Novel, acidic, and cold-adapted glycoside hydrolase family 8 endo-β-1,4-glucanase from an Antarctic lichen-associated bacterium, Lichenicola cladoniae PAMC 26568

Abstract

Endo-？-1,4-glucanase is a crucial glycoside hydrolase (GH) that takes part in the decomposition of 27 cellulosic materials. In this study, to discover a novel cold-adapted ？-1,4-D-glucan-degrading enzyme, 28 the gene coding for an extracellular endo-？-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 29 26568, a lichen (Cladonia borealis)-associated bacterium in Antarctica, was identified and 30 recombinantly expressed in Esecherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular 31 polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity 32 of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis 33 (WP_182950054). The recombinant endo-？-1,4-glucanase (rGluL: 38.0 kDa) most efficiently 34 deconstructed sodium carboxymethylcellulose (CMC) at pH 4.0 and 45oC, and showed approximately 35 23% of its maximum degradation activity even at 3oC. The biocatalytic activity of rGluL could be 36 noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl between 0.1 and 0.5 M, while 37 the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-38 bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL was a true endo-？-1,4-glucanase, which 39 could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and 40 barley ？-glucan, without additional glycoside hydrolase activities. The specific activity (15.1 U mg-1) 41 and kcat/Km value (6.35 mg-1 s-1 mL) of rGluL toward barley ？-glucan were approximately 1.8- and 2.2-42 fold higher compared to its specific activity (8.3 U mg-1) and kcat/Km value (2.83 mg-1 s-1 mL) toward 43 CMC. Enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-44 cellobiose, accompanying with the formation of D-glucose, D-cellotriose, and D-cellotetraose. However, 45 the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-46 cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 47 endo-？-1,4-glucanase with high specific activity, which can be employed as a promising candidate in 48 the textile and food processes being performed at low temperatures.