Metagenomic exploration of cold-active enzymes for detergent applications: Characterization of a novel, cold-active and alkali-stable GH8 endoglucanase from ikaite columns in SW Greenland

Abstract

Microbial communities from extreme environments are largely understudied, but are essential as producers of metabolites, including enzymes, for industrial processes. As cultivation of most microorganisms remains a challenge, culture-independent approaches for enzyme discovery in the form of metagenomics to analyse the genetic potential of a community are rapidly becoming the way forward. This study focused on analysing a metagenome from the cold and alkaline ikaite columns in Greenland, identifying 282 open reading frames (ORFs) that encoded putative carbohydrate-modifying enzymes with potential applications in, for example detergents and other processes where activity at low temperature and high pH is desired. Seventeen selected ORFs, representing eight enzyme families were synthesized and expressed in two host organisms, Escherichia coli and Aliivibrio wodanis. Aliivibrio wodanis demonstrated expression of a more diverse range of enzyme classes compared to E. coli, emphasizing the importance of alternative expression systems for enzymes from extremophilic microorganisms. To demonstrate the validity of the screening strategy, we chose a recombinantly expressed cellulolytic enzyme from the metagenome for further characterization. The enzyme, Cel240, exhibited close to 40% of its relative activity at low temperatures (4°C) and demonstrated endoglucanase characteristics, with a preference for cellulose substrates. Despite low sequence similarity with known enzymes, computational analysis and structural modelling confirmed its cellulase-family affiliation. Cel240 displayed activity at low temperatures and good stability at 25°C, activity at alkaline pH and increased activity in the presence of CaCl₂, making it a promising candidate for detergent and washing industry applications.

FIGURE 1

Maximum likelihood phylogenetic tree of Cel240 and its closest relatives based on amino acid sequences, showing its phylogenetic relationship to the GH8 family enzymes (Bootstrap n = 100). All enzyme sequences were analysed using InterPro, and GH8 family enzyme sequences wrongly annotated as GH5 are marked with an asterisk.

FIGURE 2

Alignment of Cel240 to related sequences from the blastp (nr_nt) and blastp (pdb) searches. Alignment was performed using Clustal Omega multiple sequence alignment. The alignment showed conserved regions (highly conserved in black and somewhat conserved in grey) and circles indicate the catalytic residues as determined in the 1KWF cellulase from Clostridium thermocellum.

FIGURE 3

In silico alignment of modelled Cel240 (yellow) with structures determined with X‐ray crystallography of endoglucanases Cel10 (pdb id 5GY3, blue), Komagataeibacter sucrofermentans (6VC5, cyan), Pseudomonas putida (4Q2B, salmon) and Paenibacillus sp.X4 (5XD0, grey) (A). Identified the catalytic (red, sticks) (B) and sugar‐recognition residues (purple, sticks) (C) of Cel240 through the alignment with Cel10 and CtCelA (pdb id 1KWF) and the model substrate cellopentaose on the cleft of the enzymes obtained from PDB entry 1KWF (green, sticks). Electrostatic surface potential for the endoglucanases Cel240, Cel10 and CtCelA were calculated with the APBS plugin in PyMOL (D).

FIGURE 4

Activity of cellulase Cel240 and TrCel7A from Trichoderma reesei in different types of cellulosic substrates at 25°C. BMCC, bacterial microcrystalline cellulose; CMC, carboxymethyl cellulose; RAC, regenerated amorphous cellulose; pNPL, p‐nitrophenyl‐β‐d‐lactopyranoside; pNPG, p‐nitrophenyl‐β‐d‐glucopyranoside.

FIGURE 5

pH and temperature profile of Cel240 (A, B) and TrCel7B (B). Thermal stability of Cel240 and TrCel7B at 25°C (C) and 50°C (D). Activity in presence of CaCl₂ (E) and NaCl (F). Thermal unfolding (T _m) values for Cel240 and TrCel7B in presence of CaCl₂ (G). The activity of Cel240 and TrCel7B at 8°C (H). The CMC (0.5%) was used as substrate in all the assays.