Characterization of an eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia Yezoensis

Abstract

Background: Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis.Methods: The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated.Results: PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage.Conclusion: This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.

Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability

“…This activity was comparable with the maximum activity of PyAly at 35°C and pH 8 (39), and it was ϳ2.5% that of FlAlyA at 30°C and pH 8 (48). When comparing the maximum activity of rNitAly with other PL enzymes, abalone alginate lyase HdAly belonging to PL-14 has about 1.3-fold higher activity at 35°C and pH 8 (49).…”

“…The mutant proteins of NitAly (rNitAlyC80A, rNitAlyC232A, and rNitAlyC80A/C232A) were purified using the same method as that described for rNitAly in the previous section. Wild-type rPyAly and its mutant proteins (rPyAlyG79C, rPyAlyD230C, and rPyAlyG79C/D230C) were purified using the same method as that used for rPyAly, as reported previously (39).…”

“…All assays were repeated three times, and the data are shown as mean Ϯ S.D. activity ceased at incubation temperatures above 50°C (39). Alignment of the amino acid sequences of PyAly and NitAly (Fig.…”

“…(34), Klebsiella pneumoniae (35), Zobellia galactanivorans (36), Saccharophagus degradans (37), and an uncultured bacterium (38). Within the last year, a novel alginate lyase, PyAly belonging to the PL-7 family, was identified from the red alga Pyropia yezoensis (39). This discovery was unexpected because P. yezoensis is not considered to have any alginate utilization systems.…”

Cloning, expression, and characterization of a new pH‐ and heat‐stable alginate lyase from Pseudoalteromonas carrageenovora ASY5

“…A double bond is then formed between nonreducing ends, namely C4 and C5, where the glycosidic bond is located, and an oligomeric oligosaccharide with a 4‐deoxy‐L‐erythro‐hex‐4‐enepyranosyluronate structure is simultaneously produced (Wong, Preston, & Schiller, ). Alginate lyases can be obtained from various sources, such as marine and terrestrial bacteria (Yu, Zhu, Wang, Tan, & Yin, ; Zhu, Ni, Sun, & Yao, ), fungus (Cao et al, ; Singh et al, ), virus (Ichiro et al, ), marine mollusks (Hata et al, ; Rahman, Inoue, Tanaka, & Ojima, ), and algae (Inoue et al, ). They can be classified into two groups, namely, polyG specific (polyG lyase, EC 4.2.2.11) and polyM specific (polyM lyase, EC 4.2.2.3), because of their different effects on substrates (Zhu & Yin, ).…”