Harnessing glucan branching enzymes for amylose-based starch nanoparticle formation: A novel bottom-up approach

Abstract

The activity of glucan branching enzyme (GBE) was investigated in the production of starch nanoparticles (SNPs). An amylose-enriched fraction (AEF; amylose purity ≥98%) was prepared from purified field pea starch, and used in the production of SNPs by a "bottom-up" nanoprecipitation process. The dual action of GBE, involving both hydrolysis and subsequent branching, was performed at various time intervals over a 24h period. Scanning electron microscopy (SEM) images showed that AEF alone without GBE branching failed to produce small, spherically uniform and discrete starch nanoparticles. However, GBE (1%)-to-substrate (AEF) activity successfully formed SNPs after 20h of enzymatic treatment followed by nanoprecipitation. Notably, a sonication step was required prior to enzyme addition to facilitate SNP formation. The SEM images showed that performing sonication after branching, followed by nanoprecipitation, did not promote SNPs formation. This suggests that the timing and sequence of sonication relative to enzymatic branching are critical for successful SNP formation. Furthermore, the HPSEC data indicated that the sonication induced fragmentation of amylose likely enhances enzymatic branching. This is possibly due to creation of optimal chain lengths of amylose for GBE to effectively recognize and facilitate the initiation of its hydrolyzing activity. Thus, the starch molecules' solubility state as well as the presence of a mere level of short-chain branches were found to be significant factors facilitating the nanoprecipitation dynamics in the SNP formation. This study demonstrates the first attempt to utilize GBE in the bottom-up approach to form amylose-based nanoparticles from purified pea starch.