A Novel Member of GH16 Family Derived from Sugarcane Soil Metagenome

Abstract

Glycoside hydrolases (GHs) are enzymes found in all living kingdoms that are involved in multiple physiological functions. Due to their multiple enzymatic activities, GHs are broadly applied in bioethanol, food, and paper industry. In order to increase the productivity of these industrial processes, a constant search for novel and efficient enzymes has been proved to be necessary. In this context, metagenomics is a powerful approach to achieve this demand. In the current study, we describe the discovery and characterization of a novel member of GH16 family derived from the sugarcane soil metagenome. The enzyme, named SCLam, has 286 amino acid residues and displays sequence homology and activity properties that resemble known laminarases. SCLam is active against barley beta-glucan, laminarin, and lichenan (72, 33, and 10 U mg(-1), respectively). The optimal reaction conditions were identified as 40 °C and pH 6.5. The low-resolution structure was determined using the small-angle X-ray scattering technique, revealing that SCLam is a monomer in solution with a radius of gyration equal to 19.6 Å. To the best of our knowledge, SCLam is the first nonspecific (1,3/1,3:1,4)-β-D-glucan endohydrolase (EC 3.2.1.6) recovered by metagenomic approach to be characterized.

Biochemical and biophysical properties of a metagenome-derived GH5 endoglucanase displaying an unconventional domain architecture

“…Following incubation, the soluble fractions from the reactions were recovered and derivatized with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS, Sigma-Aldrich) by reductive amination [33]. Separations were performed using a P/ACE MQD Instrument (Beckman Coulter) equipped with laser induced fluorescence detection at the same conditions as described in Alvarez et al [34].…”

Insights into the dual cleavage activity of the GH16 laminarinase enzyme class on β-1,3 and β-1,4 glycosidic bonds

“…As described previously (14), SCLam degradation of barley β-glucan resulted in accumulation of mainly glucose and oligosaccharides with the degree of polymerization (DP) ranging from two to four glucose units (DP2 to DP4). Barley β-glucan contains a ratio of β-1,4/β-1,3 linkages of 2.4 (16) and, thus, the accumulation of glucose indicates that SCLam was able to cleave both β-1,3 and β-1,4 glycosidic bonds.…”

“…The cloning and heterologous expression of SClam was performed as described (14). The mutant of SCLam, named SCLam E144S , resulted from the substitution of glutamic acid in position 144 for serine (E144S).…”

“…Previously we described the identification and biochemical characterization of a (1,3/1,3;1,4)-β-D-glucan endohydrolase derived from the soil metagenome, named as SCLam (14). Of interest, SCLam is able to cleave cellohexaose, which contains only 1,4-β-D-glycosyl linkages.…”

Characterisation of three novel β-1,3 glucanases from the medically important house dust mite Dermatophagoides pteronyssinus (airmid)

“…βglucans form 50-60% of the fungal cell wall with β-1,3 contributing 65-90% of the β-glucan content (Fesel and Zuccaro, 2016). Enzymes from the Glyco Hydrolase family 16 (GH16) exhibit hydrolytic activity against a variety of polysaccharides, including β-1,3 glucans commonly found in fungal cell walls (Alvarez et al, 2015). Enzymes capable of hydrolysing β-1,3 glucans exhibit endo-β-1,3 glucanase (EC 3.2.1.39), endo-β-1,3-1,4 glucanase (EC 3.2.1.6) or exo-β-1,3 glucanase activity (EC 3.2.1.58) (Song et al, 2010).…”