Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium

Abstract

The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

Cold adapted Pseudomonas: ecology to biotechnology

“…Other factors responsible for the flexibility of such enzymes include enhanced surface, core hydrophobicity and the arrangement of aminoacids. Some of the latest reports strongly support that the presence and orientation of specific amino acids within the structure of enzyme affects the stability and activity, for example, the catalytic triads Ser, His and Asp in lipase encoding genes found in P. marinensis were observed to be necessary for preserving enzyme functions ( Guo et al, 2021 ). Öten et al (2022) observed a boost in the adaptation of enzymes to cold conditions by proline (Pro) and glycine (Gly) residues modifying conformational changes in the three-dimensional (3-D) structure of proteins.…”

“… Zhang and Zeng (2011) reported the identification of the amylase gene from the psychrotolerant P. stutzeri strain 7,193. Numerous other Pseudomonas species isolated from the regions like Indian Himalaya ( Salwan et al, 2010 ; Jain and Pandey, 2016 ; Pandey et al, 2019 ; Jain et al, 2020 ), Ny-Alesund, Svalbard, Arctic ( Srinivas et al, 2009 ), Humboldt Glacier, Mount Humboldt, Andean region of Venezuela ( Ball et al, 2014 ) have been studied for production of industrially important enzymes such as amylases, proteases, RNA polymerase and lipases ( Uma et al, 1999 ; Zhang and Zeng, 2011 ; Jain et al, 2017 ; Salwoom et al, 2019 ; Guo et al, 2021 ). Beside mountains, the oceans represent an untapped potential that exists in the microbiome to produce enzymes with distinctive properties.…”

“…Beside mountains, the oceans represent an untapped potential that exists in the microbiome to produce enzymes with distinctive properties. For example P. marinensis studied by Guo et al (2021) possess two novel genes encoding for cold-active lipases. Such enzymes are also reported to survive and remain active at alkaline pH ( Salwoom et al, 2019 ; Salwan et al, 2020 ).…”

Novel Microbial Enzymes with Industrial Applications

“…The potential of this bacterial genus for several industrial applications was revealed. Guo et al [3] isolated bacterial strains from the sediment of deep-sea cold seep, finding a novel strain that exhibited high cold-active (4 • C) lipase activity. This strain was proposed to constitute a new species of Pseudomonas, and was named P. marinensis.…”