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

Chenchen Guo,Ruining Cai,Rikuan Zheng,Shimei Wu,Chaomin Sun

doi:10.3390/microorganisms9040802

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.

Highlights

The plates were incubated at 28 ◦ C for 48 h, and bacteria producing an obvious halo-forming zone were evaluated as potential lipase-producing strains
The Pseudomonas genus represents a kind of Gram-negative, aerobic bacteria, which belongs to the family Pseudomonadaceae of class Gammaproteobacteria [44]
A large number of novel Pseudomonas species have been isolated from different environments such as soil, fresh and saline waters, plants, and clinical specimens [45], while relatively few species have been reported from the deep-sea cold seep

Summary

Introduction

Cold environments such as those in the deep sea [1], glaciers, and mountain regions are one of the most abundant environments for microorganisms on the Earth’s surface. Even in the cold environments, where the temperatures are close to 5 ◦ C, these regions are still colonized successfully by numerous microorganisms [2]. In order to cope with the harsh effects of such environments, these microorganisms have evolved various adaptation strategies, such as producing enzymes with high specific activity at low temperatures, collectively termed cold-active enzymes [3]. Cold-active enzymes usually have high catalytic activities at temperatures below 25 ◦ C, which gives them great advantages in detergent, textile, and food industries because of the energy savings they provide [4]

Methods

Results

Conclusion