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Title: Interrelationships between cellulase activity and cellulose particle morphology

Abstract

It is well documented that the enzymatic hydrolysis of cellulose follows a reaction pattern where an initial phase of relatively high activity is followed by a gradual slow-down over the entire course of the reaction. This phenomenon is not readily explained by conventional factors like substrate depletion, product inhibition or enzyme instability. It has been suggested that the underlying reason for the loss of enzyme activity is connected to the heterogeneous structure of cellulose, but so far attempts to establish quantitative measures of such a correlation remain speculative. Here, we have carried out an extensive microscopy study of Avicel particles during extended hydrolysis with Hypocrea jecorina cellobiohydrolase 1 (CBH1) and endoglucanase 1 and 3 (EG1 and EG3) alone and in mixtures. We have used differential interference contrast microscopy and transmission electron microscopy to observe and quantify structural features at um and nm resolution, respectively. We implemented a semi-automatic image analysis protocol, which allowed us to analyze almost 3000 individual micrographs comprising a total of more than 300,000 particles. From this analysis we estimated the temporal development of the accessible surface area throughout the reaction. We found that the number of particles and their size as well as the surface roughnessmore » contributed to surface area, and that within the investigated degree of conversion (<30 %) this measure correlated linearly with the rate of reaction. Lastly, based on this observation we argue that cellulose structure, specifically surface area and roughness, plays a major role in the ubiquitous rate loss observed for cellulases.« less

Authors:
 [1];  [2];  [3];  [1]; ORCiD logo [4]
  1. Roskilde Univ. (Denmark). Dept. of Science, Systems and Models
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center
  3. Novozymes A/S, Bagsvaerd (Denmark)
  4. National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1289432
Report Number(s):
NREL/JA-5100-66700
Journal ID: ISSN 0969-0239
Grant/Contract Number:
AC36-08GO28308; 11-116772; 14-4639; 14-3925
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Cellulose
Additional Journal Information:
Journal Volume: 23; Journal Issue: 4; Journal ID: ISSN 0969-0239
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; imaging; transmission electron microscopy; cellulose surface structure; cellulase; cellobiohydrolase; endoglucanase

Citation Formats

Olsen, Johan P., Donohoe, Bryon S., Borch, Kim, Westh, Peter, and Resch, Michael G. Interrelationships between cellulase activity and cellulose particle morphology. United States: N. p., 2016. Web. doi:10.1007/s10570-016-0979-x.
Olsen, Johan P., Donohoe, Bryon S., Borch, Kim, Westh, Peter, & Resch, Michael G. Interrelationships between cellulase activity and cellulose particle morphology. United States. doi:10.1007/s10570-016-0979-x.
Olsen, Johan P., Donohoe, Bryon S., Borch, Kim, Westh, Peter, and Resch, Michael G. 2016. "Interrelationships between cellulase activity and cellulose particle morphology". United States. doi:10.1007/s10570-016-0979-x. https://www.osti.gov/servlets/purl/1289432.
@article{osti_1289432,
title = {Interrelationships between cellulase activity and cellulose particle morphology},
author = {Olsen, Johan P. and Donohoe, Bryon S. and Borch, Kim and Westh, Peter and Resch, Michael G.},
abstractNote = {It is well documented that the enzymatic hydrolysis of cellulose follows a reaction pattern where an initial phase of relatively high activity is followed by a gradual slow-down over the entire course of the reaction. This phenomenon is not readily explained by conventional factors like substrate depletion, product inhibition or enzyme instability. It has been suggested that the underlying reason for the loss of enzyme activity is connected to the heterogeneous structure of cellulose, but so far attempts to establish quantitative measures of such a correlation remain speculative. Here, we have carried out an extensive microscopy study of Avicel particles during extended hydrolysis with Hypocrea jecorina cellobiohydrolase 1 (CBH1) and endoglucanase 1 and 3 (EG1 and EG3) alone and in mixtures. We have used differential interference contrast microscopy and transmission electron microscopy to observe and quantify structural features at um and nm resolution, respectively. We implemented a semi-automatic image analysis protocol, which allowed us to analyze almost 3000 individual micrographs comprising a total of more than 300,000 particles. From this analysis we estimated the temporal development of the accessible surface area throughout the reaction. We found that the number of particles and their size as well as the surface roughness contributed to surface area, and that within the investigated degree of conversion (<30 %) this measure correlated linearly with the rate of reaction. Lastly, based on this observation we argue that cellulose structure, specifically surface area and roughness, plays a major role in the ubiquitous rate loss observed for cellulases.},
doi = {10.1007/s10570-016-0979-x},
journal = {Cellulose},
number = 4,
volume = 23,
place = {United States},
year = 2016,
month = 6
}

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  • The initial rate of hydrolysis of six commercially available native (type 1) celluloses was determined for the crude cellulase complexes of the thermophilic anaerobic bacterium C. thermocellum and the mesophilic fungus T. reesei. These rates were then compared with certain physical features of the substrates in an attempt to determine the role of cellulose structure in its degradability. Within the substrate series tested, the Clostridium system showed a greater relative range in rate of enzymatic hydrolysis than did the Trichoderma system. Average correlation coefficients for the kinetic rates from bacterial and fungal cellulases, respectively, and the following physical parameters weremore » obtained: relative crystallinity index (RCI) from acid hydrolysis, -0.61 and -0.85; RCI from x-ray diffraction, -0.75 and -0.89; accessibility to formylation at 4 degrees C, +0.49 and +0.60; nonaccessibility to formylation at 65 degrees, -0.40 and - 0.73; fiber saturation point, +0.83 and +0.85. Kinetic and pore volume distribution data suggest that the rate-limiting components of both the bacterial and fungal cellulase systems are of similar size, approximately 43 Angstroms along one axis. 32 references.« less
  • The activity of components of the extracellular cellulase system of the thermophilic fungus Sporotrichum thermophile showed appreciable differences between strains; ..beta..-glucosidase was the most variable component. Although its endoglucanase and exoglucanase activities were markedly lower, S. thermophile degraded cellulose faster than Trichoderma reesei. The production of ..beta..-glucosidase lagged behind that of endoglucanase and exoglucanase. The latter activities were produced during active growth. When growth was inhibited by cycloheximide treatment, the hydrolysis of cellulose was lower than in the control in spite of the presence of both endoglucanase and exoglucanase activities in the culture medium. Degradation of cellulose was a growth-associatedmore » process, with cellulase preparations hydrolyzing cellulose only to a limited extent. The growth rate and cell density of S. thermophile were similar in media containing cellulose or glucose. A distinctive feature of fungal development in media incorporating cellulose or lactose (inducers of cellulase activity) was the rapid differentiation of reproductive units and autolysis of hyphal cells to liberate propagules which were capable of renewing growth immediately.« less
  • Five chemically modified forms of cellulose were prepared, characterized, and tested as substrates for a homogeneous glucanohydrolase from A. niger. The relative order of reactivity at pH 4.0 was DEAE = PEI more than benzyl DEAE more than cellulose more than P more than CM. This indicates that positively charged cellulose substrates are more susceptible to hydrolysis by the cellulase. This observation strengthens an earlier proposal that carboxyl groups on the enzyme are involved in substrate binding and catalytic action. Chemical modification is suggested as a method to increase the rate of enzymatic hydrolysis of cellulose, a process now inmore » the commercial development stage. (Refs. 27).« less
  • Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H/sub 2/SO/sub 4/, Cadoxen, and DMSO-P-formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymaticmore » hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hours by Aspergillus niger cellulase containing mainly 1,4-Beta-glucan glucanohydrolase without action of 1,4-beta-glucan cellobiohydrolase. On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.« less
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