilon-amino group of Lys residues and N terminus); some proteins may not possess a high amount of this residue on the surface, so that in these cases, the CLEA preparation is not straightforward. This has been solved by using a feeder (which can be a Lys-rich protein [186][187][188][189][190][191][192][193] or an aminated polymer [194,195]), by a chemical amination of the enzyme [196][197][198] or by using an alternative crosslinking group in the enzyme (e.g., carboxylic groups) [199]. Usually, the crosslinking reagent is glutaraldehyde [182,184] due to its good properties as crosslinking reagent [200,201].…”

Preparation of Magnetic Cross-Linked Amyloglucosidase Aggregates: Solving Some Activity Problems

“…Moreover, magnetic CLEAs prepared CLEAs prepared under the same conditions, replacing magnetic nanoparticles with bovine serum albumin as the co-feeder, showed good performance (Figure 11b) regarding the recovered activity (around 35%), DE in the starch hydrolysis (around 60), and residual activity (above 60%). Although this co-feeder is widely used in the preparation of CLEAs [20,26,27,30,50], the replacement by magnetic nanoparticles is advantageous because of the ease of capture by an external magnetic field [40], avoiding the formation of clusters usually observed in the separation of CLEAs co-aggregated with bovine serum albumin by centrifugation [45]. Moreover, magnetic CLEAs prepared in the presence of polyethyleneimine and crosslinking with 500 mM glutaraldehyde (CLEA DCNP-N+PEI) showed similar performance (recovered activity around 40%, DE in the starch hydrolysis around 60, and residual activity around 60%), so were selected to be kinetically characterized and used in the hydrolysis of starch under industrial conditions.…”

“…Thereafter, the formed aggregates are cross-linked using bifunctional (usually glutaraldehyde) or polyfunctional (dextran polyaldehyde, for example) agents [21]. CLEAs of many different enzymes have been reported [18,[22][23][24][25][26], including amylolytic enzymes, e.g., β-amylase [27] and amyloglucosidases [28,29].…”

“…However, the crosslinking step may be problematic if the enzyme is poor in external free primary amino groups. This problem has been solved using Lys-rich proteins as protein feeders [22,24,27,30], co-immobilizing enzymes and a primary amino rich polymer (e.g., polyethylenimine, PEI) [31] or even enriching the enzyme in primary amino groups via chemical modification [32]. AMG has 13 Lys, but only nine are exposed to the medium [3,5], two of which are located in the starch-binding domain and one close to the active site (see Figure 1).…”

Immobilized Biocatalysts

“…Barley β-amylase is, among others, important for maltose production. Araujo-Silva et al [34] prepared immobilized β-amylase in the form of cross-linked enzyme aggregates using bovine serum albumin or soy protein isolate as feeder proteins to reduce diffusion problems and to successfully obtain maltose from converting the residual starch contained in cassava bagasse. A two-enzyme system developed by Petrovičová and colleagues [35], consisting of ketoreductase and glucose dehydrogenase immobilized inside of polyvinyl alcohol (PVA) gel particles, served to catalyze the asymmetric reduction of keto esters to optically active hydroxy esters.…”