Cloning and expression of Trichoderma reesei Cellobiohydrolase I in Pichia pastoris

Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS−PAGE ge...

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Bibliographic Details
Published in:Biotechnology progress 1999, Vol.15 (5), p.828-833
Main Authors: Godbole, S, Decker, S.R, Nieves, R.A, Adney, W.S, Vinzant, T.B, Baker, J.O, Thomas, S.R, Himmel, M.E
Format: Article
Language:English
Subjects:
09 BIOMASS FUELS
Amino Acid Sequence
Base Sequence
Biological and medical sciences
Biotechnology
cbh1 gene
cellobiohydrolase
CELLULASE
Cellulase - biosynthesis
Cellulase - genetics
CELLULOSE
Cellulose 1,4-beta-Cellobiosidase
Chemical operations
Chromatographic analysis
Circular Dichroism
Cloning, Molecular
DNA-CLONING
Enzyme kinetics
Enzymes
Fundamental and applied biological sciences. Psychology
Gels
gene expression
GENE REGULATION
gene transfer
Genes
Genetic engineering
Genetic technics
genetic transformation
Hypocrea jecorina
Kinetics
Methods. Procedures. Technologies
Microorganisms
Modification of gene expression level
Molecular Sequence Data
Mutagenesis
Pichia - genetics
Pichia - metabolism
Pichia pastoris
PROTEIN ENGINEERING
Purification
Reaction kinetics
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Sequence Analysis, Protein
TRICHODERMA
Trichoderma - enzymology
Trichoderma - genetics
Trichoderma reesei
YEASTS
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Summary:Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS−PAGE gels. Carbohydrate content determination and SDS−PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T. reesei enzyme could be isolated chromatographically. The P. pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (i.e., pNPL and Sigmacell), although at a level significantly lower than the wild‐type enzyme. More seriously, the yeast‐expressed enzyme showed non‐wild‐type secondary structure by circular dichroism. We conclude that P. pastoris may not serve as an adequate host for the site‐directed mutagenesis of T. reesei CBH I.
ISSN:8756-7938
1520-6033
DOI:10.1021/bp9901116