Encapsulation of the reductase component of p ‐hydroxyphenylacetate hydroxylase in poly(lactide‐co ‐glycolide) nanoparticles by three different emulsification techniques

p ‐Hydroxyphenylacetate 3‐hydroxylase component 1 (C ₁) is a useful enzyme for generating reduced flavin and NAD⁺ intermediates. In this study, poly(lactide‐co‐glycolide) (PLGA) nanoparticles (NPs) were used to encapsulate the C ₁ (PLGA‐C ₁ NPs). Enzymatic activity, stability, and reusability of PLGA‐C ₁ NPs prepared using three different methods [oil in water (o/w), water in oil in water (w/o/w), and solid in oil in water (s/o/w)] were compared. The s/o/w provided the optimal conditions for encapsulation of C ₁ (PLGA‐C _1,s NPs), giving the highest enzyme activity, stability, and reusability. The s/o/w method improves enzyme activity ∼11 and 9‐fold compared to w/o/w (PLGA‐C _1,w NPs) and o/w (PLGA‐C _1,o NPs). In addition, s/o/w prepared PLGA‐C _1,s NPs could be reused 14 times with nearly 50% activity remaining, a much higher reusability compared to PLGA‐C _1,o NPs and PLGA‐C _1,w NPs. These nanovesicles were successfully utilised to generate reduced flavin mononucleotide (FMN) and supply this cofactor to a hydroxylase enzyme that has application for synthesising anti‐inflammatory compounds. Therefore, this recycling biocatalyst prepared using the s/o/w method is effective and has the potential for use in combination with other enzymes that require reduced FMN. Application of PLGA‐C _1,s NPs may be possible in additional biocatalytic processes for chemical or biochemical production.Inspec keywords: nanoparticles, enzymes, biotechnology, biochemistry, recycling, catalysts, nanofabrication, encapsulationOther keywords: reductase component, poly(lactide‐co‐glycolide) nanoparticles, emulsification techniques, p‐hydroxyphenylacetate 3‐hydroxylase component, NAD⁺ intermediates, PLGA, enzymatic activity, PLGA‐C₁ reusability, water in oil in water methods, solid in oil in water methods, oil in water methods, optimal conditions, encapsulation, enzyme stability, enzyme reusability, s/o/w method, reduced flavin mononucleotide, hydroxylase enzyme, anti‐inflammatory compounds, recycling biocatalyst, FMN, biocatalytic processes, biochemical production, chemical production