Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities

Abstract

BackgroundCold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds.ResultsA bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones.ConclusionsBglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.

Diversity of Glycosyl Hydrolase Enzymes from Metagenome and Their Application in Food Industry

“…The recombinant bgl MKg expressed in E. coli reportedly demonstrated high β‐galactosidase, β‐glucosidase, and β‐fucosidase activities. The cold adaptation of the enzyme catalyzed a reverse transglycosylation reaction (Wierzbicka‐Wos and others ). Another β‐galactosidase gene zd410 isolated by screening a soil metagenomic library was regarded as a cold‐adapted due to its optimal temperature of 38 °C and 54% residual activity at 20 °C (Wang and others ).…”

Seasonal Effects of Extreme Weather Events on Potential Extracellular Enzyme Activities in a Temperate Grassland Soil

“…β-glucosidase is responsible for the regulation of the entire cellulose hydrolysis process by easing cellobiose-mediated suppression and producing the final product glucose (Singhania et al, 2013). So far, different β-glucosidases have already been characterized from bacteria, archaea and eukaryotes in a large variety of environments (Wierzbicka-Wos et al, 2013). Thus, not surprisingly high ß-glucosidase activities were also found in arid to semiarid environments (Cañizares et al, 2011), indicating a high tolerance of this enzyme to drought stress, which is in line with the present study.…”

Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

“…This work follows on other metagenomic functional screening efforts that have discovered β-galactosidases of GH1 (Gupta et al 2012), GH42 (Wang et al 2010) (Zhang et al 2013), GH43 (Ferrer et al 2012; Wierzbicka-Wos et al 2013), and two new GH members (Beloqui et al 2010). Here we have highlighted the application of functional metagenomics for mining novel enzymes from soil microbial communities/ While the functional metagenomics strategy has potential for expanding the availability of enzymes that can be further developed for biotech applications, it is perhaps just as important to apply such strategies to the expansion of knowledge that will inform functional interpretation of DNA sequence.…”

“…The transgalactosylation activity is used to synthesize galactosylated products (Gosling et al 2010). Functional screening of metagenomic libraries resulted in discovery of a GH43 enzyme acting on multiple substrates including lactose (Ferrer et al 2012), cold-adapted or thermostable GH42 β-galactosidases (Wang et al 2010; Zhang et al 2013), a thermostable-alkalophilic or cold-active GH1 (Gupta et al 2012; Wierzbicka-Wos et al 2013), and two novel β-galactosidases without any similarity to known GHs (Beloqui et al 2010).…”