Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei

Jim E. Pitts, Jaana M. Uusitalo, Dimitris Mantafounis, Philip G. Nugent, Dominic D. Quinn, Poonsook Orprayoon, Merja E. Penttilä

Research output: Contribution to journalArticleScientificpeer-review

9 Citations (Scopus)

Abstract

The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.

Original languageEnglish
Pages (from-to)69-83
Number of pages15
JournalJournal of Biotechnology
Volume28
Issue number1
DOIs
Publication statusPublished - 1 Jan 1993
MoE publication typeA1 Journal article-refereed

Fingerprint

Chymosin
Trichoderma
Enzymes
Enzyme kinetics
Mutagenesis
Gene encoding
Fungi
Structural analysis
Escherichia coli
Peptides
Catalysis
Hydrogen bonds
Proteins
Cellulose 1,4-beta-Cellobiosidase
Substrates
Protein Engineering
Cellulase
Hydrogen Bonding
Site-Directed Mutagenesis
Aspartic Acid

Keywords

  • Aspartic proteinase
  • Chymosin
  • pH optima
  • Protein engineering
  • Site-directed mutagenesis
  • Trichoderma reesei

Cite this

Pitts, Jim E. ; Uusitalo, Jaana M. ; Mantafounis, Dimitris ; Nugent, Philip G. ; Quinn, Dominic D. ; Orprayoon, Poonsook ; Penttilä, Merja E. / Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei. In: Journal of Biotechnology. 1993 ; Vol. 28, No. 1. pp. 69-83.
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Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei. / Pitts, Jim E.; Uusitalo, Jaana M.; Mantafounis, Dimitris; Nugent, Philip G.; Quinn, Dominic D.; Orprayoon, Poonsook; Penttilä, Merja E.

In: Journal of Biotechnology, Vol. 28, No. 1, 01.01.1993, p. 69-83.

Research output: Contribution to journalArticleScientificpeer-review

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