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