The significance of hydrolase cascades on poly(aspartic) acid biodegradation assessment

Water-soluble polymers have exceptional properties and are therefore used in many applications. Many of these products end up down the drain; therefore biodegradability, especially in wastewater treatment plants, is essential to prevent accumulation in the environment. This study examined recombinant hydrolases PahZ1_KT-1 and PahZ2_KT-1 from Sphingomonas sp. KT-1 and PahZ1_KP-2 from Pedobacter sp. KP-2 for their role in biodegrading water-soluble poly(aspartic acid) (tPAA). Analysis by a turbidity assay and Gel Permeation Chromatography (GPC) revealed highest activity of PahZ1_KT-1 and PahZ1_KP-2 at pH 8 and 40 °C on tPAA, whereas PahZ2_KT-1 showed no activity on the polymer but yet on its oligopeptides with highest values at pH 7 and 55 °C. GPC analysis revealed that PahZ1_KT-1 and PahZ1_KP-2 hydrolyzed tPAA (MN > 17,000 Da) into oligopeptides (>500 Da), while PahZ2_KT-1 further degraded them to α-di(l-aspartic acid) and l-aspartic acid. Combined, these enzymes synergistically decomposed tPAA completely into l-aspartic acid within 24 h. Supplementation of hydrolases into standardized biodegradation test improved biodegradation (54 %) of tPAA after 28 d. Supplementing PahZ1_KT-1 or PahZ1_KP-2 alone achieved 52 % and 54 % tPAA biodegradation, respectively. Preincubating tPAA with hydrolases boosted degradation to 71 % in 28 d. These findings highlight enzymes' crucial role in breaking down macromolecules into lower molecular weight species for effective tPAA biodegradation.