Heterologous expression and site-directed mutagenesis studies of two Trichoderma harzianum chitinases, Chit33 and Chit42, in Escherichia coli

Harry Boer (Corresponding Author), Helena Simolin, Sylvain Cottaz, Hans Söderlund, Anu Koivula

Research output: Contribution to journalArticleScientificpeer-review

15 Citations (Scopus)

Abstract

Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, and offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.
Original languageEnglish
Pages (from-to)216-226
JournalProtein Expression and Purification
Volume51
Issue number2
DOIs
Publication statusPublished - 2007
MoE publication typeA1 Journal article-refereed

Fingerprint

Chitinases
Trichoderma
Site-Directed Mutagenesis
Protein Engineering
Escherichia coli
Carboxylic Acids
Mutagenesis
Protons
Inclusion Bodies
Energy Transfer
Hydrolases
Enzymes
Proteins

Keywords

  • Chitinase
  • Catalytic residues
  • Ice-nucleation protein
  • Chitin-oligosaccharides
  • Resonance energy transfer
  • Activity screening

Cite this

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title = "Heterologous expression and site-directed mutagenesis studies of two Trichoderma harzianum chitinases, Chit33 and Chit42, in Escherichia coli",
abstract = "Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, and offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.",
keywords = "Chitinase, Catalytic residues, Ice-nucleation protein, Chitin-oligosaccharides, Resonance energy transfer, Activity screening",
author = "Harry Boer and Helena Simolin and Sylvain Cottaz and Hans S{\"o}derlund and Anu Koivula",
year = "2007",
doi = "10.1016/j.pep.2006.07.020",
language = "English",
volume = "51",
pages = "216--226",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press",
number = "2",

}

Heterologous expression and site-directed mutagenesis studies of two Trichoderma harzianum chitinases, Chit33 and Chit42, in Escherichia coli. / Boer, Harry (Corresponding Author); Simolin, Helena; Cottaz, Sylvain; Söderlund, Hans; Koivula, Anu.

In: Protein Expression and Purification, Vol. 51, No. 2, 2007, p. 216-226.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Heterologous expression and site-directed mutagenesis studies of two Trichoderma harzianum chitinases, Chit33 and Chit42, in Escherichia coli

AU - Boer, Harry

AU - Simolin, Helena

AU - Cottaz, Sylvain

AU - Söderlund, Hans

AU - Koivula, Anu

PY - 2007

Y1 - 2007

N2 - Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, and offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.

AB - Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, and offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.

KW - Chitinase

KW - Catalytic residues

KW - Ice-nucleation protein

KW - Chitin-oligosaccharides

KW - Resonance energy transfer

KW - Activity screening

U2 - 10.1016/j.pep.2006.07.020

DO - 10.1016/j.pep.2006.07.020

M3 - Article

VL - 51

SP - 216

EP - 226

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -