Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters

Harry Boer, Tuula Teeri, Anu Koivula (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

104 Citations (Scopus)

Abstract

Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N‐glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The kcat and Km values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site‐directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

Original languageEnglish
Pages (from-to)486 - 494
Number of pages9
JournalBiotechnology and Bioengineering
Volume69
Issue number5
DOIs
Publication statusPublished - 2000
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Pichia
alcohol oxidase
Yeast
Screening
Oxidoreductases
Cellulase
Bioreactors
Substrates
Enzymes
Disulfides
Proteins
Saccharomyces cerevisiae
Yeasts
Fluorescence
Crystalline materials
Degradation
Alcohols

Cite this

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title = "Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters",
abstract = "Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N‐glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The kcat and Km values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site‐directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.",
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Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. / Boer, Harry; Teeri, Tuula; Koivula, Anu (Corresponding Author).

In: Biotechnology and Bioengineering, Vol. 69, No. 5, 2000, p. 486 - 494.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters

AU - Boer, Harry

AU - Teeri, Tuula

AU - Koivula, Anu

PY - 2000

Y1 - 2000

N2 - Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N‐glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The kcat and Km values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site‐directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

AB - Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N‐glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The kcat and Km values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site‐directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

U2 - 10.1002/1097-0290(20000905)69:5<486::AID-BIT3>3.0.CO;2-N

DO - 10.1002/1097-0290(20000905)69:5<486::AID-BIT3>3.0.CO;2-N

M3 - Article

VL - 69

SP - 486

EP - 494

JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

SN - 0006-3592

IS - 5

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