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Abstract
Laccase, an oxidative enzyme, oxidizes phenolic compounds with broad substrate specificity, including amino acids such as tyrosine and cysteine in protein. Ferulic acid in sugar beet pectin (SBP) and tyrosine in -lactoglobulin (BLG) are potential substrates for laccase catalyzed formation of homo- and hetero- conjugates. The aim of this work was to determine if laccase catalyzes the conjugation of SBP and BLG, to characterize SBP-BLG conjugates, and to determine the effect on the functional properties of homo- and hetero-conjugates. Separately prepared SBP dispersion (3%, w/v) and BLG dispersion (3%, w/v) in 100 mM sodium phosphate buffer (pH 6.5), were equilibrated with or without laccase at 30C for 20 hours. Then, BLG dispersions were heated at 80 C for 15 min and mixed with SBP treated with or without laccase. Size exclusion chromatography combined with multi angle laser light scattering, refractive index, and UV detection was performed to confirm the formation of homo- and hetero-conjugates. Laccase treated SBP had a larger molecular weight and developed a more compact, branched structure based on smaller RMS (Root Mean Square radius) as compared to the larger molecular weight than non-treated SBP. Emulsions prepared with conjugated SBP retained smaller (d43) and more uniform particle size (light microscopy) during 30 days storage than non-conjugated SBP. Conformational change in laccase treated BLG, was manifested by smaller and broader distribution of RMS, changed polymer chain shape and larger particle size, compared to non-treated BLG. Laccase catalyzed polymerization in BLG after heat treatment, which suggests that laccase might work after exposing more tyrosine when heated. Furthermore, hetero-conjugation between heat treated BLG and SBP with laccase was confirmed by UV 280: the laccase treated BLG peak eluted earlier and at greater absorbance than laccase untreated dispersions. Laccase mediated SBP-BLG conjugates showed ~35 % higher solubility at the isoelectric point of BLG and relatively constant G (loss modulus) against shear force compared to non-treated SBP-BLG samples. INDEX WORDS: Laccase; Sugar beet pectin; -lactoglobulin; Hetero- Homo- Conjugation; HPSEC-MALLS; Emulsion; Solubility