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Table 5 Nineteen GH family genes selected for evaluation of contig assembly and for cellulase expression in yeastsa

From: Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses

Cloning
sequence ID
Contig ID Contig length ORF length GH family CBM domain Signal peptideb RPKM
value
ESI-Trap LC-MS/MS Domain predictionc Enzyme activity assayd
           P. pastoris S. cerevisia
W5-CAT13 Contig8146 2,691 1,986 GH1 - Y 2,290.4 Y β-glu β-glu (S, P)e No activity
W5-16143 Contig4878 1,875 1,815 GH3 - Y 159.8 Y β-glu β-glu (S, P)e β-glu (P)f
W5-celDg AF053363 3,949 1,383 GH5 CBM 10 Y - Y EG + Xyl EG (S, P)f NA
W5C-P Contig12293 1,392 1,392 GH5 CBM 10 N 405.7   EG NA NA
W5-CAT5 Contig8755 1,582 1,479 GH6 CBM 10 Y 2,735.9   EXG No activity No activity
W5-CAT6-9 Contig8363 1,540 1,497 GH6 CBM 10 Y 203.2   EXG EXG (S, P)f EXG (P)f
W5-20147 Contig20147 1,981 1,503 GH6 CBM 10 Y 3,576.0   EXG No activity No activity
W5C-O Contig19058 1,449 1,197 GH6 CBM 1 Y 1,712.7   EXG NA NA
W5-16271 Contig18112 1,754 1,389 GH6 CBM 10 Y 760.1   EG + EXG No activity No activity
W5-01055 Contig13874 3,935 3,858 GH6 CBM 10 N 239.8   EXG No activity No activity
W5-10151-39 Contig9299 1,900 1,473 GH6 CBM 10 Y 4,397.8   EXG EXG (S, P)f EXG (P)f
W5-10151-38 Contig9839 1,415 1,341 GH6 CBM 10 Y 414.1   EXG EXG (S, P)f NA
W5-10151-5 Contig15588 1,777 1,461 GH6 CBM 10 Y 2,727.9   EXG EXG (S, P)f NA
W5-10151-6 Contig10151 1,852 1,494 GH6 CBM 10 Y 654.6   EXG EXG (S, P)f NA
W5-00992 Contig8960 2,042 2,037 GH9 CBM 10 Y 154.1 Y EG EG (S, P)e EG (P)f
W5-14806 Contig10733 2,509 2,355 GH9 CBM 10 Y 165.6   EG No activity No activity
W5-CAT24 Contig4514 1,575 1,116 GH18 CBM 10 N 88.1 Y Chitinase NA NA
W5-CAT7 Contig19103 2,619 2,262 GH43 CBM 6, CBM 10 N 747.4 Y Xyl No activity NA
W5-CAT26-68 Contig13654 2,793 2,271 GH48 CBM 10 Y 6,953.9 Y EXG EXG (S, P)e EXG (P)f
  1. aGH: glycosyl hydrolase; ORF: open reading frame. bThe predicted signal peptide could be absent due to incomplete ORF sequence at the 5' end of contig sequence. cTop score and e-value < 10-10 as putative domain: endoglucanase (EG), exoglucanase (EXG), β-glucanase (β-glu), xylanase (Xyl) and chitinase. d4-methylumbelliferyl-β-cellobiose (4-MUC) assays were used for broad cellulase activity analysis of endo-, exo-, β-glucanase and xylanase. S = supernatant; P = cell pellet. eThese cellulase constructs were chosen for subsequent enzymatic assays of dye CM-cellulose and p-nitrophenyl-β-D-glucopyranoside for their high 4-MUC activity expressed in P. pastoris (Figure 5). fLow or undetectable levels of dye-CMC and p NPG activity. gcelD is a known gene from National Center for Biotechnology Information GenBank (Xue et al. [26]). The cloned sequences were submitted to GenBank (GenBank:JF906702-GenBank:JF906719).
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