L. M. Godinho and I. de Sa´ -Nogueira AraL sugar phosphatase from B. subtilis an auto-induction regime for the overproduction of AraL recombinant proteins was used [20]. To prepare the cell-free extracts, the cells were resuspended in lysis buffer (20 mm sodium phosphate buffer, pH 7.4, 62.5 mm NaCl, 10 mm imidazole, glycerol 10%) and disrupted in the presence of lysozyme (1 mgÆmL)1) by three cycles of freezing in liquid nitrogen and thawing for 5 min at 37 C, followed by incu- bation with benzonase nuclease (Novagen , Darmstadt, Germany). After 15 min of centrifugation at 16 000 g and 4 C, the soluble and insoluble fractions of the crude extract were obtained. For overproduction and puriﬁcation of recombinant AraL-His6, E. coli BL21(DE3) pLysS cells harboring pLG11 were grown in 100 mL of auto-induction medium [20]. Cells were harvested by centrifugation at 6000 g and 4 C for 10 min. All subsequent steps were carried out at 4 C. The harvested cells were resuspended in Start Buffer (TrisHCl 100 mm buffer, pH 7.4, 62.5 mm NaCl, 10 mm imidazole, glycerol 10%) and lysed by passing three times through a French pressure cell. The lysate was centrifuged for 1 h at 13 500 g and the proteins from the supernatant were loaded onto a 1 mL Histrap Ni2+-nitrilotriacetic acid afﬁnity column (GE Healthcare Life Sciences). The bound proteins were eluted with a discontinuous imidazole gradi- ent and those fractions containing AraL that were more than 95% pure were dialysed overnight against storage buf- fer (TrisHCl 100 mm buffer, pH 7.4, 100 mm NaCl, glyc- erol 10%) and then frozen in liquid nitrogen and kept at )80 C until further use. Protein analysis Analysis of production, homogeneity and the molecular mass of the enzyme were determined by SDS ⁄ PAGE using broad-range molecular weight markers (Bio-Rad Laborato- ries, Hercules, CA, USA) as standards. The degree of puri- ﬁcation was determined by densitometric analysis of Coomassie blue-stained SDS ⁄ PAGE gels. The protein con- tent was determined by using Bradford reagent (Bio-Rad Laboratories) with BSA as standard. Enzyme assays Phosphatase activity Phosphatase activity assays were performed using the gen- eral substrate pNPP. The reaction mixture comprising 100 mm Tris–HCl buffer, pH 7, containing 15 mm MgCl2 and appropriately diluted enzyme (20 lg) was incubated at 37 C for 5 min. Addition of 20 mm pNPP started the reac- tion and the mixture was incubated for an additional 1 h. The reaction was stopped by adding 1 mL of 0.2 m NaOH, the tubes were centrifuged at 16 000 g for 1 min and 1 mL of the supernatant was recovered for measurement of A405. A calibration curve for phosphatase activity assays using pNPP as a substrate was made using various concentrations (mgÆmL)1) of p-nitrophenol, within the measuring range of the method [36]. Negative controls were made using 20 lg of BSA, and blanks had no protein added. Enzymatic activity was also determined in the presence of 15 mm EDTA, using the same conditions: 1 U of AraL hydrolyses, 1 lmol of sub- strate per min. Both optimum temperature and pH for enzy- matic activity of AraL-His6 were determined as described above. The effect of temperature was tested in 100 mm Tris– HCl buffer, pH 7, containing 15 mm MgCl2, at temperatures in the range 25–70 C. The effect of pH on the activity was assayed at 65 C in a series of Britton–Robinson buffers (0.1 m boric acid, 0.1 m acetic acid and 0.1 m phosphoric acid, pH 3–6, and Tris–HCl buffers, pH 7.0–9.0). Continuous activity assays All continuous assays were carried out at 37 C in 100 mm Tris–HCl buffer, pH 7, containing 15 mm MgCl2, unless stated otherwise. Glucose production from glucose 6-phos- phate was monitored by measurement of the glucose dehy- drogenase catalysed reduction of NADP. The initial velocity of glucose formation by dephosphorylation of glu- cose 6-phosphate in reaction solutions initially containing 20 lg of AraL, 0.7 U of glucose 6-phosphate dehydroge- nase, 0.2 mm NADP, 1–15 mm a-glucose 6-phosphate and 15 mm MgCl2 in 0.5 mL of 100 mm Tris–HCl (pH 7.5, 37 C) was determined by monitoring the increase in A340. Discontinuous assays Initial phosphate hydrolysis for all substrates used in sub- strate screening was assessed to detect total phosphate release using the Malachite Green Phosphate Detection Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. The 150 lL assay mixture comprising 100 mm Tris–HCl buffer (pH 7), con- taining 15 mm MgCl2, was incubated for 1 h at 37 C. Background phosphate levels were monitored in parallel using a control reaction without the AraL enzyme. A620 was measured. Steady-state kinetics was carried out using 20 lg of AraL with varying concentrations of substrates. Kinetic parameters were determined using the enzyme kinetics software graphpad prism, version 5.03 (GraphPad Software Inc., San Diego, CA, USA). In-frame deletion of araL in B. subtilis To create B. subtilis mutant strains with an in-frame dele- tion of araL, plasmid pLG10 was constructed using pMAD (Table 1). Regions immediately upstream and downstream of araL were ampliﬁed by two independent PCR experi- ments, from chromosomal DNA of B. subtilis 168T+, using primers ARA444 and ARA458 (PCR1) and ARA459 and ARA460 (PCR2). The products were joined by overlapping FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS 2521