Biochemical Characterization and Elucidation of the Hybrid Action Mode of a New Psychrophilic and Cold-Tolerant Alginate Lyase for Efficient Preparation of Alginate Oligosaccharides

Abstract

Alginate lyases with unique biochemical properties have irreplaceable value in food and biotechnology industries. Herein, the first new hybrid action mode Thalassotalea algicola-derived alginate lyase gene (TAPL7A) with both psychrophilic and cold-tolerance was cloned and expressed heterologously in E. coli. With the highest sequence identity (43%) to the exolytic alginate lyase AlyA5 obtained from Zobellia galactanivorans, TAPL7A was identified as a new polysaccharide lyases family 7 (PL7) alginate lyase. TAPL7A has broad substrate tolerance with specific activities of 4186.1 U/mg, 2494.8 U/mg, 2314.9 U/mg for polyM, polyG, and sodium alginate, respectively. Biochemical characterization of TAPL7A showed optimal activity at 15 °C, pH 8.0. Interestingly, TAPL7A exhibits both extreme psychrophilic and cold tolerance, which other cold-adapted alginate lyase do not possess. In a wide range of 5-30 °C, the activity can reach 80-100%, and the residual activity of more than 70% can still be maintained after 1 h of incubation. Product analysis showed that TAPL7A adopts a hybrid endo/exo-mode on all three substrates. FPLC and ESI-MS confirmed that the final products of TAPL7A are oligosaccharides with degrees of polymerization (Dps) of 1-2. This study provides excellent alginate lyase candidates for low-temperature environmental applications in food, agriculture, medicine and other industries.

Keywords: alginate lyase; biochemical characterization; cold-tolerant; endo/exolytic; psychrophilic.

Figure 1

The domain analysis and multiple amino-acid sequence alignment of TAPL7A and other alginate lyases of PL7 family. (a) The domain of TAPL7A. (b) AlyA5 (CAZ98266.1) from Zobellia galactanivorans Dsij^T. AlyA (AAA25049.1) from Klebsiella pneumoniae subsp. aerogenes, AlyB (5ZU5) from Vibrio splendidus, PA1167 (AAG04556.1) from Pseudomonas aeruginosa PAO1, AlgAT5 (5ZQI) from Defluviitalea, alyPG (BAA83339.1) from Corynebacterium sp. ALY-1, AlyA1 (CAZ95239.1) from Zobellia galactanivorans Dsij^T, AlyQ (WP_053404615.1) from Persicobacter sp. CCB-QB2, A1-Ⅱ’ (BAD16656.1) from Sphingomonas sp. A1, FlAlyA (BAP05660.1) from Sphingomonas sp. A1, PsAlg7A (VFY81779.1) from Paradendryphiella salina. Shaded yellow indicates identical and similar amino-acid residues in alginate lyase. The red boxes indicate the positions of the three conserved regions. Black triangles are catalytic bases, red triangles and green triangles represent catalytic acids of PL7 lyase in different red boxes, respectively. Black circles indicate the neutralization residues.

Figure 2

Phylogenetic analysis of TAPL7A with other alginate lyases of PL7 family. Subfamilies are separated by blue dotted lines according to the CAZy database. Enzymes for structure-solved are indicated by red five-pointed stars. Characterized enzymes are indicated by blue circles. PloyMG, PolyM, and PolyG represent the specific substrates of each enzyme, respectively. The red font is TAPL7A. The green vertical lines and fonts on the right represent the various subfamilies and PsAlg7A of eukaryotic origin.

Figure 3

SDS-PAGE analysis of the molecular mass and purification effect of TAPL7A. Lane M protein: restrained marker (Thermo Scientific, USA); lane 1: the supernatant of E. coli-pET21a-TAPL7A; lane 2: induced cell lysate of E. coli-pET21a-TAPL7A; lane 3: renatured purified TAPL7A.

Figure 4

Biochemical characterization of TAPL7A. (a) The optimal pH of TAPL7A. (b) The pH stability of TAPL7A. (c) The optimal temperature and temperature stability of TAPL7A. (d) The effects of metal ions and EDTA on activity of TAPL7A. Orange represents metal ion activation; blue represents metal ion inhibition.

Figure 5

Effect of NaCl on TAPL7A.

Figure 6

Analysis of 0–48 h products of TAPL7A by FPLC with (a) sodium alginate, (b) polyM, (c) polyG. The eluents were detected by measuring the absorbance at 235 nm.

Figure 7

Analysis of 48 h product composition of TAPL7A by ESI-MS with (a) sodium alginate, (b) polyM, (c) polyG as substrates; The oligo-uronates are commonly described by their degree of polymerization (DPx). Oligo-uronates with the unsaturated terminal uronate are denoted in this study as ΔDPx, and such an oligomer would be ΔDP1.

Figure 8

(a) Overall structure of TAPL7A; (b) The structural comparison of TAPL7A (green) and AlyA5 (cyan); (c) Comparison of loops at the ends of catalytic groove of TAPL7A (red and purple) and AlyA5 (blue); (d) Structure-based sequence alignment of TAPL7A and AlyA5. Conservative amino acids are highlighted by the red background and similar amino acids are highlighted by the red letters. α-helices are displayed as squiggles, β-strands are rendered as arrows, strict β-turns as TT letters. This figure has been generated using the program ESPRIPT 3.0.