Dataset Information

The disulphide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei.

ABSTRACT: The positions of the disulphide bridges of the 1,4-beta-glucan cellobiohydrolase (CBH I) of the fungus Trichoderma reesei have been investigated. The results can be summarized as follows. (1) The enzyme contains 12 disulphide bridges and no free cysteine residues. (2) The location of six disulphide bridges have been determined experimentally. (3) The bonding patterns of the two disulphide bridges in the C-terminal region is suggested on the basis of internal homology. (4) The remaining four disulphide bridges are put into two groups, each containing four half-cystine residues where two are adjacent. (5) A repeating bonding pattern is observed along the peptide chain and a non-local disulphide bond with an unusually long separation distance links the N-terminal and the C-terminal region. (6) The disulphide-bonded CNBr peptides of a 1,4-beta-glucan glucanohydrolase (endoglucanase II) from T. reesei have been isolated and a disulphide bonding pattern is suggested on the basis of the sequence homology between the two enzymes.

SUBMITTER: Bhikhabhai R

PROVIDER: S-EPMC1144236 | biostudies-other | 1984 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundTrichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60-70°C.ResultsA B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50-70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in Tm) and improved half-life at 60°C (t1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.ConclusionStructure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Dataset Information

The disulphide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets