Enzymes in Biomass Conversion - American Chemical Society

Chapter 26

Cellulomonasfimiβ-1,4-Glucanases

Downloaded by RUTGERS UNIV on December 26, 2017 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch026

Neil R. Gilkes, Andreas Meinke, John B. Coutinho, Edgar Ong, Jeffrey M. Greenwood, Robert C. Miller, Jr., Douglas G. Kilburn, and Antony J. Warren Department of Microbiology, The University of British Columbia, Vancouver, British Columbia V6T 1W5, Canada

The basic structural and functional organization of four β1, 4-glucanases from the cellulolytic bacterium Cellulomonas fimi has been established. Endoglucanase A (CenA) and an exoglucanase (Cex) each contain two independent structural and functional domains: a catalytic domain and an amino- or carboxyl-terminal cellulose -binding domain (CBD). The CBDs show significant sequence identity and similar structures are evident in glycanases from other bacteria. Endoglucanases Β and C (CenB and CenC, respectively) are large (>100 kDa) enzymes composed of multiple domains. A CBD is found at the carboxyl-terminus of CenB. Both enzymes contain units of unknown function comprised of repeated blocks of amino acids. Conspicuous linker sequences occur at the junctions of the domains in all four enzymes and resemble sequences seen in other bacterial glycanases. The CBDs of CenA and Cex have been fused to heterologous proteins by genetic manipulation. Examples are presented to illustrate the potential of such fusions for protein immobilization or purification. Structural & Functional Organization of Cellulomonas fimi β-1, 4Glucanases It is now apparent that many bacterial cellulases are composed of two or more structural and functional units or domains and it has been suggested that such enzymes arose by a process of domain shuffling (2). The domains may be catalytic or non-catalytic and their occurrence is often indicated in the primary protein structure by an intervening 0097-6156/91/0460-0349$06.00/0 © 1991 American Chemical Society

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

350

ENZYMES IN BIOMASS CONVERSION


sequence rich in proline and hydroxyamino acids. The catalytic domains have recently been classified into families on the basis of sequence similarities and hydrophobic cluster analysis (2,3). The noncatalytic domains comprise a more diverse group and include the cellulose-binding domains (CBDs) as well as elements of unknown function (3). Four β-l, 4-glucanases from C. fimi have been studied by us in some detail and their structural and functional organization illustrates many of these features, as summarized below. Endoglucanase A (CenA) and Exoglucanase (Cex). Two β-1, 4glucanases, CenA and Cex, are major extracellular enzymes produced when C. fimi is grown on microcrystalline cellulose. Both are glycoproteins which react with concanavalin A (4). Their apparent molecular masses, determined by SDS-polyacrylamide gel electrophoresis, are 53.0 and 49.3 kDa, respectively (5). The genes encoding CenA and Cex have been cloned and expressed in Escherichia coli (6) and their D N A sequences have been determined (7,8). The lower apparent molecular masses of the cloned gene products (48.7 and 47.3 kDa, respectively) reflect the absence of glycosyl substitution. CenA hydrolyzes carboxymethylcellulose randomly; Cex shows a preference for the hydrolysis of terminal linkages (4). Both enzymes also hydrolyze and bind tightly to insoluble cellulosic substrates (4,5,9,10). The end-products of cellulose hydrolysis for both enzymes are predominantly cellobiose and cellotriose. The basic structural and functional organization of CenA and Cex has been established and is represented in Figure 1. The primary structure of mature CenA is divided into two regions by the sequence: (PT)4 T(PT)7, the Pro-Thr box. Digestion of ngCenA (the non-glycosylated form of the CenA synthesized in Escherichia coli from recombinant C. fimi DNA) by a C. fimi protease releases a stable 'core' peptide comprising the carboxylterminal region (amino acids 135 - 443). This peptide retains enzymatic activity towards several cellulosic substrates but no longer binds to cellulose. The binding function is contained in the corresponding amino-terminal fragment (amino acids 1 - 135). Therefore, CenA is a bifunctional protein comprising a carboxyl-terminal catalytic domain joined to an amino-terminal CBD by the Pro-Thr box. Cex is similarly organized but the arrangement of its domains is reversed (5). Small-angle X-ray scattering analyses have shown CenA is tadpole-shaped (Figure 2): the catalytic domain forms the head region and the CBD and Pro-Thr box forms the extended tail region (11). The model contains an angle of 135° between the long axes of the head and tail regions. The overall dimensions of the molecule are comparable to the similarly shaped cellobiohydrolases I & II (CBH I and II) from Trichoderma reesei (12).


26.

GILKES ET A L

Cellulomonasfimifi-l 4-Glucanases

351

y

CfCenA 418

135

CfCex 336

CfCenB

443

ι ι •m 912

1012

I

CfCenC


ClfX 203

308

CtEGD

PfEndA 859

962

Figure 1. The organization of catalytic and non-catalytic domains in cellulases from C. fimi and other bacteria. CfCenA, Β and C, and CfCex are the endo- and exo-β- 1, 4-glucanases of C. fimi. ClfX is a translated open reading frame from Cellulomonas flavigena (29). CtEGD and PfEndA are endo-β-Ι, 4-glucanases from Clostridium thermocellum and Pseudomonas fluorescens, respectively (30,32). The primary structures are drawn approximately to scale and are numbered from the amino terminus of the mature protein; ClfX is numbered from the start of the open reading frame. Unshaded areas represent catalytic domains, cross-hatched areas indicate cellulose-binding domains, repeated blocks of amino acids are stippled, and black areas represent linker regions.

Figure 2. Model of CenA (non-glycosylated form) derived from small-angle X-ray scattering analyses. The structure of the catalytic domain (shaded region) was resolved by a separate scattering analysis of the isolated 'core' peptide (see text for details). Adapted from reference 21.



352


Catalytic Domains. The catalytic domain of CenA has been classified in family Β (2); this family also contains T. reesei C B H II. CenA and CBH II both catalyse hydrolysis of β-1, 4-glucosidic bonds with inversion of anomeric carbon configuration (13,14). Inversion indicates a single displacement reaction mechanism. It is not yet known whether all members of family Β are inverting enzymes or, more generally, whether all members of any given cellulase family share a common stereospecificity. Cex belongs to family F (2) and catalyses hydrolysis with retention of configuration (double displacement reaction) (13) but the stereospecificities of other members of family F have not yet been reported. The other members of family F are xylanases (2,3) and significant xylanase activity was also reported for Cex (4). Such relaxed substrate specificity is not unprecedented; several β-l, 4-glucanases with activity towards xylan, chitosan and other glycans have been described (e.g. refs. 15 and 16). For a cloned gene product, such results are unequivocal since contamination by other glycanases can be avoided. Cellulose-binding Domains. The CBDs of CenA and Cex show significant (~ 50 %) sequence identity (1). Closely related sequences occur at the amino and carboxyl termini of several bacterial endo-β-Ι, 4glucanases as well as at the amino terminus of a xylanase from Pseudomonas fluorescens (Figure 1). It is probable that each of these sequences represents a C B D , as recently demonstrated for endoglucanase Β from P.fluorescens(17). Alignment of the CenA, Cex and other CBD sequences reveals that aromatic amino acids as well as Asn and Gly residues are strongly conserved (Figure 3). Moreover, two Cys residues, towards the amino and carboxyl ends, occur in all the CBD sequences except that from Butyrivibrio. The mechanism of binding is not yet known but a motif of four rather regularly spaced tryptophan residues (corresponding to CenA Trp , T r p , T r p and Trp , Figure 3) is strongly conserved in all the CBD sequences suggesting the involvement of these hydrophobic residues in the interaction with cellulose. It is noteworthy that certain tryptophan residues are also well conserved in the analogous starch-binding domains of amylases and related enzymes (18). Cellodextrins, as well as maltodextrins, inhibit the binding of an amylase starch-binding domain (Nikolov, Z.L., this volume); in contrast, there is presently no evidence for the interaction of CBDs with a-1, 4-glucose polymers. Proteolytic truncation of ngCenA showed that affinity for cellulose was retained by products missing up to 64 amino-proximal amino acids, although the strength of the interaction was lowered (10). At present, the functional significance of the binding to cellulose is not clear. CBDs also occur at the amino or carboxyl termini of fungal cellulases (19) and the structure of the CBD for T. reesei CBH I has been resolved by 2-dimensional N M R (20). There is little sequence similarity between the bacterial and fungal CBDs although hydrophobic and 14

34

50

68



TrEGIII

Y

TNQWN GFTA V TV Ν GSS INGWTL W Y

GQRITQSWNA

V A

WNGSIP G TV FGFQGS

GS

Ρ SV

NG C

-IMNEPHDVNI-NT-WAATVQEWTA-1RNAGATSQFISLP GNDWQSAGAFISDG

S SG

TASTNGGQVSVTSLPWNGSIPTGGTASFGFNGS-WAGS-NPTPASFSLNGTTCTGTVPT TVTQSGSAVTVRNAPWNGSIPAGGTAQFGFNGS-HTGT-NAAPTAFSLNGTPCTVG* DLSTSGGNVTVRNVSWNGNVPAGGSTSFGFLGS-GTG—QL SSSI TCSAS * DWSQSGTTVTAKNAAWNGSLAAGQTVDIGFNGA-HNGT-NNKPASFTLNGATCTVG* NVTGNN-PYAASALGWNANIQPGQTAEFGFQGTKGAGSRQV-PA-VT—GSVCQ* GLSGAN-PYSATPVGWNTSIPIGSSVEFGVQGNN—GSSRAQVPAVT—GAICGGQG NVSGSN-PYSASNLSWNGNIQPGQSVSFGFQVNKNGGSAER-P-SV—GGSICSGSVA NIAEEGGYYVITPMSWNSSLEPSASVDFGIQGS GS-IGT—SV—NISVQ*

GCLS LGAYCIVDIHNYARWNGG11GQGG-PTNAQFTSL—WSQLASKYA-SQS-RVWFG

AC

APGCRVDYAVTNOWGGFGANV-TITNLG-DPVSSWkLDWTYTAGQRIQQLWKG SGPAGÇQVLWGV-NQWNTGFTANV-TVKNTSSAPVDGWTLTFSFPSGQQVTQAWSS QP PAGRACEAT YALVNQWP GGFQAEV-TVKNTG S SPINGWTVQWTLP SGQSITQLWNG TGSCKVEYNASS-WNTGFTASV-RVTNTGTTALNGWTLTFPFANGQTVQQGWSA AASGGNCQ—YVVTNQWNNGFTA-VIRVRNNGSSAINRWSVNWSYSDGSRITNSWNA AVCE—YRVTNEWGSGFTAS-IRITNNGSSTINGWSVSWNYTDGSRVTSSWNA QTATCS—YNITNEWNTGYTGD-ITITNRGSSAINGWSVNWQYAT-NRLSSSWNA VSGALK-AEYTI-NNWGSGYQV-LIKVKNDSASRVDGWTLKISKSEV-KIDSSWCV

266

109 443 456 456 308 131 961 547

214

52 389 409 254 912 78 51 502

Figure 3. Sequence alignments of the cellulose-binding domains and putative cellulose-binding domains of bacterial glycanases. CfCenA, Β and C, and CfCex are the endo- and exo-^-1, 4-glucanases of C.fimi.ClfX is a translated open reading frame from Cellulomonas flavigena (29). MbCelA is an endo-^-1, 4-glucanase from Microbispora bispora (32). PfEndA and B, and PfXynA are endo-β-Ι, 4-glucanases and a xylanase from Pseudomonasfluorescens(17,31,33). BfEnd is an endo-^-1, 4-glucanase from Butyrivibriofibrisolvens(34). Also included is a related sequence from TrEG III, endoglucanase III of Trichoderma reesei (35). The consensus sequence shows amino acid residues common to four or more bacterial CBDs. The conserved Trp motif (see the text) and cysteine residues are shaded.

215

53 390 410 255 913 79 52 503

CfCenA CfCex MbCe1A CI fX PfEndA PfEndB PfXynA BfEnd

Consensus

161

1 336 353 203 859 30 1 451

TrEGIII

Consensus

CfCenA CfCex MbCelA ClfX PfEndA PfEndB PfXynA BfEnd


354



hydroxyamino acids show a similar pattern of distribution in both types (21). It is also of interest that T. reesei endoglucanase III (EG ΙΠ, recently renamed EG Π) contains a sequence (amino acids 161 - 266), outside of its CBD, with the conserved Trp motif of the bacterial CBDs (Figure 3); other consensus residues of the bacterial CBD are also represented. The significance of this sequence in EG III is not clear, but the amino acid cluster: -Asn-Glu-Pro- contained therein (amino acids 217 - 219) is common to some 16 bacterial and fungal cellulases and may be directly involved in catalysis (22). Linker Regions. In both CenA and Cex a 20 - 23 amino acid sequence containing only proline and threonine residues (the Pro-Thr box) joins the CBD to the catalytic domain. Similar sequences, rich in proline and hydroxyamino acid residues, are evident in many of the other known cellulase primary structures (Table 1). Typically, these regions comprise 20 - 30 amino acids; exceptionally long sequences are found in some P.fluorescensglycanases. Analogous structures occur in unrelated enzymes e.g. the 2-oxo acid dehydrogenases of prokaryotes and eukaryotes (Table 1). They are believed to provide flexibility between, and critical spatial separation of, adjoining domains. In HgCenA and ngCex the amino and carboxyl junctions of the Pro-Thr box are particularly susceptible to proteolysis suggesting an exposed location. In the native, glycosylated forms of these enzymes, sugar substitution at nearby sites is assumed to provide a degree of protection (9). Endoglucanase Β (CenB) and Endoglucanase C (CenC). The gene encoding CenB was isolated from the original shotgun cloning of C. fimi genomic D N A (6). CenC was purified from C. fimi culture supernatant and its gene subsequently cloned using a D N A probe based on the determined amino terminus (23). Both enzymes are large (> 100 kDa) endo-β-Ι, 4-glucanases that bind to cellulose (23,24). The organization of their domains, deduced from recently determined primary structures, is shown in Figure 1. Catalytic Domains. The catalytic domain of CenB (amino acids 1 - 608) shows 35% sequence identity with an endoglucanase from Persea americana (avocado); it has therefore been placed in family E, subfamily 2 (2,3). The CenC catalytic domain (amino acids 299-809) shows 28% sequence identity with Clostridium thermocellum endoglucanase D (EGD) and 43% identity with P. fluorescens endoglucanase A (End A), both members of family E, subfamily 1 (2,3). Non-catalytic Domains. CenB contains a 100 amino acid carboxyl-terminal sequence with significant sequence identity to the other bacterial CBDs shown in Figure 1. Nevertheless, when this is



Trichoderma reesei Human Human

endoglucanase immunoglobulin 2-oxo acid dehydrogenase complex

Egffl IgAl E2

CelE CelB

endoglucanase Clostridium thermocellum endoglucanase Bacillus sp. strain N4

CenA Cex ClfX ClfA CelA CelB

(ii)

(0

(ii)

(0

(ii)

(0

Designation

EndA

Protein

endoglucanase Cellulomonas fimi exoglucanase Cellulomonas fimi Cellulomonas flavigena Cellulomonas flavigena Microbispora bispora endoglucanase Caldocellum saccharolyticum bifunctional exo-endoglucanase endoglucanase Pseudomonas fluorescens

Organism

3

35 39 40

37 38

31

7 8 29 29 32 36

Reference

PT2S(PT)4T(PT)7VTPQPT (PT) T(PT)3T(PT)3S PDPTDEPTEDPT(DDPT)5EDPT PDPTD2PTQDPTD2PT P2TYSPSPTPST(PS)3QSDPGS(PS)3 T2S2(PT)4(VT)2(PT)5VTAT(PT)3PVSTPAT PAPTMTVAPTAT(PT)2l^PTV(TP)2APTQTAI(PT)2LTPN(PT)2 Si 1VPVS7I2PS6IQPS6MPS8V2AS5VS S4ASNINS12AIVS5V2S6 PLVS(PT)3LMPTPSPTVT P2SDPTP2SDPDPGEPDPTP2SDPGEYP P2SEPSDP4SEPE(PDPGE)3PDPTP2SDPEYP PGAT2lT2STRP2SGPT4RA(TS)2S2TP2TS2 PVPSTP2TPSPSTP2T PQ VP3TP3 V A2 VP2TPQPL APTPS APCPATPAGP

Linker Sequence

Table 1. Linker Sequences from Bacterial β-l, 4-Glucanases and other Proteins




356

removed by genetic deletion (24), the enzyme retains significant affinity for cellulose. This indicates the presence of an additional, aminoproximal CBD with a rather different primary structure to those so far identified. A triple repeat sequence occurs between the catalytic domain and the carboxyl-terminal CBD (Figure 1). Each repeated unit contains approximately 98 amino acids. These are separated from one another by linker sequences resembling the Pro-Thr boxes of CenA and Cex. It is interesting that the translated open reading frame ClfX from Cellulomonas flavigena contains a putative CBD at its carboxyl terminus (Figure 3) and that this is preceded by a single copy of a sequence with 50 % sequence identity to the CenB repeats (Figure 1). The CenC catalytic domain is flanked at both its amino and carboxyl ends by double repeat sequences. Neither pair correspond to any other cellulase sequences reported to date. Double repeat structures occur in several endo-p-1, 4-glucanases from C. thermocellum, for example EGD (Figure 1), but these are much smaller than those of CenC. The functional significance of the repeated sequences is not yet clear in any of these examples. The conserved duplicated region of the C. thermocellum endoglucanases resembles the Ca -binding site of several Ca -binding proteins but deletion of this region from C . thermocellum endoglucanase D did not influence Ca -binding kinetics (25). Possible functions for the repeated regions of bacterial cellulases include their involvement in the protein-protein interaction required for enzyme complex formation. There is as yet no evidence for such complexes in Cellulomonas spp. but the association of several enzymes and other proteins into the cellulosome is now firmly established for the cellulolytic Clostridia (26). ++

++

++

Application of the C. fimi Cellulose-binding Domains The construction of hybrid proteins containing bacterial CBDs may provide a cheap generic method for enzyme immobilization and/or purification using cellulosic matrices. The CBD can be fused at the amino or carboxyl terminus, as in the parent cellulase, to suit individual applications. We have constructed model fusion proteins using the C. fimi CBDs to demonstrate this potential. TnphoA insertional mutagenesis was used to generate a series of CenA' 'PhoA fusions. Those polypeptides which contained the entire CBDcenA were readily purified from E. coli periplasmic extracts by binding to filter paper (27). Similarly, CBDcex was fused to the carboxyl terminus of an Agrobacterium β-glucosidase (Abg), in this case by direct ligation of appropriate gene fragments (21,28). The Abg CBDcex hybrid could be purified from crude cell extracts by adsorption to a cellulose column. After extraneous proteins were washed off with phosphatebuffered 1 M NaCl, Abg CBDcex could be eluted with H 0 (28). Removal of the CBD from the hybrid would require further engineer ing: the introduction of a specific cleavage site at the fusion junction. 2


26. GILKES ET AL.

10.0

357

Cellulomonasfimifi-l,4-Glucanases

h

E


(0

[P]

1

(mg.mg" )

ad

Figure 4. Adsorption of AbgCBD to microcrystalline cellulose. The main panel shows a Scatchard analysis of the experimental data (open circles). Data were resolved into two classes of binding interactions of relatively high and low affinity (straight lines). The respective adsorption parameters derived from these linear plots are given in the text. Closed circles show data obtained by substitution of these derived parameters into a Langmuir-type equation. The inset shows the same data plotted as an adsorption isotherm. (Reproduced with permission from reference 41. Copyright 1991 Butterworth Heinemann.) c

The adsorption of Abg CBDcex to Avicel (microcrystalline cellulose) in 50 m M potassium phosphate, p H 7, at 4° is described by the adsorption isotherm shown in Figure 4 (inset). Abg itself has no significant affinity for Avicel. The adsorption data may be resolved by Scatchard analysis as a two- site model ( K ^ i = 1.5 χ 10~ mg protein.ml , [Plad, max,l = 1-3 χ 10" mg protein.mg" Avicel; Kd,2 = 5.2 χ 10" mg. ml- ; [Plad, max,2 = 2.7 χ 10~ mg.mg" ) (Figure 4, main panel). Abg CBDcex shows quasi-irreversible adsorption to cellulosic substrates as illustrated in Figure 5. In this experiment identical columns containing chopped cotton fibers were loaded with approximately 1 mg Abg CBDcex per g cotton. The columns were continously eluted at 37° or 50° with 50 m M potassium phosphate, pH7, containing p-nitrophenol-p-D-glucoside. Hydrolysis of the chromogenic substrate to p-nitrophenolate was determined from the absorption of the eluate at 400 nm. The fusion protein remained bound to the cotton matrix for at least 10 days during continuous operation at 37°. The enzyme was inactivated after 3 days at 50° (Figure 5) although there was no loss of protein from the column. 3

2

1

2

-1

2

1


1


358 •

37°C

1.0

o o .o 0.5

o >

0.0


-

50°C

1.0

0.5

0.0

0

5 Time (days)

10

Figure 5. Performance of the fusion protein AbgCBD immobilized on a column containing chopped cotton fiber at 37 ° and 50 °. (Reproduced with permission from reference 41. Copyright 1991 Butterworth Heinemann.) c

Fusions to thermostable enzymes will allow us to evaluate adsorption at higher temperatures. When a column containing Abg-CBDcex/ adsorbed to cellulose at p H 7.0, was eluted with an increasing or decreasing p H gradient (constant ionic strength), protein (enzymatically inactive) was eluted above p H 9, but there was no desorption evident at low pH. (Ong, E.; Gilkes, N.R.; Miller, R.C., Jr.; Warren, R.A.J.; Kilburn, D.G. Enzyme Microb. Technol, in press). These results demonstrate several useful properties of the C. fimi CBDs for enzyme immobilization and purification. Presumably, the CBDs of other bacterial cellulases (Figure 3) can be used in similar ways. It remains to be seen whether these differ significantly in their affinity or capacity for adsorption; if so, certain CBDs may be preferable for specific purposes. Acknowledgments. Our work on C. fimi cellulases has been generously supported by the Natural Sciences and Engineering Research Council of Canada. Literature Cited 1. Warren, R.A.J.; Beck, C.F.; Gilkes, N.R.; Langsford, M.L.; Miller, R.C., Jr.; O'Neill, G.P.; Scheuffens, M . ; Wong, W.K.R. Proteins . 1986, 1, 335-341. Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

GILKES ET A L

2. 3. 4. 5. 6.


7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Cellulomonas fimi fi-l,4-Glucanases

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Henrissat, B.M.; Claeyssens, M.; Tomme, P.; Lemesle, L.; Mornon, J.-P. Gene. 1989, 81, 83-95. Béguin, P. Annu. Rev. Microbiol. 1990, 44, 219-248. Gilkes, N.R.; Langsford, M.L.; Kilburn, D.G.; Miller, R.C., Jr.; Warren, R.A.J. J. Biol. Chem. 1984, 259, 10455-10459. Gilkes, N.R.; Warren, R.A.J.; Miller, R.C., Jr.; Kilburn, D.G. J. Biol. Chem. 1988, 263, 10401-10407. Gilkes, N.R.; Kilburn, D.G.; Langsford, M.L.; Miller, R.C., Jr.; Wakarchuk, W.W.; Warren, R.A.J.; Whittle, D.J.; Wong, W.K.R. J. Gen. Microbiol. 1984, 130, 1377-1384. Wong, W.K.R.; Gerhard, B.; Guo, Z.M.; Kilburn, D.G.; O'Neill, G.P.; Goh, S.H.; Warren, R.A.J.; Miller, R.C., Jr. Gene. 1986, 44, 315-324. O'Neill, G.P.; Goh, S.H.; Warren, R.A.J.; Kilburn, D.G.; Miller, R.C., Jr. Gene. 1986, 44, 325-330. Langsford, M.L.; Gilkes, N.R.; Singh, B.; Moser, B.; Miller, R.C. Jr.; Warren, R.A.J.; Kilburn, D.G. FEBS Lett. 1987, 225, 163-167. Gilkes, N.R.; Kilburn, D.G.; Miller, R.C., Jr.; Warren, R.A.J. J. Biol. Chem. 1989, 264, 17802-17808. Pilz, I.; Schwarz, E.; Kilburn, D.G.; Miller, R.C., Jr.; Warren, R.A.J.; Gilkes, N.R. Biochem. J. 1990, 271, 277-280. Abuja, P.M.; Pilz, I.; Claeyssens, M.; Tomme, P. Biochem. Biophys. Res. Commun. 1988, 156, 180-185. Withers, S.G.; Dombroski, D.; Berven, L.A.; Kilburn, D.G.; Miller, R.C., Jr.; Warren, R.A.J.; Gilkes, N.R. Biochem. Biophys. Res. Commun. 1986, 139, 487-494. Knowles, T.K.C.; Lentovaara, P.; Murray, M.; Sinnott, M.L. J. Chem. Soc. Chem. Commun. 1988, 1401-1402. Shoemaker, S.; Watt, K.; Tsitovsky, G.; Cox, R. Bio/technology 1983, 1, 687-690. Hedges, Α.; Wolfe, R.S. J. Bacteriol. 1974, 120, 844-853. Gilbert, H.J.; Hall, J.; Hazelwood, G.P.; Ferreira, L.M.A. Molec. Microbiol. 1990, 4, 759-767. Svensson, B.; Jespersen, H.; Sierks, M.R.; Macgregor, E.A. Biochem. J. 1989, 264, 309-311. Tomme, P.; van Tilbeurgh, H.;Pettersson, G.; Van Damme, J.; Vandekerckhove, J.; Knowles, J.; Teeri, T.; Claeyssens, M . Eur. J. Biochem. 1988, 170, 575-581. Kraulis, P.J.; Clore, G.M.; Nilges, M.; Jones, T.A.; Pettersson, G.; Knowles, J.; Gronenborn, A . M . Biochemistry. 1989, 28, 72417257. Ong, E.; Greenwood, J.M.; Gilkes, N.R.; Kilburn, D.G.; Miller, R.C., Jr.; Warren, R.A.J. Trends Biotechnol. 1989, 7, 239-243. Baird, S.D.; Hefford, M.A.; Johnson, D.A.; Sung, W.L.; Yaguchi, M.; Seligy, V.L. Biochem. Biophys. Res. Commun. 1990, 169, 1035-1039.


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Enzymes in Biomass Conversion - American Chemical Society

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