Characterization and Application of a Thermostable HAD Phosphatase From Thermophilibacter mediterraneus for...

Glucosamine (GlcN), a high-value nutraceutical, is currently produced via environmentally detrimental chitin hydrolysis or inefficient microbial fermentation. While acidic hydrolysis of crustacean chitin raises environmental and allergen concerns, microbial fermentation faces challenges in strain engineering and byproduct formation. One-pot production of GlcN from maltodextrin by an in vitro synthetic enzymatic biosystem (ivSEB) containing glucosamine 6-phosphate phosphatase, which dephosphorylates glucosamine 6-phosphate (GlcN6P) to GlcN, was developed recently. In this study, we identified a thermostable haloacid dehalogenase (HAD) phosphatase, TmHAD, from Thermophilibacter mediterraneus through database mining. Biochemical characterization revealed its remarkable dephosphorylation specificity for GlcN6P, exhibiting 27.6- and 138.0-fold higher activity toward GlcN6P compared to glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), respectively. The enzyme demonstrated Mg²⁺-dependent activity and moderate thermal stability with a half-life of 6.6 h at 45°C. When incorporated into an ivSEB (phosphorylation, isomerization, amination, and dephosphorylation), TmHAD enabled GlcN production from maltodextrin with a molar yield of 44.5%. This biosystem represented an effective complement to current GlcN production methods, with the exceptional substrate specificity and thermal stability of TmHAD making it particularly promising for industrial-scale GlcN manufacturing in vitro.