Fig. 4

Characterization of HaSGNH1. a SDS-PAGE analysis of HaSGNH1. b Native-PAGE analysis and zymographic analysis of HaSGNH1. After running native-PAGE, the resulting gel was stained with Coomassie Brilliant Blue R-250 (lane 1) or and 4-methylumbelliferone (4-MU) acetate (lane 2). c Hydrolysis of 4-methylumbelliferyl (4-MU) acetate and phosphate. HaSGNH1 only (lane 1), 4-MU acetate with HaSGNH1 (lane 2), and 4-MU phosphate with HaSGNH1 (lane 3). Hydrolysis of 4-MU acetate in an Eppendorf tube containing HaSGNH1 was observed as strong fluorescence after UV illumination at 254 nm. d The hydrolysis of p-nitrophenyl ester derivatives by wild-type HaSGNH1 (black) and its S18A variant (gray). pNA: p-nitrophenyl acetate; pNB: p-nitrophenyl butyrate; pNH: p-nitrophenyl hexanoate; pNO: p-nitrophenyl octanoate; pNDe: p-nitrophenyl decanoate; pNDo: p-nitrophenyl dodecanoate. e The hydrolysis of naphthyl ester derivatives by HaSGNH1. The symbols * and ** represent significant differences (p-value < 0.001 (*) or p-value < 0.0001 (**), respectively), determined by Student’s t tests. (F) pH stability of HaSGNH1. HaSGNH1 was incubated at different pH between 3 and 10. Hydrolase activities were determined by measuring the amount of p-nitrophenol released during hydrolysis at 405 nm using a VersaMax 680 microplate reader (Bio-Rad Laboratories, CA, USA). In these experiments, the enzyme activity of HaSGNH1 under standard assay condition was defined as 100%