The enhancement of the cellulolytic activity of cellobiohydrolase I and endoglucanase by the addition of cellulose binding domains derived from Trichoderma reesei

ABSTRACT Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.

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Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis

Biochemical Journal ◽

10.1042/bj3030817 ◽

1994 ◽

Vol 303 (3) ◽

pp. 817-823 ◽

Cited By ~ 84

Author(s):

B Nidetzky ◽

W Steiner ◽

M Claeyssens

Keyword(s):

Mass Transfer ◽

Trichoderma Reesei ◽

Filter Paper ◽

Cellulose Hydrolysis ◽

Solid Substrate ◽

Bulk Liquid ◽

Cellobiohydrolase I ◽

Irreversible Adsorption ◽

Core Proteins ◽

Cellulose Binding

Separate binding of several purified cellulolytic components of Trichoderma reesei on to filter paper was studied and concomitant hydrolysis rates evaluated. Enhancement of mass transfer from the bulk liquid to the solid substrate by agitation has two different effects on adsorption depending on the type of enzyme: (i) the fraction of cellobiohydrolase II (CBH II) and endoglucanase III (EG III) bound at equilibrium is increased, whereas (ii) the rate but not the extent of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) adsorption is affected. The adsorption of CBH I core, a component lacking the cellulose-binding domain (CBD), is, however, not significantly influenced by mass transfer. The CBH I interdomain peptide (present in CBH I core b) does not participate in adsorption but enhances stability. The adsorption of CBH I core proteins is a fully reversible process whereas that of the intact CBH I is not. Thus, the interaction of the CBD with filter paper apparently accounts for the mass-transfer-limited binding rate and also for the irreversible adsorption of intact CBH I. Adsorption isotherms at 50 degrees C indicate very similar relative association constants for the intact cellulases (0.24-0.30 l/g of cellulose), but drastically reduced values for CBH I core proteins (0.03 l/g of cellulose). The specific activities of adsorbed CBH I and of its core proteins are identical and a linear relationship between adsorption and rates of hydrolysis is found only for these enzymes. Thus, non-productive binding on to cellulose seems evident in the case of CBH II and EG III but not CBH I.

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Adsorption Characteristics of Fungal Family 1 Cellulose-Binding Domain from Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose: Negative Cooperative Adsorption via a Steric Exclusion Effect

Langmuir ◽

10.1021/la302352k ◽

2012 ◽

Vol 28 (40) ◽

pp. 14323-14329 ◽

Cited By ~ 19

Author(s):

Naohisa Sugimoto ◽

Kiyohiko Igarashi ◽

Masahisa Wada ◽

Masahiro Samejima

Keyword(s):

Trichoderma Reesei ◽

Binding Domain ◽

Crystalline Cellulose ◽

Cellulose Binding Domain ◽

Cellobiohydrolase I ◽

Adsorption Characteristics ◽

Cooperative Adsorption ◽

Steric Exclusion ◽

Cellulose Binding ◽

Exclusion Effect

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Analogs of the cellulose binding domain of cellobiohydrolase I from Trichoderma reesei: Synthesis and binding

Peptides 1992 ◽

10.1007/978-94-011-1470-7_358 ◽

1993 ◽

pp. 780-781

Author(s):

Gunnar Lindeberg ◽

Per Hansen ◽

Jerry Ståhlberg ◽

Göran Pettersson

Keyword(s):

Trichoderma Reesei ◽

Binding Domain ◽

Cellulose Binding Domain ◽

Cellobiohydrolase I ◽

Cellulose Binding

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Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei

European Journal of Biochemistry ◽

10.1046/j.1432-1327.1999.00455.x ◽

1999 ◽

Vol 262 (3) ◽

pp. 637-643 ◽

Cited By ~ 58

Author(s):

Geraldine Carrard ◽

Markus Linder

Keyword(s):

Trichoderma Reesei ◽

Binding Domains ◽

Cellulose Binding

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Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

Applied and Environmental Microbiology ◽

10.1128/aem.68.5.2503-2508.2002 ◽

2002 ◽

Vol 68 (5) ◽

pp. 2503-2508 ◽

Cited By ~ 20

Author(s):

Markus Linder ◽

Jadwiga Winiecka-Krusnell ◽

Ewert Linder

Keyword(s):

Trichoderma Reesei ◽

Cyst Wall ◽

Binding Assay ◽

Pneumocystis Carinii ◽

Staining Reaction ◽

Entamoeba Dispar ◽

Microscopy Imaging ◽

Binding Domains ◽

Unicellular Organisms ◽

Cellulose Binding

ABSTRACT Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent β-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.

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