The β galactosidase A and B from Adsuki bean cotyledons were isolated by ammonium sulfate fractionation and DEAE\|Sepharose FF ion exchange chromatography.The A form was further purified by CM Sepharose FF ion exchan...The β galactosidase A and B from Adsuki bean cotyledons were isolated by ammonium sulfate fractionation and DEAE\|Sepharose FF ion exchange chromatography.The A form was further purified by CM Sepharose FF ion exchange chromatography and Sephadex G 150 gel filtration.Purified β galactosidase A showed one protein band on PAGE.It was also a single protein band on SDS\|PAGE and its Mr was 3 3×10 4. Mr of A and B determined by gradient PAGE was the same as 1 4×10 5.It is suggested that β galactosidase A comprised four identical subunits.The p I of β galactosidase A and B estimated by isoelectric focusing PAGE were 7 6 and 4 6 respectively.The apparent K m of A and B were 2.08 mmol/L and 2.45 mmol/L(ONPGal),0.75 mmol/L and 1.24 mmol/L(PNPGal),44 mmol/L and 25 mmol/L(lactose).Their activation energies were 43.9 kJ/mol and 34.3 kJ/mol using ONPGal as substrate.Their optimum pH all were 4.0.Their optimum temperatures were 50℃ and 55℃ respectively.Stability ranges of pH were pH 4.0～6.0 and pH 3.5～5.0 separately.Galactose and lactose were reversibly competitive inhibitors. K i for A were 6.21 mmol/L(galactose)and 329 mmol/L(lactcse), K i for B were 3.93 mmol/L(galactose )and 169 mmol/L(lactose).Melibiose and raffinose were reversibly uncompetitive inhibitors against the enzymes.Metal ions and some chemical agents also showed inhibition against the enzymes.展开更多
文摘The β galactosidase A and B from Adsuki bean cotyledons were isolated by ammonium sulfate fractionation and DEAE\|Sepharose FF ion exchange chromatography.The A form was further purified by CM Sepharose FF ion exchange chromatography and Sephadex G 150 gel filtration.Purified β galactosidase A showed one protein band on PAGE.It was also a single protein band on SDS\|PAGE and its Mr was 3 3×10 4. Mr of A and B determined by gradient PAGE was the same as 1 4×10 5.It is suggested that β galactosidase A comprised four identical subunits.The p I of β galactosidase A and B estimated by isoelectric focusing PAGE were 7 6 and 4 6 respectively.The apparent K m of A and B were 2.08 mmol/L and 2.45 mmol/L(ONPGal),0.75 mmol/L and 1.24 mmol/L(PNPGal),44 mmol/L and 25 mmol/L(lactose).Their activation energies were 43.9 kJ/mol and 34.3 kJ/mol using ONPGal as substrate.Their optimum pH all were 4.0.Their optimum temperatures were 50℃ and 55℃ respectively.Stability ranges of pH were pH 4.0～6.0 and pH 3.5～5.0 separately.Galactose and lactose were reversibly competitive inhibitors. K i for A were 6.21 mmol/L(galactose)and 329 mmol/L(lactcse), K i for B were 3.93 mmol/L(galactose )and 169 mmol/L(lactose).Melibiose and raffinose were reversibly uncompetitive inhibitors against the enzymes.Metal ions and some chemical agents also showed inhibition against the enzymes.
