The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes

Abstract

Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5°C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

“…These chemicals included 11 model esters (7 pNP esters and 4 triacylglycerols) (Fig. 4), as well as a battery of 120 structurally different esters (3,6,27,28,37,53) (Fig. 5).…”

“…Based on a calculation of the apparent enantiomeric ratios for separate enantiomers (6), the enantiomeric ratios and substrate preferences are tentatively the following: Substrate fingerprints: comparison with known enzymes. The substrate profiles of the enzymes reported in this study against a set of 131 ester substrates (see the section on materials and methods in the supplemental material) were compared to a set of wellcharacterized commercial preparations with high substrate versatility (including CalA, CalB, Novozym 388 L, and PLE) and a set of carboxyl esterases obtained via genomic mining of marine bacteria (ABO_1197, ABO_1251, CCSP0084, CCSP0211, CCSP0528, and CCSP2178) or via metagenomic approaches of communities from marine (27,28) and brackish sediment samples (6).…”

“…Therefore, understanding the characteristics of other deep-sea enzymes, particularly esterases for which multiple biochemical data are available compared to other deep-sea enzymes (3), might help in understanding the phenomenon of protein versatility and adaptability to different polyextremes. Second, esterases were selected because they are ubiquitous enzymes that are widespread in nature (at least one per genome), multiple commercial preparations that are used at industrial scale are available, and biochemical data of such enzymes from a number of marine habitats (3,27,28) have been reported recently, which collectively may assist to perform an extensive comparative analysis. Finally, they are of great interest as biocatalysts for chemical synthesis (29).…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…Obvious examples are coldadapted (Berlemont, Pipers et al 2011, Jansson and Tas 2014, Martinez-Martinez, Lores et al 2014, Tchigvintsev, Tran et al 2014) and heat-adapted (Xia, Ju et al 2013 enzymes that have been identified and isolated from extremely cold and hot habitats, respectively. The isolated extremozymes have definite industrial applications ).…”

Discovery of Polyesterases from Moss-Associated Microorganisms

“…It was demonstrated that metagenomes are also promising sources for novel enzymes (23). However, there are only seven examples of polyester-hydrolyzing enzymes from metagenomic sources, including the LC-cutinase from a leaf-branch compost metagenomic library, which is capable of hydrolyzing not only cutin but also PET (24), the PLA depolymerase PlaM4, which is also from a compost metagenome (25), and five esterases from marine metagenomic libraries with hydrolytic activities toward several polyester substrates, including PLA (26). The potential of plant-associated metagenomes as a source of novel enzymatic activities has been less well explored (27).…”