Article Figures & Data
Figures
- Fig. 1.
The yvdE to yvdM region from 3,545 to 3,558 kb of B. subtilis. The yvdL gene (black arrow, now named malL) encoding the α-glucosidase is depicted below the DNA. Restriction sites used for malLinactivation by the aphA3 cassette are listed. Potential transcription terminators as proposed in the SubtiList data bank (15, 32) are denoted at the ends of the DNA.
- Fig. 2.
Growth phase-dependent MalL activity. B. subtilis strains were grown in minimal media containing 10 mM maltose. Aliquots were harvested at the indicated times, and MalL activity was determined with PNPG as a substrate and expressed in nanomoles of product formed minute−1 milligram of crude protein extract−1 for the wild-type (○) and the MalL− strain MD193 (▵). The corresponding OD600s of the cultures are indicated for the wild-type (•) and the MalL− strain MD193 (▴). The growth and MalL activities of strains MD192 and MD193 were identical.
- Fig. 3.
Elution profile in Ni2+ HiTrap chelating column and purification of MalL. Protein absorption at 280 nm (solid line) and the imidazole gradient as the percentage of buffer B (dashed line) are presented as a function of the column volume (in milliliters). Analysis of selected fractions (indicated by numbers on the elution profile) on SDS-PAGE is shown in the insert. Pure MalL is in fraction 4. Molecular mass standards with the indicated sizes in kilodaltons (kD) are shown in lane st. mAU, milli-absorption units.
Tables
- Table 1.
Bacterial strains and plasmids used
Strain or plasmid Relevant genotype or phenotype Reference or sourcea Strains Bacillus subtilis 168 trpC2 (wild type) BGSC, 1A1 MD192 trpC2 malL::aphA3 pMalLK2 tf> 168 MD193 trpC2 malL::aphA3 pMalLK1 tf> 168 Escherichia coli RB791 F′ (lacIq L8)hsdR+hsdM 2 Plasmids pDG792 pMTL23 derivative containing theaphA3 antibiotic cassette 11 pMalL pQE-9 derivative containing malL fused in frame to the His-tag coding region under tac promoter control This work pMalLK1 pMalL derivative carryingmalL::aphA3 This work pMalLK2 pMalL derivative carryingmalL::aphA3 This work pQE-9 Expression vector for His-tag fusions underT5 promoter control 21 ↵a tf> indicates transformation of DNA mentioned. BGSC, Bacillus Genetic Stock Center, Ohio State University, Columbus.
- Table 2.
MalL substrate specificity and kinetic parameters
Carbohydrate Systematic name of carbohydrate Vmax (μmol min−1mg of protein−1) Km (mM) Sucrose 1-O-α-d-glucopyranosyl-β-d-fructofuranoside 357 10.2 Isomaltose 6-O-α-d-glucopyranosyl-d-glucose 162 0.455 Maltose 4-O-α-d-glucopyranosyl-d-glucose 65 0.135 PNPG p-Nitrophenyl-α-d-glucopyranoside 3.3 0.210
