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.