Figure 1.
SDS-PAGE analysis of recombinant AlgMsp expression and purification.
Lane 1 is the Precision Plus protein standard (Bio-Rad). Lane 2 is the cell lysate. Lane 3 is the flow-through from the nickel column. Lane 4 is the elution from the nickel column. Lane 5 is the elution from the S-200 gel filtration column.
Figure 2.
Effects of pH and NaCl on alginate lyase activity of AlgMsp.
(A) Effect of pH in the absence of NaCl on activity in 20 mM Tris (circles) or BP buffer (triangles). (B) Effect of NaCl on activity in 20 mM Tris buffer pH 8. (C) Effect of pH on activity in the presence of 200 mM NaCl in 20 mM Tris (circles) or BP buffer (triangles). Activity determined by the initial velocity of increase in absorbance at 235 nm over a 10 minute incubation at 25°C. Activity was normalized to 100% for the most active sample.
Figure 3.
Effect of temperature on alginate lyase activity and stability of activity.
(A) Effect of temperature on activity with (filled circles) and without NaCl (open circles). Activity was determined, after 10 minutes at the target temperature, by the increase in absorbance at 235 nm over the absorbance of the control reaction without enzyme. (B) Thermostability of alginate lyase activity. AlgMsp was incubated at 50°C (circles), 37°C (squares), and 25°C (triangles) for 24 hours in 20 mM Tris, 200 mM NaCl, pH 8. Samples were tested at 0, 0.5, 1, 2, 4, 8, and 24 hours for residual activity as above. Activity was normalized to 100% for the most active sample.
Table 1.
Effects of divalent cations on AlgMsp activity.
Table 2.
Kinetics of AlgMsp.
Figure 4.
Circular dichroism analysis of stability and structure of AlgMsp.
(A) Far-UV CD analysis of AlgMsp structure, representative graph displaying the calculated secondary structure composition. (B) CD-melting curve of AlgMsp. The enzyme was heated at 1°C/min in 20 mM sodium phosphate buffer pH 8 at a protein concentration of 0.1 mg/ml.
Figure 5.
FPLC fractionation and analysis of products from AlgMsp-cleavage of alginate.
AlgMsp-cut and uncut alginate are represented by a solid black line and a dashed red line respectively in panels A to D. (A) Real-time FPLC UV-detector trace from absorbance at 254 nm of products eluting from the Superdex Peptide HR10/30 column. (B) Testing of 0.25 ml FPLC fractions for unsaturated uronates by absorbance at 235 nm. (C) Testing of 0.25 ml FPLC fractions for all uronic acids by the carbazole assay with an increase in absorbance at 530 nm. (D) Testing of 0.25 ml FPLC fractions for reducing sugars by the DNS assay with an increase in absorbance at 535 nm.
Figure 6.
ESI-mass spectrometry analyses of peak fractions from FPLC separation of the oligo-uronates from AlgMsp digestion of alginate.
DPx is degree of polymerization. DPx and ΔDPx represent saturated and unsaturated oligo-uronates, respectively. (A) Mass spectrum of FPLC fraction 1, the 13.4 ml elution peak, showing predominance of ΔDP5 eluent. Inset: MS/MS product ion spectrum of m/z 439 peak, showing the methodology used to elucidate the structure of all observed ESI-MS anion peaks. The m/z 703 and m/z 721 peaks are indicative of the double charge of the precursor anion, m/z 439. The annotated peak series, {[ΔDPx-1H]−, x = 1–4}, and the peaks denoted with asterisks ([DP4-2H]2− (m/z 360), {[DP3-H]− (m/z 545), and {[DP4-H]− (m/z 721), confirm that the composition of the m/z 439 precursor anion, is consistent with [ΔDP5-2H]2−. (B) Mass spectrum of FPLC fraction 2, the 14.0 ml elution peak, showing predominance of ΔDP4 eluent. (C) Mass spectrum of FPLC fraction 3, the 14.7 ml elution peak, showing predominance of ΔDP3 eluent. (D) Mass spectrum of FPLC fraction 4, the 15.6 ml elution peak, showing predominance of ΔDP2 eluent.