Sensitive high-throughput screening for the detection of reducing sugars

Andrea Mellitzer, Anton Glieder, Roland Weis, Christoph Reisinger, Karlheinz Flicker

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2.

Original languageEnglish
Pages (from-to)155-62
Number of pages8
JournalBiotechnology Journal
Volume7
Issue number1
DOIs
Publication statusPublished - Jan 2012

Fingerprint

Hydrolysis
Cellulose 1,4-beta-Cellobiosidase
Endo-1,4-beta Xylanases
Trichoderma
Pichia
Biofuels
Enzyme Assays
Enzymes
Acids
4-hydroxybenzoic acid hydrazide
lignocellulose

Keywords

  • Biofuels
  • Carbohydrate Metabolism
  • Carbohydrates
  • Cellulose 1,4-beta-Cellobiosidase
  • Endo-1,4-beta Xylanases
  • High-Throughput Screening Assays
  • Hydrolysis
  • Hydroxybenzoates
  • Lignin
  • Pichia
  • Sensitivity and Specificity
  • Trichoderma
  • beta-Mannosidase
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Sensitive high-throughput screening for the detection of reducing sugars. / Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz.

In: Biotechnology Journal, Vol. 7, No. 1, 01.2012, p. 155-62.

Research output: Contribution to journalArticleResearchpeer-review

Mellitzer, Andrea ; Glieder, Anton ; Weis, Roland ; Reisinger, Christoph ; Flicker, Karlheinz. / Sensitive high-throughput screening for the detection of reducing sugars. In: Biotechnology Journal. 2012 ; Vol. 7, No. 1. pp. 155-62.
@article{214762a58244437d88dbe1870a0f2c49,
title = "Sensitive high-throughput screening for the detection of reducing sugars",
abstract = "The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8{\%}. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2.",
keywords = "Biofuels, Carbohydrate Metabolism, Carbohydrates, Cellulose 1,4-beta-Cellobiosidase, Endo-1,4-beta Xylanases, High-Throughput Screening Assays, Hydrolysis, Hydroxybenzoates, Lignin, Pichia, Sensitivity and Specificity, Trichoderma, beta-Mannosidase, Journal Article, Research Support, Non-U.S. Gov't",
author = "Andrea Mellitzer and Anton Glieder and Roland Weis and Christoph Reisinger and Karlheinz Flicker",
note = "{\circledC} 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.",
year = "2012",
month = "1",
doi = "10.1002/biot.201100001",
language = "English",
volume = "7",
pages = "155--62",
journal = "Biotechnology Journal",
issn = "1860-6768",
publisher = "Wiley-VCH",
number = "1",

}

TY - JOUR

T1 - Sensitive high-throughput screening for the detection of reducing sugars

AU - Mellitzer, Andrea

AU - Glieder, Anton

AU - Weis, Roland

AU - Reisinger, Christoph

AU - Flicker, Karlheinz

N1 - © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PY - 2012/1

Y1 - 2012/1

N2 - The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2.

AB - The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2.

KW - Biofuels

KW - Carbohydrate Metabolism

KW - Carbohydrates

KW - Cellulose 1,4-beta-Cellobiosidase

KW - Endo-1,4-beta Xylanases

KW - High-Throughput Screening Assays

KW - Hydrolysis

KW - Hydroxybenzoates

KW - Lignin

KW - Pichia

KW - Sensitivity and Specificity

KW - Trichoderma

KW - beta-Mannosidase

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1002/biot.201100001

DO - 10.1002/biot.201100001

M3 - Article

VL - 7

SP - 155

EP - 162

JO - Biotechnology Journal

JF - Biotechnology Journal

SN - 1860-6768

IS - 1

ER -