Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

Abstract

Background: The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns.

Results: A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities.

Conclusions: Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low temperature, suggesting a possibly use in the dairy industry for this enzyme. The two different approaches complemented each other by targeting different microbial communities, highlighting the usefulness of combining methods for bioprospecting. Finally, we document here that ikaite columns constitute an important source of cold- and/or alkaline-active enzymes with industrial application potential.

Figure 1

Changes in microbial diversity during library preparation. Diversity shifts at phylum (left) and class (right) level of samples introduced during preparation of DNA for the expression library. Total DNA represents DNA extracted directly before manipulation, Intact Cells represents DNA from extracted cells, and MDA DNA represents DNA after MDA treatment.

Figure 2

Temperature and pH profiles of the α-amylase. Temperature (left) and pH (right) profile of crude extract of the IKA3C6 α-amylase using amylopectin as substrate. Error bars indicate standard deviations from triplicate experiments.

Figure 3

Temperature and pH profiles of β-galactosidases. Temperature (left) and pH (right) profile of crude extracts of the two β-galactosidases BGal_I3H5 (dotted line) and BGal_I17E2 (solid line) with ortho-nitrophenyl-β-galactoside (ONPG) as substrate. Error bars show standard deviations from triplicate experiments.

Figure 4

TLC analysis of lactose hydrolysis at 5°C and 37°C. (A) Controls of Lactose, Galactose and Glucose (each 5 mg/ml). (B) Lactose (5 mg/ml) incubated with crude extract of E. coli carrying the empty pET21b vector. (C) Lactose (5 mg/ml) incubated with crude extract of BGal_I17E2. (D) Lactose (5 mg/ml) incubated with crude extract of BGal_I3H5.