AlgM4: A New Salt-Activated Alginate Lyase of the PL7 Family with Endolytic Activity

Abstract

Alginate lyases are a group of enzymes that catalyze the depolymerization of alginates into oligosaccharides or monosaccharides. These enzymes have been widely used for a variety of purposes, such as producing bioactive oligosaccharides, controlling the rheological properties of polysaccharides, and performing structural analyses of polysaccharides. The algM4 gene of the marine bacterium Vibrio weizhoudaoensis M0101 encodes an alginate lyase that belongs to the polysaccharide lyase family 7 (PL7). In this study, the kinetic constants Vmax (maximum reaction rate) and Km (Michaelis constant) of AlgM4 activity were determined as 2.75 nmol/s and 2.72 mg/mL, respectively. The optimum temperature for AlgM4 activity was 30 °C, and at 70 °C, AlgM4 activity dropped to 11% of the maximum observed activity. The optimum pH for AlgM4 activity was 8.5, and AlgM4 was completely inactive at pH 11. The addition of 1 mol/L NaCl resulted in a more than sevenfold increase in the relative activity of AlgM4. The secondary structure of AlgM4 was altered in the presence of NaCl, which caused the α-helical content to decrease from 12.4 to 10.8% and the β-sheet content to decrease by 1.7%. In addition, NaCl enhanced the thermal stability of AlgM4 and increased the midpoint of thermal denaturation (Tm) by 4.9 °C. AlgM4 exhibited an ability to degrade sodium alginate, poly-mannuronic acid (polyM), and poly-guluronic acid (polyG), resulting in the production of oligosaccharides with a degree of polymerization (DP) of 2–9. AlgM4 possessed broader substrate, indicating that it is a bifunctional alginate lyase. Thus, AlgM4 is a novel salt-activated and bifunctional alginate lyase of the PL7 family with endolytic activity.

Purification and Characterization of a Novel Endolytic Alginate Lyase from Microbulbifer sp. SH-1 and Its Agricultural Application

“…AlgSH7 contains 642 amino acids with a theoretical molecular mass of 66.4 kDa and theoretical pI 4.71. AlgSH7 belongs to the PL7 family because it contains the SA3 domain (RTELR), the SA4 domain (YFKAGVYNQ), and the SA5 catalytic domain (QIH), which are conserved in the aligned catalytic modules of PL7 family alginate lyase [21,25]. Among the characterized alginate lyases derived from the Microbulbifer genus, AlgSH7 is 83.6% homologous with alginate lyase AlgMsp (BAJ62034.1) and 73.22% homologous with alginate lyase AlyM.…”

“…It has been reported that the conserved regions of polyM-preference, polyG-preference, and polyMG-preference alginate lyases contain QVH, QIH, and QIH amino acid residues, respectively [24]. For example, AlgNJU-03, AlgM4, and AlyPI contain QIH in the conserved region and show activities towards polyM and polyG [21,34,35]. The polyG-preference alginate lyases, such as ALG-5, AlyM, and Alg2A all contain an QIH sequence [23,36,37].…”

“…Alginate lyases can be affected by various metal ions [21]. Na + functions as an activator for many alginate lyases.…”

“…In the presence of 0.4 mol/L NaCl, the enzyme activity of AlgSH7 is increased by 1.75-fold, suggesting AlgSH7 is a salt-activated enzyme. The enhancing effect of Na + may due to altering the secondary structure of AlgSH7, which enhances the affinity of the enzyme for its substrates and facilitates enzymolysis [21,25]. The activation effects of metal ions on alginate lyases, such as K + , Ca 2+ , and Mg 2+ , have been widely reported [45].…”

“…Multiple sequences alignments of AlgSH7 and related alginate lyases from the PL7 family are shown in Figure 3A. PL7 family alginate lyases are identified by containing three highly conserved domains, SA3 (RXEXR), SA4 (YXKAGXYXQ), and SA5 (QXH) [21]. Three highly conserved regions RTELR, YFKAGVYNQ, and QIH are identified in the amino acid sequence of AlgSH7, suggesting that AlgSH7 is a new member of the PL7 family.…”