Product inhibition of five Hypocrea jecorina cellulases

Leigh Murphy, Peter Westh, Christina Bohlin, Martin Johannes Baumann, Søren Nymand Olsen, Trine Holst Sørensen, Lars Anderson, Kim Borch

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC50 = 19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC50 = 60–80 mM), and weakly inhibited by glucose (IC50 = 350–380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).
Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC50 = 19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC50 = 60–80 mM), and weakly inhibited by glucose (IC50 = 350–380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).
LanguageEnglish
JournalEnzyme and Microbial Technology
Volume52
Issue number3
Pages163-169
ISSN0141-0229
DOIs
StatePublished - 5 Mar 2013

Cite this

Murphy, L., Westh, P., Bohlin, C., Baumann, M. J., Olsen, S. N., Sørensen, T. H., ... Borch, K. (2013). Product inhibition of five Hypocrea jecorina cellulases. Enzyme and Microbial Technology, 52(3), 163-169. DOI: 10.1016/j.enzmictec.2013.01.002
Murphy, Leigh ; Westh, Peter ; Bohlin, Christina ; Baumann, Martin Johannes ; Olsen, Søren Nymand ; Sørensen, Trine Holst ; Anderson, Lars ; Borch, Kim. / Product inhibition of five Hypocrea jecorina cellulases. In: Enzyme and Microbial Technology. 2013 ; Vol. 52, No. 3. pp. 163-169
@article{2cf9ee9ea71e416fb9d7528203beffe5,
title = "Product inhibition of five Hypocrea jecorina cellulases",
abstract = "Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50{\%} activity-loss in 19 mM cellobiose (IC50 = 19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC50 = 60–80 mM), and weakly inhibited by glucose (IC50 = 350–380 mM). The highest resistance to both products was found for Cel5A, which retained about 75{\%} of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).",
author = "Leigh Murphy and Peter Westh and Christina Bohlin and Baumann, {Martin Johannes} and Olsen, {S{\o}ren Nymand} and S{\o}rensen, {Trine Holst} and Lars Anderson and Kim Borch",
year = "2013",
month = "3",
day = "5",
doi = "10.1016/j.enzmictec.2013.01.002",
language = "English",
volume = "52",
pages = "163--169",
journal = "Enzyme and Microbial Technology",
issn = "0141-0229",
publisher = "Elsevier Inc.",
number = "3",

}

Murphy, L, Westh, P, Bohlin, C, Baumann, MJ, Olsen, SN, Sørensen, TH, Anderson, L & Borch, K 2013, 'Product inhibition of five Hypocrea jecorina cellulases' Enzyme and Microbial Technology, vol. 52, no. 3, pp. 163-169. DOI: 10.1016/j.enzmictec.2013.01.002

Product inhibition of five Hypocrea jecorina cellulases. / Murphy, Leigh; Westh, Peter; Bohlin, Christina; Baumann, Martin Johannes; Olsen, Søren Nymand; Sørensen, Trine Holst; Anderson, Lars; Borch, Kim.

In: Enzyme and Microbial Technology, Vol. 52, No. 3, 05.03.2013, p. 163-169.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Product inhibition of five Hypocrea jecorina cellulases

AU - Murphy,Leigh

AU - Westh,Peter

AU - Bohlin,Christina

AU - Baumann,Martin Johannes

AU - Olsen,Søren Nymand

AU - Sørensen,Trine Holst

AU - Anderson,Lars

AU - Borch,Kim

PY - 2013/3/5

Y1 - 2013/3/5

N2 - Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC50 = 19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC50 = 60–80 mM), and weakly inhibited by glucose (IC50 = 350–380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).

AB - Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC50 = 19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC50 = 60–80 mM), and weakly inhibited by glucose (IC50 = 350–380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).

U2 - 10.1016/j.enzmictec.2013.01.002

DO - 10.1016/j.enzmictec.2013.01.002

M3 - Journal article

VL - 52

SP - 163

EP - 169

JO - Enzyme and Microbial Technology

T2 - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

SN - 0141-0229

IS - 3

ER -

Murphy L, Westh P, Bohlin C, Baumann MJ, Olsen SN, Sørensen TH et al. Product inhibition of five Hypocrea jecorina cellulases. Enzyme and Microbial Technology. 2013 Mar 5;52(3):163-169. Available from, DOI: 10.1016/j.enzmictec.2013.01.002