1
Table S1: PCR primers used to amplify the Cel5A and Cel5B genes from fosmids isolated from the
cow rumen metagenome library
Primer Name
Oligonucleotide sequencea
Restriction
enzyme
Cel5AF
5’ TACATATGAAGAGAATTCTACTC 3’
NdeI
Cel5AR
5’ TAAAGCTTTTTCATAACATATTTTTTCC 3’
HindIII
Cel5BF
5’ TACATATGCAAAGTTTTGAATCTGC 3’
NdeI
Cel5BR
5’ TAAAGCTTTCTGGCTATATATCTC 3’
HindIII
a
The underlined sequences indicate the engineered NdeI and HindIII restriction sites.
2
Table S2: Enzyme characteristics of various cellulolytic enzymes derived from rumen bacterial metagenome
Enzyme
Optimum pH
Optimum
Specific activity (U/mg)
temperature
KM
Reference
(mg/ml)
13.6 (0.5% CMC)
Cel5A (Bovine)
7-9 (CMC)
69ºC (CMC)
This Study
153.3 (0.5% birchwood xylan)
0.39 (xylan)
127.4 (0.5% CMC)
Cel5B (Bovine)
9 (CMC)
65ºC (CMC)
This Study
42.3 (0.5% birchwood xylan)
2.25 (xylan)
RuCelA (Yak)
5.0 (CMC)
50ºC (CMC)
54.3 (1% CMC)
2.71 (CMC)
Xylanase/endoglucanase
7.0 (xylan)
65ºC (xylan)
264 (1% birchwood xylan)
0.58 (xylan)
73 (1% CMC)
36.8 (CMC)
4.5 (CMC)
45ºC (CMC)
[1]
C67-1 (Buffalo)
Cellulase
[2]
14.4 (1% birchwood xylan)
DC9-2 (Buffalo)
7 (CMC)
45ºC (CMC)
Not reported
[2]
4.5 (CMC)
45ºC (CMC)
Not reported
[2]
Cellulase
M8-2 (Buffalo)
Cellulase
1.
Chang L, Ding M, Bao L, Chen Y, Zhou J, Lu H (2011) Characterisation of a bifunctional xylanase/endoglucanase from yak rumen microorganism. Appl.
Microbiol. Biotechnol. 90: 1933-1942.
2.
Duan C, Xian L, Zhao G, Feng Y, Pang H, Bai X, Tang J, Ma Q, Feng J (2009) Isolation and partial characterization of novel genes encoding acidic cellulases from
metagenomes of buffalo rumens. J. Appl. Microbiol. 107: 245 – 256.
3
4
Figure S1: Figure 1: Multiple sequence alignment of Cel5A and Cel5B versus other glycoside hydrolase family 5 proteins. Cel5A and Cel5B, from this study, are compared
to other cellulolytic enzymes (ACA61132.1, ACA61135.1, ACA61137.1, ACA61140.1 [2]; ADK55024 [1], EU282863 [3]).
1.
Chang L, Ding M, Bao L, Chen Y, Zhou J, Lu H (2011) Characterisation of a bifunctional xylanase/endoglucanase from yak rumen microorganism. Appl.
Microbiol. Biotechnol. 90: 1933-1942.
2.
Duan C, Xian L, Zhao G, Feng Y, Pang H, Bai X, Tang J, Ma Q, Feng J (2009) Isolation and partial characterization of novel genes encoding acidic cellulases from
metagenomes of buffalo rumens. J. Appl. Microbiol. 107: 245 – 256.
3.
Wang F, Li F, Chen G, Liu W (2009) Isolation and characterization of novel cellulase genes from uncultured microorganisms in different environmental niches.
Microbiol. Res. 164: 650-657.
5
A
B
Figure S2: Histidine tagged purification of Cel5A and Cel5B
Cel5A is shown in lane 4 (A), with a standard protein marker in lane M, followed by the crude cell extract in
lane 1 and finally, two wash fractions in lanes 2 and 3. Cel5B is similarly shown in lane 4 (B) with the same
configuration in the other lanes.
6
120%
Relative Activity (%)
100%
80%
60%
40%
20%
0%
4
5
6
7
8
9
10
11
60
70
80
90
pH
120%
Relative Activity (%)
100%
80%
60%
40%
20%
0%
20
30
40
50
Temperature ( C)
Figure S3: pH (A) and Temperature (B) optima experiments using Cel5A () and Cel5B (). Data shown is
the average of quadruplicate assays using 2.0% CMC as substrate over 10 min.