Immunoaffinity Chromatographic Purification of Cellobiohydrolase II Mutants from RecombinantTrichoderma reeseiStrains Devoid of Major Endoglucanase Genes

Koivula, Anu; Lappalainen, Arja; Virtanen, Susanna; Mäntylä, Arja; Suominen, Pirkko; Teeri, Tuula T.

doi:10.1006/prep.1996.0116

Cited by 17 publications

(14 citation statements)

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“…reesei Cel6A wild-type was purified from a T. reesei strain lacking the endogenous genes for the major endoglucanases Cel5A and Cel7B. The presence of contaminating cellulolytic activities was ruled out by measuring the activities towards MeUmb(Glc) 1 , MeUmb(Glc) 2 , and hydroxyethyl cellulose (HEC), as described earlier (27). Concentrations of purified wild-type protein were determined from UV absorbance at 280 nm using the molar extinction coefficient ε = 104,000 M -1 cm -1 , based on the result of total amino acid analysis (28).…”

Section: Methodsmentioning

confidence: 99%

Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface

Igarashi

Uchihashi

Koivula

et al. 2011

Science

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518

517

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A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

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Section: Methodsmentioning

confidence: 99%

Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface

Igarashi

Uchihashi

Koivula

et al. 2011

Science

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517

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“…Cellobiohydrolase I (CBHI) produced by fermentation on lactose from T. reesei strain ALKO2877 [14] was obtained in pure form from Primalco Biotec Ltd, Finland. CBHI was generally used as a stock solution of 9.5 mg/ml in water, and stored at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

Modified glycosylation of cellobiohydrolase I from a high cellulase‐producing mutant strain of Trichoderma reesei

Harrison¹,

Nouwens²,

Jardine³

et al. 1998

European Journal of Biochemistry

141

142

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Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solidphase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known. Keywords : cellobiohydrolase I; cellulase; fungal glycosylation; Trichoderma; N-acetylglucosamine.Cellobiohydrolase I (CBHI) is the major family C glycoprotein secreted by the filamentous fungus Trichoderma reesei, where it operates in synergy with other exo-glucanases and endo-glucanases to achieve the degradation of cellulose [1]. CBHI is typically secreted in the order of several grams/litre, and many aspects of its structure and biology are well characterised. Like most other fungal cellulolytic enzymes characterised, CBHI conforms to a well-defined multi-domain structure, typified by a relatively large catalytic (core) domain spatially removed from a smaller cellulose-binding domain (CBD) by a highly O-glycosylated linker domain [2]. Presumably, the CBD serves as a form of anchor, and the linker a form of torsional leash, providing the catalytic region with access to a wide range of potential (and perhaps otherwise inaccessible) sites [3]. Although the catalytic domain of CBHI by itself is sufficient for cellulose binding and full catalytic activity at low enzyme to cellulose ratios, a functional CBD with sufficient spatial separation from the catalytic region is necessary for cellulolytic activity at higher enzyme-to-cellulose ratios [3]. The importance of the linker domain in maintaining spatial orientation of catalytic Correspondence to N. H. Packer,

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“…The Cel6A mutant constructions were transformed into Trichoderma strain ALKO2877, devoid of the cel6A and egl7A genes, and the mutant proteins W272A and W272D were produced in the Trichoderma essentially as described earlier [18]. Desalted culture supernatant was purified with ion‐exchange, affinity chromatography [8] and finally with immunoaffinity chromatography [25]. Cel6A wild type (wt) was produced and purified as described earlier [18].…”

Section: Methodsmentioning

confidence: 99%

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A

et al. 1998

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Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.z 1998 Federation of European Biochemical Societies.

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Immunoaffinity Chromatographic Purification of Cellobiohydrolase II Mutants from RecombinantTrichoderma reeseiStrains Devoid of Major Endoglucanase Genes

Cited by 17 publications

References 0 publications

Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface

Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface

Modified glycosylation of cellobiohydrolase I from a high cellulase‐producing mutant strain of Trichoderma reesei

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A

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