Characterization of esterases acting on hemicelluloses

Dissertation

Maija Tenkanen

Research output: ThesisDissertationCollection of Articles

2 Citations (Scopus)

Abstract

Xylans and mannans contain different esterified substituents such as acetyl, feruloyl and p-coumaroyl side groups. The properties and function of hemicellulose-acting esterases of Trichoderma reesei and Aspergillus oryzae were studied in this work. Both fungi produce multiple esterases. Two almost identical acetyl xylan esterases of pI 7.0 (AXE I) and pI 6.8 (AXE II) were purified from T. reesei. Both enzymes were monomeric glycoproteins with apparent molecular masses of 34 kDa and they seemed to be coded by a single gene. The purified enzyme(s) efficiently liberated esterified acetic acid from polymeric xylans and were even capable of removing acetyl groups from xylan on the surface layers of ground birchwood. Acetyl xylan esterase(s) had high specificity for acetylated xylan: the enzyme was unable to remove acetyl substituents from softwood galactoglucomannan or phenolic substituents from wheat straw arabinoxylan. T. reesei also produces another type of esterase which was shown to have activity only towards short oligomeric and monomeric acetates. This enzyme showed clear regional specificity, removing mainly O-3' linked acetyl substituents from xylobiose. Even though acetyl xylan esterase had high activity towards polymeric xylan it was unable to liberate all the acetyl substituents. Acetyl esterase was needed to remove these residual acetyl groups, which were most probably located near to the other substituent, 4-O-methylglucuronic acid. In addition to acetylated xylooligomers, the acetyl esterase exhibited activity towards acetylated oligomers derived from galactoglucomannan. Athough T. reesei is an efficient producer of different xylanolytic and mannanolytic enzymes, it was not found to produce esterases which were able to efficiently liberate esterified ferulic acid from wheat straw xylan, or acetic acid from softwood galactoglucomannan. These esterases were therefore purified from another fungus, Aspergillus oryzae. Both feruloyl esterase and acetyl glucomannan esterase were small, acidic monomeric proteins with apparent molecular masses of 30 and 36 kDa and isolelectric points of 3.6 and 4.6, respectively. Feruloyl esterase was able to liberate most of the feruloyl substituents from wheat straw xylan but the reaction was enhanced by the presence of xylanase. This esterase had a broad substrate specificity. In addition to feruloyl substituents it was active towards p-coumaroyl and acetyl side groups. Feruloyl esterase was equally efficient in the deacetylation of xylan as the acetyl xylan esterase of T. reesei and it could also remove most of the acetyl substituents from galactoglucomannan. The acetyl glucomannan esterase was more specific than the feruloyl esterase. It did not show any feruloyl esterase activity and its specific acetyl glucomannan esterase activity was eight times higher and specific acetyl xylan esterase activity four times lower than those of feruloyl esterase. Although the acetyl glucomannan esterase had high activity towards polymeric galactoglucomannan, the maximum amount of acetic acid liberated was less than with feruloyl esterase. The activity of acetyl glucomannan esterase was clearly enhanced by addition of mannanase and a-galactosidase, whereas no significant synergism between these two glycanases and the feruloyl esterase was observed. The simultaneous enzymatic liberation of acetyl groups from O-acetyl-4-O-methylglucuronoxylan of hardwood and O-acetyl-galactoglucomannan of softwood clearly enhanced the action of other xylanolytic and mannanolytic enzymes. If deacetylation (chemical or enzymatic) was performed before hydrolysis with endoxylanase the extent of the hydrolysis was lower, due to the decreased solubility of the deacetylated substrate. Removal of feruloyl groups from wheat straw arabinoxylan had little effect on the action of endoxylanase and a-arabinosidase, which is most probably due to the low amount of these side groups in xylans of gramineous plants. Esterases with high activity towards polymeric substrates have several potential applications. They could be used to facilitate the study of the functional role of these side groups in biopolymers and may provide new possibilities for the modification of carbohydrates. Enzymatic deacetylation of dissolved O-acetyl-galactoglcuomannan was found to increase the yield of the TMP process by as much as 1%. Similarly the effluent load was reduced. The esterases may also have potential use in synthetic chemistry for the preparation of specific sugar acetates which are difficult to manufacture by traditional methods.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • Helsinki University of Technology
Supervisors/Advisors
  • Poutanen, Kaisa, Advisor
  • Viikari, Liisa, Advisor, External person
Award date22 Sep 1995
Place of PublicationEspoo
Publisher
Print ISBNs951-38-4780-2
Publication statusPublished - 1995
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

hemicellulose
esterases
xylan
Trichoderma reesei
wheat straw
softwood
enzymes
endo-1,4-beta-xylanase
acetic acid
Aspergillus oryzae
arabinoxylan
hydrolysis
acetates
mannans
molecular weight
alpha-N-arabinofuranosidase
galactosidases
beta-mannosidase
fungi
feruloyl esterase

Keywords

  • esterases
  • enzymes
  • hemicelluloses
  • polysaccharides
  • fungi
  • purification
  • degradation
  • plant cells
  • production

Cite this

Tenkanen, M. (1995). Characterization of esterases acting on hemicelluloses: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Tenkanen, Maija. / Characterization of esterases acting on hemicelluloses : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1995. 162 p.
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abstract = "Xylans and mannans contain different esterified substituents such as acetyl, feruloyl and p-coumaroyl side groups. The properties and function of hemicellulose-acting esterases of Trichoderma reesei and Aspergillus oryzae were studied in this work. Both fungi produce multiple esterases. Two almost identical acetyl xylan esterases of pI 7.0 (AXE I) and pI 6.8 (AXE II) were purified from T. reesei. Both enzymes were monomeric glycoproteins with apparent molecular masses of 34 kDa and they seemed to be coded by a single gene. The purified enzyme(s) efficiently liberated esterified acetic acid from polymeric xylans and were even capable of removing acetyl groups from xylan on the surface layers of ground birchwood. Acetyl xylan esterase(s) had high specificity for acetylated xylan: the enzyme was unable to remove acetyl substituents from softwood galactoglucomannan or phenolic substituents from wheat straw arabinoxylan. T. reesei also produces another type of esterase which was shown to have activity only towards short oligomeric and monomeric acetates. This enzyme showed clear regional specificity, removing mainly O-3' linked acetyl substituents from xylobiose. Even though acetyl xylan esterase had high activity towards polymeric xylan it was unable to liberate all the acetyl substituents. Acetyl esterase was needed to remove these residual acetyl groups, which were most probably located near to the other substituent, 4-O-methylglucuronic acid. In addition to acetylated xylooligomers, the acetyl esterase exhibited activity towards acetylated oligomers derived from galactoglucomannan. Athough T. reesei is an efficient producer of different xylanolytic and mannanolytic enzymes, it was not found to produce esterases which were able to efficiently liberate esterified ferulic acid from wheat straw xylan, or acetic acid from softwood galactoglucomannan. These esterases were therefore purified from another fungus, Aspergillus oryzae. Both feruloyl esterase and acetyl glucomannan esterase were small, acidic monomeric proteins with apparent molecular masses of 30 and 36 kDa and isolelectric points of 3.6 and 4.6, respectively. Feruloyl esterase was able to liberate most of the feruloyl substituents from wheat straw xylan but the reaction was enhanced by the presence of xylanase. This esterase had a broad substrate specificity. In addition to feruloyl substituents it was active towards p-coumaroyl and acetyl side groups. Feruloyl esterase was equally efficient in the deacetylation of xylan as the acetyl xylan esterase of T. reesei and it could also remove most of the acetyl substituents from galactoglucomannan. The acetyl glucomannan esterase was more specific than the feruloyl esterase. It did not show any feruloyl esterase activity and its specific acetyl glucomannan esterase activity was eight times higher and specific acetyl xylan esterase activity four times lower than those of feruloyl esterase. Although the acetyl glucomannan esterase had high activity towards polymeric galactoglucomannan, the maximum amount of acetic acid liberated was less than with feruloyl esterase. The activity of acetyl glucomannan esterase was clearly enhanced by addition of mannanase and a-galactosidase, whereas no significant synergism between these two glycanases and the feruloyl esterase was observed. The simultaneous enzymatic liberation of acetyl groups from O-acetyl-4-O-methylglucuronoxylan of hardwood and O-acetyl-galactoglucomannan of softwood clearly enhanced the action of other xylanolytic and mannanolytic enzymes. If deacetylation (chemical or enzymatic) was performed before hydrolysis with endoxylanase the extent of the hydrolysis was lower, due to the decreased solubility of the deacetylated substrate. Removal of feruloyl groups from wheat straw arabinoxylan had little effect on the action of endoxylanase and a-arabinosidase, which is most probably due to the low amount of these side groups in xylans of gramineous plants. Esterases with high activity towards polymeric substrates have several potential applications. They could be used to facilitate the study of the functional role of these side groups in biopolymers and may provide new possibilities for the modification of carbohydrates. Enzymatic deacetylation of dissolved O-acetyl-galactoglcuomannan was found to increase the yield of the TMP process by as much as 1{\%}. Similarly the effluent load was reduced. The esterases may also have potential use in synthetic chemistry for the preparation of specific sugar acetates which are difficult to manufacture by traditional methods.",
keywords = "esterases, enzymes, hemicelluloses, polysaccharides, fungi, purification, degradation, plant cells, production",
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note = "Project code: B5SU0072",
year = "1995",
language = "English",
isbn = "951-38-4780-2",
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publisher = "VTT Technical Research Centre of Finland",
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Tenkanen, M 1995, 'Characterization of esterases acting on hemicelluloses: Dissertation', Doctor Degree, Helsinki University of Technology, Espoo.

Characterization of esterases acting on hemicelluloses : Dissertation. / Tenkanen, Maija.

Espoo : VTT Technical Research Centre of Finland, 1995. 162 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Characterization of esterases acting on hemicelluloses

T2 - Dissertation

AU - Tenkanen, Maija

N1 - Project code: B5SU0072

PY - 1995

Y1 - 1995

N2 - Xylans and mannans contain different esterified substituents such as acetyl, feruloyl and p-coumaroyl side groups. The properties and function of hemicellulose-acting esterases of Trichoderma reesei and Aspergillus oryzae were studied in this work. Both fungi produce multiple esterases. Two almost identical acetyl xylan esterases of pI 7.0 (AXE I) and pI 6.8 (AXE II) were purified from T. reesei. Both enzymes were monomeric glycoproteins with apparent molecular masses of 34 kDa and they seemed to be coded by a single gene. The purified enzyme(s) efficiently liberated esterified acetic acid from polymeric xylans and were even capable of removing acetyl groups from xylan on the surface layers of ground birchwood. Acetyl xylan esterase(s) had high specificity for acetylated xylan: the enzyme was unable to remove acetyl substituents from softwood galactoglucomannan or phenolic substituents from wheat straw arabinoxylan. T. reesei also produces another type of esterase which was shown to have activity only towards short oligomeric and monomeric acetates. This enzyme showed clear regional specificity, removing mainly O-3' linked acetyl substituents from xylobiose. Even though acetyl xylan esterase had high activity towards polymeric xylan it was unable to liberate all the acetyl substituents. Acetyl esterase was needed to remove these residual acetyl groups, which were most probably located near to the other substituent, 4-O-methylglucuronic acid. In addition to acetylated xylooligomers, the acetyl esterase exhibited activity towards acetylated oligomers derived from galactoglucomannan. Athough T. reesei is an efficient producer of different xylanolytic and mannanolytic enzymes, it was not found to produce esterases which were able to efficiently liberate esterified ferulic acid from wheat straw xylan, or acetic acid from softwood galactoglucomannan. These esterases were therefore purified from another fungus, Aspergillus oryzae. Both feruloyl esterase and acetyl glucomannan esterase were small, acidic monomeric proteins with apparent molecular masses of 30 and 36 kDa and isolelectric points of 3.6 and 4.6, respectively. Feruloyl esterase was able to liberate most of the feruloyl substituents from wheat straw xylan but the reaction was enhanced by the presence of xylanase. This esterase had a broad substrate specificity. In addition to feruloyl substituents it was active towards p-coumaroyl and acetyl side groups. Feruloyl esterase was equally efficient in the deacetylation of xylan as the acetyl xylan esterase of T. reesei and it could also remove most of the acetyl substituents from galactoglucomannan. The acetyl glucomannan esterase was more specific than the feruloyl esterase. It did not show any feruloyl esterase activity and its specific acetyl glucomannan esterase activity was eight times higher and specific acetyl xylan esterase activity four times lower than those of feruloyl esterase. Although the acetyl glucomannan esterase had high activity towards polymeric galactoglucomannan, the maximum amount of acetic acid liberated was less than with feruloyl esterase. The activity of acetyl glucomannan esterase was clearly enhanced by addition of mannanase and a-galactosidase, whereas no significant synergism between these two glycanases and the feruloyl esterase was observed. The simultaneous enzymatic liberation of acetyl groups from O-acetyl-4-O-methylglucuronoxylan of hardwood and O-acetyl-galactoglucomannan of softwood clearly enhanced the action of other xylanolytic and mannanolytic enzymes. If deacetylation (chemical or enzymatic) was performed before hydrolysis with endoxylanase the extent of the hydrolysis was lower, due to the decreased solubility of the deacetylated substrate. Removal of feruloyl groups from wheat straw arabinoxylan had little effect on the action of endoxylanase and a-arabinosidase, which is most probably due to the low amount of these side groups in xylans of gramineous plants. Esterases with high activity towards polymeric substrates have several potential applications. They could be used to facilitate the study of the functional role of these side groups in biopolymers and may provide new possibilities for the modification of carbohydrates. Enzymatic deacetylation of dissolved O-acetyl-galactoglcuomannan was found to increase the yield of the TMP process by as much as 1%. Similarly the effluent load was reduced. The esterases may also have potential use in synthetic chemistry for the preparation of specific sugar acetates which are difficult to manufacture by traditional methods.

AB - Xylans and mannans contain different esterified substituents such as acetyl, feruloyl and p-coumaroyl side groups. The properties and function of hemicellulose-acting esterases of Trichoderma reesei and Aspergillus oryzae were studied in this work. Both fungi produce multiple esterases. Two almost identical acetyl xylan esterases of pI 7.0 (AXE I) and pI 6.8 (AXE II) were purified from T. reesei. Both enzymes were monomeric glycoproteins with apparent molecular masses of 34 kDa and they seemed to be coded by a single gene. The purified enzyme(s) efficiently liberated esterified acetic acid from polymeric xylans and were even capable of removing acetyl groups from xylan on the surface layers of ground birchwood. Acetyl xylan esterase(s) had high specificity for acetylated xylan: the enzyme was unable to remove acetyl substituents from softwood galactoglucomannan or phenolic substituents from wheat straw arabinoxylan. T. reesei also produces another type of esterase which was shown to have activity only towards short oligomeric and monomeric acetates. This enzyme showed clear regional specificity, removing mainly O-3' linked acetyl substituents from xylobiose. Even though acetyl xylan esterase had high activity towards polymeric xylan it was unable to liberate all the acetyl substituents. Acetyl esterase was needed to remove these residual acetyl groups, which were most probably located near to the other substituent, 4-O-methylglucuronic acid. In addition to acetylated xylooligomers, the acetyl esterase exhibited activity towards acetylated oligomers derived from galactoglucomannan. Athough T. reesei is an efficient producer of different xylanolytic and mannanolytic enzymes, it was not found to produce esterases which were able to efficiently liberate esterified ferulic acid from wheat straw xylan, or acetic acid from softwood galactoglucomannan. These esterases were therefore purified from another fungus, Aspergillus oryzae. Both feruloyl esterase and acetyl glucomannan esterase were small, acidic monomeric proteins with apparent molecular masses of 30 and 36 kDa and isolelectric points of 3.6 and 4.6, respectively. Feruloyl esterase was able to liberate most of the feruloyl substituents from wheat straw xylan but the reaction was enhanced by the presence of xylanase. This esterase had a broad substrate specificity. In addition to feruloyl substituents it was active towards p-coumaroyl and acetyl side groups. Feruloyl esterase was equally efficient in the deacetylation of xylan as the acetyl xylan esterase of T. reesei and it could also remove most of the acetyl substituents from galactoglucomannan. The acetyl glucomannan esterase was more specific than the feruloyl esterase. It did not show any feruloyl esterase activity and its specific acetyl glucomannan esterase activity was eight times higher and specific acetyl xylan esterase activity four times lower than those of feruloyl esterase. Although the acetyl glucomannan esterase had high activity towards polymeric galactoglucomannan, the maximum amount of acetic acid liberated was less than with feruloyl esterase. The activity of acetyl glucomannan esterase was clearly enhanced by addition of mannanase and a-galactosidase, whereas no significant synergism between these two glycanases and the feruloyl esterase was observed. The simultaneous enzymatic liberation of acetyl groups from O-acetyl-4-O-methylglucuronoxylan of hardwood and O-acetyl-galactoglucomannan of softwood clearly enhanced the action of other xylanolytic and mannanolytic enzymes. If deacetylation (chemical or enzymatic) was performed before hydrolysis with endoxylanase the extent of the hydrolysis was lower, due to the decreased solubility of the deacetylated substrate. Removal of feruloyl groups from wheat straw arabinoxylan had little effect on the action of endoxylanase and a-arabinosidase, which is most probably due to the low amount of these side groups in xylans of gramineous plants. Esterases with high activity towards polymeric substrates have several potential applications. They could be used to facilitate the study of the functional role of these side groups in biopolymers and may provide new possibilities for the modification of carbohydrates. Enzymatic deacetylation of dissolved O-acetyl-galactoglcuomannan was found to increase the yield of the TMP process by as much as 1%. Similarly the effluent load was reduced. The esterases may also have potential use in synthetic chemistry for the preparation of specific sugar acetates which are difficult to manufacture by traditional methods.

KW - esterases

KW - enzymes

KW - hemicelluloses

KW - polysaccharides

KW - fungi

KW - purification

KW - degradation

KW - plant cells

KW - production

M3 - Dissertation

SN - 951-38-4780-2

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Tenkanen M. Characterization of esterases acting on hemicelluloses: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1995. 162 p.