Chapter 3
Structure—Function Studies of Endo-1,4-β-D-glucanase E2 from Thermomonospora fusca M i k e Spezio, P . A n d r e w K a r p l u s , D i a n a I r w i n , a n d D a v i d B . W i l s o n
Downloaded by UNIV LAVAL on October 27, 2015 | http://pubs.acs.org Publication Date: October 7, 1994 | doi: 10.1021/bk-1994-0566.ch003
Section of Biochemistry, M o l e c u l a r and C e l l Biology, C o r n e l l U n i v e r s i t y , Ithaca, NY 14853
Six different cellulases have been purified to homogeneity from the culture supernatant of a protease negative mutant of the soil bacterium, Thermomonospora fusca (1,2). Three of these enzymes are endoglucanases, two are exocellulases and one is an exocellulase with some endoglucanase activity (3). All six enzymes contain a cellulose binding domain joined by a flexible linker to a catalytic domain. When the distributions of reducing sugars between the solution and filter paper in filter paper assays of E 2 and E2cd were determined, there were two soluble sugars produced for each insoluble sugar with E 2 while with E2cd there was only one soluble sugar produced for each insoluble sugar (3). This result seems reasonable since the cellulose binding domain should attach the enzyme to a specific site on the substrate for a longer time than would the catalytic domain alone.
The six cellulases isolated from T. fusca culture broth comprise a well-studied bacterial cellulase system. E 2 is the smallest T. fusca cellulase and has a molecular weight of 42,000. It contains an N-terminal catalytic domain of 31,000 joined by a 26 amino acid linker to a C-terminal cellulose binding domain (4). E 2 has specific activities of 370,170, and 0.85 pmoles/min/umole on carboxymethylcellulose ( C M C ) , swollen cellulose and filter paper respectively (3) as assayed by the procedure of Ghose (5). A proteolytic derivative of E 2 , the E 2 catalytic domain (E2cd), which is missing the cellulose binding domain and linker region has specific activities of 344, 113 and 0.50 umoles/min/pmole on the same set of substrates but on filter paper it cannot give 5% digestion so the specific activity was calculated from the activity given by 0.6 nmoles of enzyme. E 2 has been shown to invert the configuration of the anomeric carbon during hydrolysis (6). Selected properties of E 2 and other T. fusca cellulase enzymes are given in Table I.
0097-6156/94/0566-0066$08.00/0 © 1994 American Chemical Society
In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
3.
SPEZIO ET AL.
Structure—Function
Studies of Endoglucanase E2
61
Table I. Properties of Individual Cellulases*
Downloaded by UNIV LAVAL on October 27, 2015 | http://pubs.acs.org Publication Date: October 7, 1994 | doi: 10.1021/bk-1994-0566.ch003
Protein
MW (kD) a
Extinction coefficients (molar) b
Stereo chemistry c
Activity Specific Activity C M C (umol ceUobiose/min-umol) swollen filter p-NPcellulose paper cellobiose
El
101.2
208,000
inversion
5410.0
362.0
0.182 d
40.4
E2
43.0
80,000
inversion
369.0
168.0
0.846
8
E2cd
30.0
57,600
inversion
344.0
113.0
0.501 d
8
E3
65.0
145,000
/
1.6
0.303 d
8
E4
90.2
214,000
/
122.0
34.9
0.565 d
8
E5
46.3
97,000
retention
2840.0
90.4
0.832
14.8
E5cd
34.4
70,300
retention
2480.0
85.3
0.573 d
14.4
106.0
385,000
/
83.5
0.863 e
0.04
E6
1.3 d
64.2 e
a
M W s for E 3 and E 6 were estimated from S D S polyacrylamide gels. E x t i n c t i o n coefficients were calculated from the number of trp and tyr residues in proteins. E3 and E 6 extinction coefficients were estimated from amino acid analysis. ref. 3. Target % digestion could not be achieved. In this case, filter paper specific activities were calculated using digestion achieved by 0.6 nmoles of enzyme in 16 hours. Contaminating C M C activity as determined by C M C overlays of native page gels. Not determined. Activity below detectable limits (i.e.,
