5) and frozen at -20°C for 15 min. After thawing at room temperature, the samples were centrifuged
at 10,000 × g. The supernatant containing the desired protein was applied onto affnity matrix of agarose coupled with p-aminobenzyl-1-thio-β-D-galactopyranoside (PABTG-agarose, Sigma) (10 ml column) equilibrated with four volumes of buffer A. The column was washed with 300 ml of the buffer A, and the recombinant β-D-galactosidase was eluted three times with 10 ml of 0.05 M sodium borate (pH 10.0) buffer at a flow rate of 0.5 ml/min. Active fractions containing the β-D-galactosidase were collected and dialyzed three times LY3039478 mouse against 3 L of buffer D (100 mM NH4HCO3). In case of the purification of the extracellular produced β-D-galactosidase in P. pastoris cultures, the yeast
cells were separated from the post-culture medium through centrifugation. Next, the ammonium sulphate was added to the post-culture medium to 60% w/w, at 4°C. The precipitated proteins were centrifugated at 20,000 × g, dissolved in buffer A and dialyzed overnight against the same buffer. For β-D-galactosidase purification the dissolved sample was applied further directly onto affnity matrix of agarose coupled with p-aminobenzyl-1-thio-β-D-galactopyranoside and purified as described above for bacterial system. The concentration of purified protein was determined by the Bradford method using bovine serum albumin (BSA) as a find more standard. β-D-galactosidase activity assays The activity of purified Arthrobacter sp. 32c β-D-galactosidase was determined by the use of chromogenic substrates as described elsewhere [4, 14]. The PRN1371 cost o-nitrophenol released from 10 mM of o-nitrophenyl-β-D-galactopyranoside (ONPG) by β-D-galactosidase at 0–70°C and pH range 4.5–9.5 (0.02 M citrate buffer for pH 4.5 and 5.5; 0.02 M K2HPO4-KH2PO4 for pH 6.5 and 7.0 and 0.02 M Tris-HCl for pH selleck products 8.5 and 9.5) was measured
at 405 nm. The reaction was stopped after 10 min with 1 M Na2CO3. One unit is defined as one micromolar of o-nitrophenol released per minute. Substrate specifiCity was estimated using 1 mM solution of chromogenic substrates: o-nitrophenyl-β-D-galactopyranoside (ONPG), p-nitrophenyl-β-D-galactopyranoside (PNPG), o-nitrophenyl-β-D-glucopyranoside (ONPGlu) and p-nitrophenyl-β-D-glucopyranoside (PNPGlu). Activity determination was carried out under standard conditions in 0.02 M K2HPO4-KH2PO4 (pH 6.5) buffer at 10, 20, 30, 40 or 50°C. The activity of the β-D-galactosidase towards lactose was monitored by HPLC analysis (column Bio-rad, Aminex HPX-87H) where 1% solutions of lactose, glucose, fructose and galactose were used as standards. In the combined enzyme assay glucose isomerase from Streptomyces murinus (Sigma G4166) was used in the amount of 0.01 g/ml of 5% w/v solution of lactose (0.02 M K2HPO4-KH2PO4, pH 6.5). The Arthrobacter sp. 32c β-D-galactosidase was used at concentration of 200 U/ml of the mixture.