Journal: Frontiers in Microbiology

Article Title: The Function of CBM32 in Alginate Lyase VxAly7B on the Activity on Both Soluble Sodium Alginate and Alginate Gel

doi: 10.3389/fmicb.2021.798819

Figure Lengend Snippet: Purification and enzymatic characteristics of recombinant VxAly7B and its truncated mutants. (A) Domain structure of full long VxAly7B and its two truncated mutants. (B) Purified recombinant VxAly7B-FL, VxAly7B-CM, and VxAly7B-CBM were resolved by SDS-PAGE. Lane M, molecular weight markers; Lane 1, purified recombinant VxAly7B-FL; Lane 2, recombinant VxAly7B-CM; Lane 3, recombinant VxAly7B-CBM. (C) Optimum pH for recombinant VxAly7B-FL and VxAly7B-CM were determined by measuring the activity at 40 or 45°C in 50 mM Na 2 HPO 4 -citric acid buffer (pH 4.6–7.0, inverted triangles), 50 mM Na 2 HPO 4 -NaH 2 PO 4 buffer (pH 6.6–7.6, regular triangles), 50 mM Tris-HCl buffer (pH 7.6–8.6, circles) and 50 mM Gly-NaOH buffer (pH 8.6–9.6, squares). The red line represented the recombinant VxAly7B-FL, and the blue line represented the recombinant VxAly7B-CM. (D) To determine pH stability, the residual activity of recombinant VxAly7B-FL and VxAly7B-CM were measured at 40 or 45°C in 20 mM Na 2 HPO 4 -NaH 2 PO 4 buffer (pH 7.0) and Tris-HCl buffer (pH 7.6) after incubation in 50 mM Na 2 HPO 4 -citric acid buffer (pH 3.0–7.0, inverted triangles), 50 mM Na 2 HPO 4 -NaH 2 PO 4 buffer (pH 6.6–7.3, regular triangles), 50 mM Tris-HCl buffer (pH 7.6–8.6, circles) and 50 mM Gly-NaOH buffer (pH 8.6–10.6, squares) at 4°C for 12 h, respectively. (E) Optimal temperature for recombinant VxAly7B-FL and VxAly7B-CM were determined by measuring the activity at 0–60°C. (F) To determine thermostability, the enzyme was incubated at 0–70°C for 1 h. Residual activity was determined at 40 and 45°C, respectively. (G) Effect of the NaCl concentration on recombinant VxAly7B-FL and VxAly7B-CM activity. (H) Substrate specificity of recombinant VxAly7B-FL and VxAly7B-CM toward alginate, polyM, and polyG.

Article Snippet: PolyM and polyG were obtained from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China).

Techniques: Purification, Recombinant, SDS Page, Molecular Weight, Activity Assay, Incubation, Concentration Assay