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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).