A high throughput profiling method for cutinolytic esterases

Pasi Halonen (Corresponding Author), Tapani Reinikainen, Antti Nyyssölä, Johanna Buchert

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

A procedure for identifying and profiling cutinolytic esterases was developed by combining traditional plate screen assays with an automated robotic system. In the first phase, the micro-organisms were screened on agar plates with cutin or the model substrate polycaprolactone as the sole carbon sources. In the second phase, p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The variables used were pH and the carbon chain length of the fatty acid moiety of the p-nitrophenyl substrate. Finally, 3H-labelled cutin was used as a specific substrate to verify the positive hits and to validate the screening procedure. With pH as the variable in the automatic screen, esterase production of cutinase positive strains typically proceeded in two stages: first an esterase with neutral activity optimum was produced, after which a strong esterolytic response in the alkaline range was detected. With carbon chain length of the fatty acid as the variable best correlation with cutinase production was obtained with strains showing a high ratio of activities towards p-nitrophenyl-butyrate and p-nitrophenyl-palmitate.
Original languageEnglish
Pages (from-to)394-399
Number of pages6
JournalEnzyme and Microbial Technology
Volume44
Issue number6-7
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

Esterases
Fatty Acids
Carbon
Fatty acids
Throughput
Substrates
Chain length
Assays
Palmitates
Robotics
Polycaprolactone
Agar
Culture Media
Esters
Screening
Liquids
cutinase
cutin

Keywords

  • Cutinases
  • cutin
  • screening
  • high throughput
  • esterases

Cite this

Halonen, Pasi ; Reinikainen, Tapani ; Nyyssölä, Antti ; Buchert, Johanna. / A high throughput profiling method for cutinolytic esterases. In: Enzyme and Microbial Technology. 2009 ; Vol. 44, No. 6-7. pp. 394-399.
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A high throughput profiling method for cutinolytic esterases. / Halonen, Pasi (Corresponding Author); Reinikainen, Tapani; Nyyssölä, Antti; Buchert, Johanna.

In: Enzyme and Microbial Technology, Vol. 44, No. 6-7, 2009, p. 394-399.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - A high throughput profiling method for cutinolytic esterases

AU - Halonen, Pasi

AU - Reinikainen, Tapani

AU - Nyyssölä, Antti

AU - Buchert, Johanna

PY - 2009

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N2 - A procedure for identifying and profiling cutinolytic esterases was developed by combining traditional plate screen assays with an automated robotic system. In the first phase, the micro-organisms were screened on agar plates with cutin or the model substrate polycaprolactone as the sole carbon sources. In the second phase, p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The variables used were pH and the carbon chain length of the fatty acid moiety of the p-nitrophenyl substrate. Finally, 3H-labelled cutin was used as a specific substrate to verify the positive hits and to validate the screening procedure. With pH as the variable in the automatic screen, esterase production of cutinase positive strains typically proceeded in two stages: first an esterase with neutral activity optimum was produced, after which a strong esterolytic response in the alkaline range was detected. With carbon chain length of the fatty acid as the variable best correlation with cutinase production was obtained with strains showing a high ratio of activities towards p-nitrophenyl-butyrate and p-nitrophenyl-palmitate.

AB - A procedure for identifying and profiling cutinolytic esterases was developed by combining traditional plate screen assays with an automated robotic system. In the first phase, the micro-organisms were screened on agar plates with cutin or the model substrate polycaprolactone as the sole carbon sources. In the second phase, p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The variables used were pH and the carbon chain length of the fatty acid moiety of the p-nitrophenyl substrate. Finally, 3H-labelled cutin was used as a specific substrate to verify the positive hits and to validate the screening procedure. With pH as the variable in the automatic screen, esterase production of cutinase positive strains typically proceeded in two stages: first an esterase with neutral activity optimum was produced, after which a strong esterolytic response in the alkaline range was detected. With carbon chain length of the fatty acid as the variable best correlation with cutinase production was obtained with strains showing a high ratio of activities towards p-nitrophenyl-butyrate and p-nitrophenyl-palmitate.

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KW - cutin

KW - screening

KW - high throughput

KW - esterases

U2 - 10.1016/j.enzmictec.2008.12.012

DO - 10.1016/j.enzmictec.2008.12.012

M3 - Article

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JO - Enzyme and Microbial Technology

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