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

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsMany 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.

Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis

“…However, cold-adapted EglCs are generally not thermally stability and can easily be inactivated by elevated temperatures (Vester, Glaring & Stougaard, 2014) Such processes have the merits of saving production and energy costs, maintaining taste and other organoleptic characteristics and reducing the risk of contamination (Vester, Glaring & Stougaard, 2014). …”

Characterization of a Salt-Tolerant and Cold-Adapted Xylanase from Bacillus cellulosilyticus

“…3d). According to previous reports, the typical cold-adapted enzyme exhibited the optimum activity at lower temperatures and was more sensitive to heat (Cavicchioli et al 2002;Siddiqui and Cavicchioli 2006), which was a sharp contrast with the thermophilic xylanases (Petegem et al 2003;Chang et al 2004;Shi et al 2013;Vester et al 2014). Therefore, Xyn10A can be considered a cold-adapted xylanase.…”

“…Various kinds of cold-adapted enzymes have been reported, such as esterases (Novototskaya-Vlasova et al 2012), lipases (Tian et al 2014), chitinases (Ramli et al 2011), β-glucosidases (Vester et al 2014), and proteinases (Kredics et al 2008). However, there has been only limited study of cold-adapted xylanase.…”

“…Therefore, Xyn10A can be considered a cold-adapted xylanase. Recently, the research about cold-adapted enzymes has been given much attention, as the use of cold-adapted enzymes has many advantages, such as reducing energy consumption and maintaining the original food flavor (Liu et al 2014;Vester et al 2014).…”

Two-step enzymatic synthesis of 6-deoxy-l-psicose

“…13 In most instances, the conversion ratios are no more than 40%. 14 Attempts to improve aldose-ketose isomerization include the addition of borate to break the aldose-ketose reaction equilibrium, 15 the discovery of novel enzymes in nature, 16 and directed evolution 17 have also been suggested. Nevertheless, these methods still suffer from low yield, or an isomer separation manipulation, which is a labor-intensive and time-consuming process due to the similar properties of the isomer pair.…”