A Biotechnological Approach to Enzyme‐Based Fertilisers: Immobilisation of Acid Phosphatases

ABSTRACT We explore enzyme‐based technologies as sustainable alternatives to conventional chemical fertilisers, addressing the challenges associated with using enzymes in free or immobilised form for agricultural applications. We use the metagenome‐derived Class A acid phosphatase M2‐32, selected for its high activity, broad pH tolerance and thermophilic properties, and evaluated its immobilisation on clay minerals to enhance stability and applicability in soils. Several clays were tested as immobilisation supports. Bentonite caused complete enzyme inactivation, while kaolin formed aggregates and was unsuitable. In contrast, palygorskite, sepiolite and agrozeolite adsorbed more than 99% of the added enzyme. However, only a fraction of the immobilised enzyme retained catalytic activity, with optimal performance observed at moderate protein loading (40–80 μg protein). Among the tested supports, palygorskite consistently provided the highest specific activity (22,000 ± 2200 U/mg), followed by sepiolite (11,000 ± 730 U/mg), whereas agrozeolite (2250 ± 40 U/mg) showed comparatively low activity. ATR‐FTIR spectroscopy confirmed successful enzyme immobilisation without significant alteration of the clay structures. Immobilised M2‐32 preserved a broad pH range (between 4 and 8.5) and thermophilic behaviour similar to the free enzyme, remaining active up to 50°C. Immobilisation increased substrate affinity while reducing V max relative to the free enzyme. To assess environmental compatibility, the effects of free and palygorskite‐immobilised M2‐32 on soil microbial communities were evaluated using corn rhizosphere microcosms with different organic matter contents. Metabarcoding high‐throughput sequencing revealed that microbial diversity and community structure were primarily shaped by soil type, plant presence and incubation time. Enzyme application, whether free or immobilised, did not significantly alter microbial diversity or composition. Overall, these results support palygorskite‐immobilised M2‐32 as a promising, environmentally compatible candidate for enzyme‐based fertiliser development.