The Characterization and Modification of a Novel Bifunctional and Robust Alginate Lyase Derived from Marinimicrobium sp. H1

Abstract

Alginase lyase is an important enzyme for the preparation of alginate oligosaccharides (AOS), that possess special biological activities and is widely used in various fields, such as medicine, food, and chemical industry. In this study, a novel bifunctional alginate lyase (AlgH) belonging to the PL7 family was screened and characterized. The AlgH exhibited the highest activity at 45 °C and pH 10.0, and was an alkaline enzyme that was stable at pH 6.0-10.0. The enzyme showed no significant dependence on metal ions, and exhibited unchanged activity at high concentration of NaCl. To determine the function of non-catalytic domains in the multi-domain enzyme, the recombinant AlgH-I containing only the catalysis domain and AlgH-II containing the catalysis domain and the carbohydrate binding module (CBM) domain were constructed and characterized. The results showed that the activity and thermostability of the reconstructed enzymes were significantly improved by deletion of the F5/8 type C domain. On the other hand, the substrate specificity and the mode of action of the reconstructed enzymes showed no change. Alginate could be completely degraded by the full-length and modified enzymes, and the main end-products were alginate disaccharide, trisaccharide, and tetrasaccharide. Due to the thermo and pH-stability, salt-tolerance, and bifunctionality, the modified alginate lyase was a robust enzyme which could be applied in industrial production of AOS.

Keywords: PL7 family; alginate lyase; alginate oligosaccharides; bifunctional; robust enzyme.

Figure 1

Phylogenetic tree of AlyH with other reported alginate lyases. Evolutionary analysis was inferred using the Neighbor-Joining method in MEGA7.

Figure 2

Sequence analysis of AlgH from Marinimicrobium sp. H1. (A) The domain composition of AlgH. The green, yellow, blue, and red colors correspond to signal peptide, carbohydrate binding module (CBM), F5/8 type C, and catalytic domain, respectively. (B) Amino acid alignment of catalytic active region sequence of AlgH to other reported PL7 alginate lyases. AlgH from Marinimicrobium sp. H1 in this study, AlyPI (ACM89454.1) from Pseudoalteromonas sp. CY24 [28], AlyL1 (AJO61885.1) from Agarivorans sp. L11 [29], AlgNJU-03 (ASA33933.1) from Vibrio sp. NJU-03 [30] and the conserved regions of QIH and YFKAG are boxed.

Figure 3

The SDS-PAGE analysis of the purified AlgH, AlgH-I, and AlgH-II. Lane M, the protein molecular weight standard; Lane 1, purified AlgH; Lane 2, purified AlgH-II; Lane 3, purified AlgH-I.

Figure 4

Biochemical characteristics of three recombinant alginate lyases. (A) The optimal temperatures of AlgH (purple), AlgH-I (green), and AlgH-II (red). (B) The optimal pHs of AlgH (purple), AlgH-I (green), and AlgH-II (red). (C) The thermal stabilities of AlgH (purple), AlgH-I (green), and AlgH-II (red). Solid and dashed lines correspond to incubation temperatures of 40 and 50 °C, respectively. (D) The pH stabilities of AlgH (purple), AlgH-I (green), and AlgH-II (red).

Figure 5

The effects of various metal ions on the activity of the recombinant alginate lyases. The enzymatic activity without the addition of metal ions was defined as 100%. Dark gray, red, and light gray represent AlgH, AlgH-I, and AlgH-II, respectively.

Figure 6

Substrate preference of the recombinant alginate lyases. (A) The relative activities towards alginate, polyG, and polyM. (B) HPLC analyses of the degradation products of polyG and polyM.

Figure 7

The analyses of degradation products produced by AlgH-I. (A) Time course analysis of oligosaccharide products of alginate. (B) Relative contents of oligosaccharides. HPLC analyses were performed using a Superdex peptide 10/300 GL column monitored at a wavelength of 235 nm. DP1, DP2, DP3, DP4, and DP5 represent mono, di, tri, tetra, and penta-saccharide, respectively.

Figure 8

ESI-MS and ¹H-NMR spectral analysis of the purified degradation products of alginate. (A–C) represent the ESI-MS results of di, tri, and tetra-saccharide, respectively. (D) ¹H-NMR spectra of the purified disaccharide (600-MHz).