Pectinase activity determination

An early deceleration in the release of reducing sugars throws a spanner in the works!

A. Biz, F.C. Farias, F.A. Motter, D.H. De Paula, Peter Richard, N. Krieger, D.A. Mitchell (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

16 Citations (Scopus)

Abstract

Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.
Original languageEnglish
Article numbere109529
JournalPLoS ONE
Volume9
Issue number10
DOIs
Publication statusPublished - 2014
MoE publication typeA1 Journal article-refereed

Fingerprint

Polygalacturonase
Deceleration
polygalacturonase
reducing sugars
Sugars
Assays
pectins
assays
Research Personnel
Industrial Waste
researchers
biorefining
Saccharification
saccharification
industrial wastes
extracts
production costs
Costs and Cost Analysis
Temperature
pectin

Keywords

  • aspergillus
  • cellulases
  • deceleration
  • enzymes
  • fermentation
  • hydrolysis
  • pectins
  • reaction time

Cite this

Biz, A. ; Farias, F.C. ; Motter, F.A. ; De Paula, D.H. ; Richard, Peter ; Krieger, N. ; Mitchell, D.A. / Pectinase activity determination : An early deceleration in the release of reducing sugars throws a spanner in the works!. In: PLoS ONE. 2014 ; Vol. 9, No. 10.
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abstract = "Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.",
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Pectinase activity determination : An early deceleration in the release of reducing sugars throws a spanner in the works! / Biz, A.; Farias, F.C.; Motter, F.A.; De Paula, D.H.; Richard, Peter; Krieger, N.; Mitchell, D.A. (Corresponding Author).

In: PLoS ONE, Vol. 9, No. 10, e109529, 2014.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Pectinase activity determination

T2 - An early deceleration in the release of reducing sugars throws a spanner in the works!

AU - Biz, A.

AU - Farias, F.C.

AU - Motter, F.A.

AU - De Paula, D.H.

AU - Richard, Peter

AU - Krieger, N.

AU - Mitchell, D.A.

PY - 2014

Y1 - 2014

N2 - Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

AB - Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

KW - aspergillus

KW - cellulases

KW - deceleration

KW - enzymes

KW - fermentation

KW - hydrolysis

KW - pectins

KW - reaction time

U2 - 10.1371/journal.pone.0109529

DO - 10.1371/journal.pone.0109529

M3 - Article

VL - 9

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

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M1 - e109529

ER -