Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides

Atte Mikkelson, Hannu Maaheimo, Terhi Hakala (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

32 Citations (Scopus)

Abstract

In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99% of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86–95%) and total yield (23% of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes.
Original languageEnglish
Pages (from-to)60-68
Number of pages9
JournalCarbohydrate Research
Volume372
DOIs
Publication statusPublished - 2013
MoE publication typeA1 Journal article-refereed

Fingerprint

Trichoderma
Oligosaccharides
Hydrolysis
Enzymes
Amorphophallus
Mannans
Enzymatic hydrolysis
Carbohydrates
Substrates
Mannose
Substrate Specificity
Size exclusion chromatography
endoglucanase Cel5A
konjac gluco-mannan
Cellulase
Fractionation
Bioactivity
Gel Chromatography
High Pressure Liquid Chromatography
Nuclear magnetic resonance

Keywords

  • enzymatic hydrolysis
  • glucomannan
  • konjac mannan
  • oligosaccharide

Cite this

@article{663b50fc289f4c07993bb07b8767251e,
title = "Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides",
abstract = "In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99{\%} of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86–95{\%}) and total yield (23{\%} of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes.",
keywords = "enzymatic hydrolysis, glucomannan, konjac mannan, oligosaccharide",
author = "Atte Mikkelson and Hannu Maaheimo and Terhi Hakala",
year = "2013",
doi = "10.1016/j.carres.2013.02.012",
language = "English",
volume = "372",
pages = "60--68",
journal = "Carbohydrate Research",
issn = "0008-6215",
publisher = "Elsevier",

}

Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides. / Mikkelson, Atte; Maaheimo, Hannu; Hakala, Terhi (Corresponding Author).

In: Carbohydrate Research, Vol. 372, 2013, p. 60-68.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides

AU - Mikkelson, Atte

AU - Maaheimo, Hannu

AU - Hakala, Terhi

PY - 2013

Y1 - 2013

N2 - In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99% of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86–95%) and total yield (23% of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes.

AB - In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99% of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86–95%) and total yield (23% of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes.

KW - enzymatic hydrolysis

KW - glucomannan

KW - konjac mannan

KW - oligosaccharide

U2 - 10.1016/j.carres.2013.02.012

DO - 10.1016/j.carres.2013.02.012

M3 - Article

VL - 372

SP - 60

EP - 68

JO - Carbohydrate Research

JF - Carbohydrate Research

SN - 0008-6215

ER -