Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography

Medve, József; Lee, Dora; Tjerneld, Folke

doi:10.1016/s0021-9673(98)00132-0

Cited by 63 publications

(52 citation statements)

References 23 publications

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“…Fractions containing CBH I and EG I were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) (Laemmli 1970). Zymogram analysis of of EG I and CBH I were carried out according to the method described previously (Medve et al 1998). …”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Functional display of fungal cellulases from Trichoderma reesei on phage M13

Bai

et al. 2008

World J Microbiol Biotechnol

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To test whether the phage display technology could be applied in cellulase engineering, phagemids harboring the genes encoding the mature forms of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from filamentous fungus Trichoderma reesei were constructed, respectively. CBH I and EG I fused to the phage coat protein encoded by the g3 gene were expressed and displayed on phage M13. The phage-bound cellulases retained their activities as determined by hydrolysis of the corresponding substrates, Also, their binding abilities to insoluble cellulose substrate were confirmed by an ELISA method. Overall, these results demonstrate that cellulases can be displayed on phage surface while maintaining their biological function, thus providing an alternative for directed evolution and high-throughput screening for improved cellulases.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Functional display of fungal cellulases from Trichoderma reesei on phage M13

Bai

et al. 2008

World J Microbiol Biotechnol

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“…Unfortunately, many of these model substrates are not specific enough to distinguish individual enzymes. Protein chromatography techniques have also been utilized to fractionate the enzyme mixture down to its individual components [11,12]. However, this approach is laborious and, depending on the purification protocols used, the enzyme mixture may not always completely separate into its individual components [13].…”

Section: Introductionmentioning

confidence: 99%

The Development and Use of an Elisa-Based Method to Follow the Distribution of Cellulase Monocomponents During the Hydrolysis of Pretreated Corn Stover

Pribowo¹,

Hu²,

Arantes³

et al. 2015

Fuel Production From Non-Food Biomass

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Background: It is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requires the synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to the complexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack of specific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrate interaction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in a typical "cellulase mixture" need to work cooperatively to achieve effective hydrolysis, the action of one enzyme is likely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature of the lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us to follow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commercially realistic biomass substrates. Results: A high throughput immunoassay that could quantitatively and specifically follow individual cellulase enzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb, respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to specifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II (Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosic supernatants could be minimized by dilution. Conclusion: A double-antibody sandwich ELISA was able to detect and quantify individual enzymes when present in cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 -1 μg/ml), provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay was employed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, that were present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS). All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profiles observed with the ELISA method were in agreement with earlier work where more labour intensive enzyme assay techniques were used.

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“…To study hydrolysis mechanism of individual cellulase, CBHl was separated by modification made to the protocol reported by Medve et al (1998b). A vacuum manifold system was used to provide a steady flow through parallel columns to achieve scaled-up quantities of CBH 1 from Spezyme CP cellulases.…”

Section: Methodsmentioning

confidence: 99%

“…To separate CBH 1 from Spezyme CP cellulases, the initial crude enzyme sample was buffer exchanged to pH 7.6 in a 10 mM TEA (Triethanolamine)-HCI buffer by repeated ultrafiltration in 6 ml Vivaspin centrifuge tubes III preparation for chromatography as suggested by Medve et al (1998b).…”

Section: Preparation Of Spezyme Cp Samplementioning

confidence: 99%

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The impact of adsorbed cellulase inactivation on enzymatic hydrolysis kinetics.

Ye¹

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Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography

Cited by 63 publications

References 23 publications

Functional display of fungal cellulases from Trichoderma reesei on phage M13

Functional display of fungal cellulases from Trichoderma reesei on phage M13

The Development and Use of an Elisa-Based Method to Follow the Distribution of Cellulase Monocomponents During the Hydrolysis of Pretreated Corn Stover

The impact of adsorbed cellulase inactivation on enzymatic hydrolysis kinetics.

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