Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library

Abstract

BackgroundEnzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance.ResultsHere, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either pNP-β-d-galactopyranoside, pNP-β-d-xylopyranoside, pNP-α-l-arabinopyranoside or pNP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the pNP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a β-galactosidase (74%) and (2) a β-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The β-galactosidase- and β-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the β-xylosidase-like protein 2 showed activities with both pNP-Xyl and pNP-Ara in the temperature range 40–50 °C and pH range 8.0–10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential “GGDEF” catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a “GDSL” site, encoding a ‘new family’ α-glucosidase activity.ConclusionUsing a rational screening approach, we identified and characterized four thermo-alkaliphilic glycosyl hydrolases that have the potential to serve as constituents of enzyme cocktails that produce sugars from lignocellulosic plant remains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0808-y) contains supplementary material, which is available to authorized users.

Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

“…All three AFases displayed the greatest hydrolytic activity towards pNP-α-L-arabinofuranoside compared to the activity on the other pNP-glycosides. Of the 145 GH families reported to date only families GH2, 3, 43, 51, 54 and 62 are known to encode α-L-arabinofuranosidase activity with family GH51 having the largest number of α-L-arabinofuranosidases [6, 51]. Lower activity was observed on the other pNP-glycosides except for AFase-H4 which showed substantial activity on pNP-β-D-cellobioside.…”

“…It is estimated that 40 million tons of xylan-rich agro-industrial biomass is generated globally on an annual basis. The potential of xylan-rich biomass feedstock has triggered an increasing interest in bioprospecting efforts for enzymes to degrade them to monomeric sugars which can be converted or used as part of raw materials in the production of value added products such as bioplastics, biodiesel and bioethanol [6].…”

“…Using function-based metagenomic screening approaches, novel sequences from both cultivable and uncultivable microorganisms can be exploited without prior sequence information [6]. This screening approach thus provides access to previously unknown genes and their novel enzymes with unique structural and kinetic properties [12, 13].…”

Stable Cellulolytic Enzymes and Their Application in Hydrolysis of Lignocellulosic Biomass

“…Maruthamuthu and van Elsas have reported the cloning, overexpression and characterization of four novel thermo‐alkaliphilic enzymes belonging to glycosyl‐hydrolase family identified with β‐galactosidase, β‐xylosidase and α‐glucosidase activities as retrieved from metagenomic library of wheat straw degrading microbial consortia. Recombinant thermo‐alkali‐stable endoglucanase of Myceliopthora thermophila BJA (rMT‐egl) expressed in Pichia pastoris showed optimal activity at a pH 10 and 50 °C with a t 1/2 of 60 and 15 min at 90° and 100 °C, respectively .…”

Expression and characterization of a new serine protease inhibitory protein in Escherichia coli

“…Recent studies have shown that many potential protease inhibitors have been isolated from sponge-associated microorganisms [11][12][13][14] ; however, the exploration and exploitation potential PIs from microorganisms are still a big challenge because almost all microorganisms are resistant to cultivation in laboratory conditions, especially symbiotic microbes. Fortunately, new approaches (e.g., metagenomics) provide powerful tools for predicting, detecting and expressing novel bioactive genes from noncultured microorganisms [15][16][17][18][19] . This opens new avenues for discovering new bioactive compounds, including PIs, in the future.…”