Additional file 1 of Arabinan saccharification by biogas reactor metagenome-derived arabinosyl hydrolases
Creators: name: Liu, Yajing
name: Angelov, Angel
name: Feiler, Werner
name: Baudrexl, Melanie
name: Zverlov, Vladimir
name: Liebl, Wolfgang
name: Vanderhaeghen, Sonja
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Ecology
subject: 69999 Biological Sciences not elsewhere classified

description: Additional file 1: Table S1. Bacterial community compositions of the biogas fermenter consortia revealed by 16S rRNA amplicon sequencing. Table S2. Raw data summary of shotgun sequencing and assemblage of positive fosmid clones. Table S3. Detailed information about all the putative CAZymes screened from the metagenomic libraries, including the position of ORFs on the corresponding contigs, the library origin, the KEGG annotation and blastnr identifier of each putative enzyme. Table S4. Information on heterologously expressed arabinosyl hydrolases. Table S5. Primers used for PCR amplification of putative arabinosyl hydrolase-encoding genes. Figure S1. Functional screening of recombinant clones of E. coli fosmid libraries using four different chromogenic substrates. Cleavage of the chromogenic substrates results in increased color intensity in the presence of recombinantly expressed activities, including carboxymethyl cellulase (CMCase, red color), xylanase (blue color), arabinoxylanase (blue color), arabinanase (blue color). Figure S2. Gene organization of representative gene clusters encoding fibrolytic enzymes targeting different plant polysaccharides. CAZyme genes were predicted by the use of the dbCAN and KEGG databases. Figure S3. Temperature, pH dependence and thermo-resistance of enzyme activities. Relative activities were calculated from DNS assay with SBA or DA as substrate or pNP assay with pNP-AF as substrate. The maximal activity was set as 100%. Standard reactions for searching temperature and pH optimum included 1 mM pNP-AF (143.69 nM MC57GH51 and 19.86 nM MC60GH51) or with 5 g L-1 DA (875.62 nM MC60GH43, 367.05 nM MC68GH43-2 and 465.25 nM MC72GH43-2) or SBA (MC68GH43-1), assays were carried out in 25 mM citrate phosphate buffer with pH between 4.0 and 9.0 and temperature between 25° C and 80° C for 30 min, or with 1 mM pNP-AF for 10 min. For analysis of the enzymes’ thermo-resistance, the assays included 5 g L-1 of DA, 25 mM citrate phosphate buffer with 283.3 nM MC60GH43, 547.54 nM MC68GH43-2, 99.5 nM MC72GH43-2 or 5 g L-1 of SBA with 120.04 nM MC57GH51, 410.96 nM MC60GH51, 892.86 nM MC68GH43-1. The enzymes were incubated at their optimal temperature and pH for various time spans before measuring residual activity against SBA (MC57GH51, MC60GH51, MC68GH43-1) or DA (MC60GH43, MC68GH43-2, MC72GH43-2). The assays were performed in triplicates. Figure S4. Determination of kinetic parameters of arabinosyl hydrolases with SBA or DA as substrates. Standard reactions were performed by using three different concentrations of each enzyme (as indicated in figures) and various concentration of substrates (between 1 and 50 g L-1) at each enzyme’s optimal condition for different time periods according to requirement. (MC68GH43-1 and MC60GH43 for 2 h incubation, MC60GH51, MC68GH43-2, MC72GH43-2 for 40 min incubation, MC57GH51 for 1 h incubation). Error bars represent standard deviation of triplicates. Km and Vmax were calculated by using Microsoft Excel Solver, as described in material and methods.