Characterisation of novel metagenome-derived enzymes from alpine palaeosols

Abstract

There is a continuous growing demand for the discovery of novel biocatalysts usable in the fine chemical and biotechnological industries. Soil bacteria serve as reservoirs of these biocatalysts due to their diversity and biochemical versatility. However, we are grossly ignorant of bacterial life on earth, as >95% of these organisms cannot be studied using conventional microbiology. Herein, we present the discovery and characterisation of novel enzymes from Alpine palaeosols using metagenomics. Lignocellulosic biomass are said to be the major sources of alpine soil carbon, therefore, gene mining was focused on oxidoreductase enzymes involved in lignin degradation. Three types of oxidoreductase enzymes – aldehyde dehydrogenases (ALDHs), laccases, and lignin peroxidases were discovered through the gene-targeted sequence assembly of an Alpine soil metagenome sequenced on an Illumina MiSeq sequencer. An ALDH (specifically a salicylaldehyde dehydrogenase- SALD) and two-domain laccases were successfully isolated from the Alpine metagenome using designed specific primers; the genes were cloned, recombinantly expressed, purified and characterised. This novel SALD from the Alpine soil (SALDAP) is the first from the members of Alphaproteobacteria to be purified and characterised, and it shares only 48% amino acid sequence identity with SALDs from members of Gammaproteobacteria. Biochemical characterisation of SALDAP revealed its broad substrate specificity for both aromatic and aliphatic substrates. However steady-state kinetic parameters for the oxidation of several aliphatic and aromatic substrates showed its remarkable preference for aromatic substrates with large kcat/KM values (≥ 106 M-1 s-1), whereas the enzyme showed catalytic efficiencies around 103 M-1 s-1 for aliphatic substrates. Calcium and iron were found to increase the activity of SALDAP significantly. The enzyme was also found to be relatively stable in the presence of detergents, organic solvents, and potentially inhibitory compounds. Differential scanning fluorimetry (DSF) revealed the thermal stability of the enzyme in the presence and absence of its ligands; the combined presence of the cofactor (NAD+) and any of the aldehyde substrates was found to significantly (p < 0.05) increase the thermal stability of SALDAP. Furthermore, the SALDAP was crystallised, and data were collected at a resolution of 1.9 Å; its crystal structure happens to be the second solved structure of a SALD. The crystal structure was used to carry out molecular docking studies which facilitated the discussion of the structural basis of substrate specificity and proposal of a catalytic mechanism for SALDAP based on the role of four active site amino acid residues – ASN137, Arg145, GLU238 and CYS272. On the other hand, this study is also the first to discover and characterise two-domain laccases from Acidobacteria. The enzymes showed activity against two classical laccase substrates – ABTS and SGZ. We also showed that they are copper-dependent enzymes, as an increase in copper concentration in the assay mix was accompanied by significant increase in activity. The closest homologue of novel metagenome-derived laccases – a multicopper oxidase from Terriglobus roseus was purified, and it showed laccase activity. Together, this study serves as a proof-of-concept for gene-mining and has implications for bioprospecting and exploitation of targeted novel biocatalysts from the environment, especially the diverse soil microbiome.

Date of Award	Dec 2018
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Commonwealth Scholarship Commission in the UK
Supervisor	Christopher C R Allen (Supervisor) & David J. Timson (Supervisor)