Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524

Abstract

Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics.

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

“…AlgSH7 contains an QIH sequence, while, it prefers polyM blocks. This result is similar to AlyA-OU02, AlyPM, and FlAlyA [24][25][26]. Most of the reported PL7 family alginate lyases have endolytic activity.…”

“…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.…”

“…The optimal temperature of AlgSH7 was investigated over the temperature range of 25-60 • C. AlgSH7 exhibits over 80% activity between 25 and 45 • C, and the optimal reaction temperature is 40 • C ( Figure 4A). The thermostability of AlgSH7 was determined by measuring the residual activities after incubating the enzyme at 25,30,35,40,45, and 50 • C for different times, respectively. The enzyme displays over 90% of the maximum enzyme activity after 2-h incubation at 25 and 30 • C. After incubated at 35 • C for 30 min and 2 h, the residual activities are 80% and 13%, respectively.…”

“…However, AlgSH7 is contrary to this accepted rule. AlgSH7 is active towards polyM, containing the QIH sequence which is similar to AlyA-OU02 [24], AlyPM [25], and FlAlyA [26].…”

Characterization and Application of an Alginate Lyase, Aly1281 from Marine Bacterium Pseudoalteromonas carrageenovora ASY5

“…The alignment indicated that Aly1281 was most closely related to alginate Lyase (PDB ID: 4BE3_A) from Zobellia galactanivorans with an amino acid sequence identity of 42.4%. Aly1281 contained three highly conserved regions of PL family 7, namely, R(S/T)ELV(R/G), QIH, and YFK(A/L)GAYNQ, which played an important role in the substrate binding and catalytic activity of the enzymes [15,17,20]. Moreover, catalytic (Q173, H175, and Y315) and substrate binding (R104, E194, K197, E216, D228, and W327) sites were predicted in Aly1281 on the basis of previous research on alginate lyase (AlyA5) from Z. galactanivorans [21].…”