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Table 3 Substrate specificity of Smlt2602 mutants

From: Engineering broad-spectrum digestion of polyuronides from an exolytic polysaccharide lyase

  Specific activity (ΔA235nm min−1 mg−1)
Alginate Poly-ManA Poly-GulA Poly-MG Poly-GlcA
WT 328.3 ± 27.0 661.9 ± 29.1 190.3 ± 8.3 289.3 ± 9.8 2.8 ± 0.4
Possible involvement in β-elimination mechanism
 Y455F 3.6 ± 0.3 4.0 ± 0.4 15.3 ± 1.4 11.5 ± 0.1 0.2 ± 0.1
Enhanced poly-GlcA activity
 H208A 48.2 ± 3.5 83.2 ± 4.2 6.3 ± 0.8 3.6 ± 0.4 3.1 ± 0.8
 H208W 185.3 ± 11.8 490.8 ± 4.1 48.5 ± 1.0 117.1 ± 7.8 7.1 ± 0.6
 H208F 88.4 ± 4.0 189.7 ± 4.1 9.5 ± 0.8 23.3 ± 1.0 77.2 ± 1.2
Additional putative substrate-binding residues
 R143L 224.1 ± 13.9 245.4 ± 15.3 95.5 ± 2.5 159.0 ± 9.2 1.1 ± 0.3
 Q153A 129.0 ± 3.5 456.4 ± 26.4 30.3 ± 3.5 73.6 ± 4.4 0.2 ± 0.1
 Q153 N 113.8 ± 9.9 323.7 ± 17.1 83.4 ± 2.6 108.7 ± 10.7 0.9 ± 0.2
 L155A 158.5 ± 9.5 597.6 ± 46.9 28.4 ± 0.9 89.6 ± 1.4 0.3 ± 0.1
 L155 N 125.6 ± 12.6 512.7 ± 23.2 42.4 ± 2.9 131.9 ± 4.8 0.3 ± 0.2
 N201L 380.1 ± 9.2 915.8 ± 31.9 138.6 ± 12.9 255.3 ± 9.9 3.1 ± 0.7
 H206F 73.9 ± 2.3 147.8 ± 3.9 57.0 ± 2.9 73.2 ± 0.2 0.4 ± 0.1
 Y258F 173.4 ± 9.1 346.2 ± 16.1 88.7 ± 6.0 127.7 ± 9.5 0.5 ± 0.1
 Y263F 36.1 ± 0.4 83.5 ± 1.7 5.3 ± 0.6 13.5 ± 1.4 0.2 ± 0.1
  1. Purified wild-type and mutant Smlt2602 was added to 1 mg/mL of each substrate in 20 mM sodium phosphate buffer at pH 8.5 for alginate-based polysaccharides and pH 8 for poly-GlcA. Enzymatic activity was monitored by absorbance at 235 nm. In addition to the substrates listed below, each enzyme was tested against poly-GalA, heparin, heparan sulfate, and hyaluronic acid, with no detectable activity. All reactions were performed in triplicate, and error is reported as standard deviation